Animal Breeding and Drug Treatments Covington et al., 2009 Covington 3rd, H.E.

Maze I.

LaPlant Q.C.

Vialou V.F.

Ohnishi Y.N.

Berton O.

Fass D.M.

Renthal W.

Rush 3rd, A.J.

Wu E.Y.

et al. Antidepressant actions of histone deacetylase inhibitors. Vialou et al., 2010 Vialou V.

Robison A.J.

Laplant Q.C.

Covington 3rd, H.E.

Dietz D.M.

Ohnishi Y.N.

Mouzon E.

Rush 3rd, A.J.

Watts E.L.

Wallace D.L.

et al. DeltaFosB in brain reward circuits mediates resilience to stress and antidepressant responses. We produced the progeny for each line using in vitro fertilization (IVF) and embryo transfer (ET) techniques, to produce a number of animals sufficient for the behavioral tests and other animal experiments. SMARCA3 KO mice were bred using heterozygous male and female. We carried out all the animal experiments using age (10-15 weeks)- and gender (male)-matched littermates. The BAC-[p11]-eGFP transgenic mice were bred against C57BL/6 mice (Taconics) to obtain hemizygote and used at an adult age (12-18 weeks). Food and water were provided ad libitum. For chronic drug treatments, mice were housed 1-2 per cage and SSRIs (fluoxetine or escitalopram) were administered either by the subcutaneous implantation of time-release fluoxetine pellets (Innovative Research of America; Cat. No. X-999; 10 mg/kg/day) for 2 weeks, or by the drinking water in a mixture of fluoxetine (0.160 mg/ml) and β-cyclodextrine (0.45%) up to 4 weeks, or daily I.P. injection (Escitalopram, 10 mg/kg/day) up to 4 weeks. Control groups were administered placebo pellet or vehicle only (). Drug solution in the amber water bottles was replaced with fresh drug solution every 3 days.

Plasmid Construction and Mutagenesis To generate the plasmid encoding p11 (pIRESneo-p11-Flag-HA), the p11 CDS with Flag-HA epitopes in the C terminus was subcloned into pIRES-neo3 vector (Clontech) at the two restriction enzyme cleavage sites (NheI, NotI) by PCR tagging. The WT p11 construct was used as a template to generate the mutants including C83Q, C83S and D60A using the mutagenesis kit (QuikChange II, Agilent). The plasmid encoding AnxA2 (pEGFP-N3-AnxA2-myc-His) was generated by cloning the AnxA2 CDS with myc-6xHis plus stop codon into pEGFP-N3 vector using PCR tagging with enzyme cleavage (BamHI, SmaI/EcoRV). The WT construct was used as a template to generate the mutants including L8A, L11A, and L13A. The plasmids encoding SMARCA3 (pAAV-CBA-WPRE-SMARCA3-V5) were generated by cloning the SMARCA3 CDS into pAAV-CBA-WPRE vector (In lab) with insertion of V5 epitope in the C terminus using PCR tagging with enzyme cleavage (NheI, NotI). The WT SMARCA3-V5 construct was used as a template to generate the mutants including P35A and F37Y. For the PAI1-promoter-luciferase construct, PAI-1 promoter region ranging from −791 to +38 was amplified using primer pairs (5′-ggt acc agg ctg ctg tac tgg ttc ttg-3′/5′-gat atc gtc ctc ggg gct ctg ct-3′) from the mouse genomic DNA, cloned into pCR2.1 TOPO vector. The sequence-verified insert was cloned into pGL4.10 luciferase reporter vector (Promega) with enzyme cleavage (KpnI, EcoRV). For recombinant protein production in the bacterial system, p11 CDS was amplified using the primer pair (5′-gcc cat atg cca tcc caa atg gag cac gcc-3′/5′-cgg gaattc cta ttt ctt ccc ctt ctg ctt cat-3′), cloned into both pGEX-6P1 vector (GE healthcare) and pET29b+ vector (Novagen) for GST-tag (pGEX-6P1-p11) and 6xHis tag (pET29b-p11), respectively by using the restriction enzyme cut (NdeI, EcoRI). For recombinant AnxA2 production, AnxA2 CDS was amplified using the primer pair (5′-gct cat atg tct act gtc cac gaa atc ctg tg-3′/5′-cgt gcg gcc gct cag tca tcc cca cca cac ag-3′), cloned into pET29b+ vector (Novagen) to generate the pET29b+AnxA2 construct (NdeI, NotI). For expression of recombinant SMARCA3 protein, the coding region for the N-terminal fragment of SMARCA3 (Met1-350) was amplified using the primer pair (5′-ccctctagaaataatgtcctggatgttcaagagggat-3′/5′-ggcaagctttgccttttcactggtattgtttcc-3′) and inserted into pET29b(+) vector (XbaI, HindIII). SMARCA3 deletion constructs were generated using PCR with the primer pairs shown below, followed by cloning into pCR2.1 TOPO vector (Invitrogen), and used for in vitro translation ( Figures S1 B–S1D). Primer pairs (forward/reverse) for PCR: SMARCA3 full length (cacc agg cgc tcc tct tgt catc/ taa gtc aat taa tgt tct gat ttc at), Δ854-C (cacc agg cgc tcc tct tgt catc/ttt tat gtt ggg att ctt ctt tct), Δ744-C (cacc agg cgc tcc tct tgt catc/ctt ctt tct cag ttc ttc agg tgt), Δ631-C (cacc agg cgc tcc tct tgt catc/aat tcc tcc ttc atc tcc cat t), Δ231-C (cacc agg cgc tcc tct tgt catc/cat ttc atg ggt ttt atc atc ttc), ΔN1-122 (cac caa agt aaa caa tgt gaa tgg aaa tc/taa gtc aat taa tgt tct gat ttc at), ΔN1-155 (cac cat gaa tag aaa agc ggt ttc aga tca gt/taa gtc aat taa tgt tct gat ttc at), ΔN1-230 (cac cat ggt tct agc ttg gat ggt gtc/taa gtc aat taa tgt tct gat ttc at).

Quantitative RT-PCR Total RNA was extracted with standard method using Trixol-LS reagent (Invitrogen), precipitated with sodium acetate, treated with DNase I (QIAGEN), and purified with RNeasy kit (QIAGEN). RNA quantity was measured with a Nanodrop 1000 spectrophotometer and quality was assayed with RNA nanochip kit (Agilent 2100 Bioanalyzer). cDNA was synthesized from 1 μg of total RNA using ImProm-II Reverse Transcription System (Promega) with Oligo dT primer according to manufacturer’s protocol. 10-50 ng of cDNA was used for each qPCR reaction and all samples were run in triplicate. Q-PCR was carried out in Applied Biosystems 7900HT system following standard cycling conditions (50°C for 2 min, 95°C for 10 min, then 40 cycle of 95°C for 15 s and 60°C for 1 min). All Taqman assays were purchased from Applied Biosystems as follows. Tagman assay ID for each gene; p11 (Mm00501457_m1), AnxA2 (Mm00500307_m1), SMARCA3 (Mm00447302_m1). All data were normalized to TaqMan Rodent GAPDH Control, and relative expression levels between conditions were calculated by the comparative C T (2-ΔΔCT) method, and all statistical analyses were done using Student’s t test.

Western Blot Analysis Cell pellets were lysed in lysis buffer (30 mM HEPES, pH7.2, 140 mM NaCl, 1 mM EDTA, 2 mM MgCl 2 and 0.2% Triton X-100) supplemented with a protease inhibitor cocktail (Complete-EDTAfree; Roche) and a phosphatase inhibitor cocktail (PhosStop, Roche). The lysates were incubated on ice for 30 min with intermittent vortex mixing every 5 min. The hippocampal tissues were added in 5 times excess volume of the lysis buffer, sonicated with probe-type sonicator (Branson) for 10 s, twice. The cell or tissue lysate was centrifuged and the protein level in the supernatant was measured by BCA. The protein samples were boiled in standard protein sample buffer, and subjected to SDS-PAGE followed by protein transfer onto a nitrocellulose membrane. Immunoblotting was performed with a standard protocol using the following antibodies: anti-p11 (mouse monoclonal, 1:1000, BD bioscience), anti-p11 (goat polyclonal, 1:200, R&D systems), anti-SMARCA3 (goat polyclonal, 1:200, NOVUS), anti-SMARCA3 (rabbit polyclonal, 1:500), anti-AnxA2 (mouse monoclonal, Santa Cruz), anti-myc (mouse monoclonal, 9E10, Sigma-Aldrich), anti-V5 (rabbit polyclonal, Chemicon), anti-GAPDH (mouse monoclonal, Chemicon), anti-AnxA1 (rabbit polyclonal, Zymed), anti-S100B (mouse monoclonal, BD transduction lab), anti-SPT6 (rabbit polyclonal, Bethyl lab), anti-AHNAK1 (mouse monoclonal, NOVUS), anti-lamin-B (rabbit polyclonal, Abcam), Anti-HA (rat monoclonal, Roche), and anti-actin (rabbit polyclonal, Cytoskeleton).

Immunoprecipitation Lysates from cultured cells or brain tissue were prepared as described above. After centrifugation at 12,000 x g for 10 min, the supernatant was precleared with protein A/G agarose beads (Pierce), and incubated with antibody-coupled agarose beads for 3 hr at 4°C, with constant rotation. After washing three times with the lysis buffer and one time with the lysis buffer without Triton X-100, the bound proteins were eluted by boiling in the protein sample buffer for 5 min, and subjected to SDS-PAGE. Antibody-coupled beads were as follows: anti-p11 antibody-coupled protein A/G agarose gel (mouse polyclonal anti-p11 antibody), anti-SMARCA3 antibody-coupled protein A/G agarose gel (rabbit polyclonal anti-SMARCA3 antibody I or II), anti-Flag affinity agarose gel (mouse monoclonal anti-Flag M2 antibody; Sigma-Aldrich), anti-Myc affinity agarose gel (goat polyclonal anti-Myc antibody; Bethyl laboratory), and anti-V5 affinity agarose gel (mouse monoclonal anti-V5 antibody, Sigma-Aldrich).

Antibody Generation Czernik et al., 1991 Czernik A.J.

Girault J.A.

Nairn A.C.

Chen J.

Snyder G.

Kebabian J.

Greengard P. Production of phosphorylation state-specific antibodies. Mouse polyclonal antisera against mouse p11 were obtained by immunizing recombinant mouse p11 into p11 homozygous KO mice. p11 homozygous KO mice at 6-7 weeks old were immunized subcutaneously with recombinant p11 (50 μg) protein emulsified with TiterMax adjuvant (CytRx Co.) according to the manufacturer’s instruction. The mice were boosted more than 6 times with emulsified antigen at 3 week intervals prior to the final boosting with the intravenous injection of soluble antigen (100 μg). A week later, sera were collected with retro-orbital bleeding under deep anesthesia, and tested by immunoblotting and immunoprecipitation. Rabbit polyclonal antibody was purified as reported () with minor modification. The 14-mer peptides derived from SMARCA3 (rabbit polyclonal-I; DIIPPDDFLTSDEE) and (rabbit polyclonal-II; EERKIYQSVKNEGK) were synthesized with C-terminal cysteine residue, coupled to the KLH protein using Sulfo-MBS cross-linker (Pierce Biotech). The peptide-KLH conjugates were desalted using a PD-10 column (GE healthcare), emulsified with TiterMax adjuvant (CytRx Co.), and immunized into rabbits (Cocalico Inc.). The IgG fraction was purified using protein A/G affinity chromatography from the sera of the immunized rabbit and the specific antibody fraction was further purified using peptide-coupled resins (SulfoLink Coupling Resins and Immobilization Kits, Pierce Biotech).

In Vitro Binding Analysis GST-p11 Pull-Down Assay 2 and 0.2% Triton X-100) supplemented with the protease inhibitor cocktail (Roche). After washing out the unbound AnxA2 with the binding buffer, GST, GST-p11 or GST-p11/AnxA2 complex were incubated with either full-length SMARCA3 or its fragments which were generated by in vitro transcription/translation with [35S-Methionine] labeling (TnT Quick Coupled Transcription/Translation System, Promega). After 3 hr incubation at 4°C with constant rotation, the protein complexes on the beads were eluted using the sample buffer, and were subjected to SDS-PAGE. The 35S-labeled SMARCA3 proteins on the dried gels were visualized using PhosphoImager ( GST or GST-p11 (5 μg) was immobilized on GSH-agarose (GE healthcare), and preincubated with AnxA2 (15 μg or as indicated) for 1 hr at 4°C to produce p11/AnxA2 complex in the binding buffer (30 mM HEPES, pH7.2, 140 mM NaCl, 1 mM EDTA, 2 mM MgCland 0.2% Triton X-100) supplemented with the protease inhibitor cocktail (Roche). After washing out the unbound AnxA2 with the binding buffer, GST, GST-p11 or GST-p11/AnxA2 complex were incubated with either full-length SMARCA3 or its fragments which were generated by in vitro transcription/translation with [S-Methionine] labeling (TnT Quick Coupled Transcription/Translation System, Promega). After 3 hr incubation at 4°C with constant rotation, the protein complexes on the beads were eluted using the sample buffer, and were subjected to SDS-PAGE. TheS-labeled SMARCA3 proteins on the dried gels were visualized using PhosphoImager ( Figures S1 B–S1D). Peptide Pull-Down Assay Short peptides derived from SMARCA3 (aa26-42) or AHNAK1 (aa5654-5671) were synthesized with a biotin-tag at the C terminus, and immobilized onto streptavidin-coupled magnetic beads (Dynabead M-280 Streptavidin, Invitrogen). The peptide-coupled beads were incubated with p11/AnxA2 complex (1 μg of the complex) in the binding buffer. After three times washing, the bound protein was eluted by boiling in the sample buffer, and subjected to SDS-PAGE followed by western blot analysis using the antibodies indicated ( Figure 2 F). Oligonucleotide Pull-Down Assay Herlt et al., 1988 Herlt M.

Schwarz H.P.

Neumann R.

Eggers H.J. Detection of DNA-protein binding in western blots by phosphorus-labeled and biotinylated DNA probes. Ding et al., 1996 Ding H.

Descheemaeker K.

Marynen P.

Nelles L.

Carvalho T.

Carmo-Fonseca M.

Collen D.

Belayew A. Characterization of a helicase-like transcription factor involved in the expression of the human plasminogen activator inhibitor-1 gene. Oligonucleotide pull-down assay was performed as described () with minor modification. Briefly, the streptavidin magnetic beads (Dynabeads MyOne Streptavidin T1, Invitrogen) were preabsorbed with the blocking/coupling buffer for 30 min at room temperature with constant rotation. The preabsorbed beads were dispensed into individual reaction tubes, coupled with 1 μg of the biotinylated oligonucleotide duplex (tandem B-box from mouse PAI-1 promoter: 5′-TTA GAA AGT GGG TGG GGC TGG AAC ATG TTA GAA AGT GGG TGG GGC TGG AAC ATG-3′/5′- CAT GTT CCA GCC CCA CCC ACT TTC TAA CAT GTT CCA GCC CCA CCC ACT TTC TAA-3′-Biotin) (), and washed three times with 1 × oligonucleotide binding buffer to remove the unbound oligonucleotide duplex. The washed beads were incubated with the protein solution prediluted in the reaction buffer, for 3 hr at 4°C with constant rotation. The protein complex bound to the magnetic beads was washed three times with 1 × oligonucleotide binding buffer, eluted by boiling in the protein sample buffer, and subjected to SDS-PAGE followed by quantitative western blot analysis (Odyssey Infrared System, LI-COR). Oligonucleotide binding buffer (3 × ): 36 mM HEPES, pH 7.9, 12 mM TRIS, 450 mM KCl, 36% glycerol, 3 mM EDTA, 3 mM DTT. Blocking/coupling buffer: 1 × oligonucleotide binding buffer supplemented with 2 mg/ml BSA (DNase- and lipid-free; Calbiochem), 20 μg/ml poly dI-dC (poly deoxyinosinic-deoxycytidylic acid; Sigma-Aldrich), 200 μg/ml sheared salmon sperm DNA (Invitrogen), and 0.2% NP-40 (Sigma-Aldrich). Reaction buffer: 1 × oligonucleotide binding buffer supplemented with 2 mg/ml BSA, 20 μg/ml poly dI-dC, and 0.2% NP-40.

Protein Expression and Purification Réty et al., 1999 Réty S.

Sopkova J.

Renouard M.

Osterloh D.

Gerke V.

Tabaries S.

Russo-Marie F.

Lewit-Bentley A. The crystal structure of a complex of p11 with the annexin II N-terminal peptide. Rezvanpour et al., 2009 Rezvanpour A.

Phillips J.M.

Shaw G.S. Design of high-affinity S100-target hybrid proteins. 600 reached around 0.8. Then the medium was cooled and 0.4 mM IPTG was added to the culture to induce protein expression overnight at 25°C. The cell extract was prepared using a French Press in buffer A (50 mM TRIS, pH 7.5, 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol and 5% glycerol). After centrifugation, the supernatant was loaded onto a Ni-NTA affinity column. The eluted target protein was exchanged in buffer A by dialysis overnight, with Ulp1 protease added for removal of the Sumo-tag. Digested protein was loaded onto a Ni-NTA column again to remove the 6xHis-Sumo tag and the His-tagged protease. The flow through containing target protein was further purified by using a Hiload 16/60 G75 column in buffer B (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1 mM EGTA and 5% glycerol). The p11-AnxA2 fusion protein was concentrated to about 20 mg/ml for crystallization trials. The p11-AnxA2 peptide cassette [p11(1-92) - QENLYFQGD linker - AnxA2 peptide (2-15)] was designed according to a published protocol (). The gene fragment encoding p11-AnxA2 peptide cassette was inserted into a sumo-pRSFDuet-1 vector (modified pRSFDuet-1 vector with 6xHis plus yeast Sumo as the N-terminal fusion tag). The protein was expressed in BL21 (DE3) RIL E. coli cells in LB medium. Cells were grown at 37°C till ODreached around 0.8. Then the medium was cooled and 0.4 mM IPTG was added to the culture to induce protein expression overnight at 25°C. The cell extract was prepared using a French Press in buffer A (50 mM TRIS, pH 7.5, 400 mM NaCl, 20 mM imidazole, 5 mM β-mercaptoethanol and 5% glycerol). After centrifugation, the supernatant was loaded onto a Ni-NTA affinity column. The eluted target protein was exchanged in buffer A by dialysis overnight, with Ulp1 protease added for removal of the Sumo-tag. Digested protein was loaded onto a Ni-NTA column again to remove the 6xHis-Sumo tag and the His-tagged protease. The flow through containing target protein was further purified by using a Hiload 16/60 G75 column in buffer B (10 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1 mM EGTA and 5% glycerol). The p11-AnxA2 fusion protein was concentrated to about 20 mg/ml for crystallization trials. We used the coexpression strategy to prepare the complex of full-length p11 and full-length AnxA2. The gene fragments encoding p11 and AnxA2 were inserted into the first MCS and the second MCS of the modified pRSFDuet-1 vector, respectively. All the expression and purification conditions were the same as for the p11-AnxA2 peptide cassette except for using a heparin column to remove free p11 protein from the complex. The complex of full-length p11 and full-length AnxA2 was concentrated to about 20 mg/ml in buffer B.

Crystallization Crystals of p11-AnxA2 peptide cassette in complex with AHNAK1 peptide were obtained by mixing at a cassette:peptide molar ratio of 1:2 on ice for 30 min, followed by mixing with an equal volume of reservoir buffer containing 100 mM Tris, pH 7.4, and 30% PEG6000 at 20°C. The complex of the p11-AnxA2 peptide cassette with SMARCA3 peptide was also prepared by mixing the cassette with peptide at a 1:2 molar ratio. When adding the SMARCA3 peptide, precipitation was found in the mixture. By incubating the sample at 37°C for 10 min, the precipitation almost disappeared. Crystals were obtained by mixing 1 μl complex with 1 μl crystallization buffer containing 100 mM MES, pH 5.0 and 1.6 M (NH 4 ) 2 SO 4 . The crystals of full-length p11 and full-length AnxA2 in complex with SMARCA3 peptide were obtained by mixing at a full-length complex:peptide molar ratio of 1:2 on ice for 30 min, followed by mixing with an equal volume of crystallization buffer containing 100 mM citric acid, pH 3.7 and 25% PEG3350.

Structure Determination McCoy et al., 2007 McCoy A.J.

Grosse-Kunstleve R.W.

Adams P.D.

Winn M.D.

Storoni L.C.

Read R.J. Phaser crystallographic software. Emsley and Cowtan, 2004 Emsley P.

Cowtan K. Coot: model-building tools for molecular graphics. Adams et al., 2002 Adams P.D.

Grosse-Kunstleve R.W.

Hung L.W.

Ioerger T.R.

McCoy A.J.

Moriarty N.W.

Read R.J.

Sacchettini J.C.

Sauter N.K.

Terwilliger T.C. PHENIX: building new software for automated crystallographic structure determination. X-ray diffraction data sets for complexes of p11-AnxA2 peptide cassette with AHNAK1 and SMARCA3 peptides were collected at the Advanced Photon Source NE-CAT 24ID-E beam line. The data sets were indexed, integrated and scaled using HKL2000. The structure of p11-AnxA2 peptide cassette - AHNAK1 peptide complex was solved by the molecular replacement method in PHASER () using the structure of p11 and AnxA2 complex (PDB 1BT6 ) as the search model. Further modeling of AHNAK1 peptide was carried out using COOT (), and model refinement was performed in PHENIX (). The final model of the complex was refined to 2.0 Å. The structure of p11-AnxA2 peptide cassette - SMARCA3 peptide complex was solved by molecular replacement method in PHASER using the 2.0 Å structure of the p11-AnxA2 peptide cassette - AHNAK1 peptide complex as the search model. The SMARCA3 peptide was then modeled in COOT and the whole structure of the complex was refined using PHENIX. The final model of the complex was refined to 3.0 Å resolution. Data collection statistics and structural refinement parameters for complexes of p11-AnxA2 peptide cassette with bound AHNAK1 and bound SMARCA3 peptides are summarized in Table S1 The structure of the ternary complex of full-length p11/full-length AnxA2/SMARCA3 peptide was solved by the molecular replacement method in PHASER using the 3.0 Å structure of the p11-AnxA2 peptide cassette bound to SMARCA3 peptide and also free AnxA2 (PDB 1W7B ) as search models. The missing parts were then modeled in COOT and the entire structure of the complex was refined using PHENIX. Data collection statistics and structural refinement parameters for the p11/AnxA2/SMARCA3 peptide ternary complex are summarized in Table S2

Nuclear Matrix Preparation van Steensel et al., 1995 van Steensel B.

Brink M.

van der Meulen K.

van Binnendijk E.P.

Wansink D.G.

de Jong L.

de Kloet E.R.

van Driel R. Localization of the glucocorticoid receptor in discrete clusters in the cell nucleus. 4 ) 2 SO 4 in digestion buffer for 10 min, and washed twice with the CSK buffer. For western blot analysis, the nuclear matrix preparation was solubilized with 1 × RIPA buffer and scraped. For the in situ immunocytochemistry, the whole cells and the nuclear matrices on coverslips (BD BioCoat) were fixed for 10 min with 2% PFA solution. The cells were incubated with PBS containing 0.5% NP-40, followed by 5 min incubation in PBS containing 100 mM glycine. The washed whole cells or the nuclear matrices were subjected to the standard immunocytochemistry procedures using anti-p11 antibody (mouse monoclonal, 1:500, BD bioscience) and anti-SMARCA3 antibody (goat polyclonal, 1:200, Novus biological). Nuclear matrix was prepared as reported with minor modification (). After washing two times with ice-cold PBS, cells were incubated with a cell-permeable cross-linker, 1 mM DSP (Pierce), in 1 × PBS for 1 hr on ice. The cells were washed twice in CSK buffer, incubated with CSK buffer supplemented with 0.5% NP-40, and 0.5 mM sodium tetrathionate (Sigma-Aldrich), and incubated 3 min with CSK buffer supplemented with 0.5 mM sodium tetrathionate. After washing twice with CSK buffer and once with digestion buffer, the chromatin was digested with 200 μg/ml RNase-free DNase I (Sigma-Aldrich) in the digestion buffer for 30 min, extracted with 0.25 M (NHSOin digestion buffer for 10 min, and washed twice with the CSK buffer. For western blot analysis, the nuclear matrix preparation was solubilized with 1 × RIPA buffer and scraped. For the in situ immunocytochemistry, the whole cells and the nuclear matrices on coverslips (BD BioCoat) were fixed for 10 min with 2% PFA solution. The cells were incubated with PBS containing 0.5% NP-40, followed by 5 min incubation in PBS containing 100 mM glycine. The washed whole cells or the nuclear matrices were subjected to the standard immunocytochemistry procedures using anti-p11 antibody (mouse monoclonal, 1:500, BD bioscience) and anti-SMARCA3 antibody (goat polyclonal, 1:200, Novus biological). CSK buffer: 10 mM PIPES, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 1 mM EGTA, protease inhibitor cocktail (Complete-EDTAfree; Roche). Digestion buffer: 10 mM PIPES, pH 6.8, 50 mM NaCl, 300 mM sucrose, 3 mM MgCl 2 , 1 mM EGTA, protease inhibitor cocktail.

Luciferase Reporter Assay PAI-1 promoter-driven luciferase activity (pGL4.10-mPAI1 promoter) was measured using the Dual-Luciferase Reporter Assay System (Promega) with Renilla (pGL4.73, Promega) as a reference. The reporter plasmid mixture of Firefly luciferase/Renilla luciferase vectors (50 ng/0.5 ng) was cotransfected with SMARCA3 (0.2 μg), p11 and AnxA2 expression plasmids (0.4 μg each) into N2a cells. At 24 hr posttransfection, the luciferase activity in the transfected cells was assayed according to the manufacturer’s manual. For p11 silencing, COS-7 cells were transfected with either p11 siRNA duplex (5′-UUG CCA UCU CUA CAC UGG UCC AGGU-3′/5′-ACC UGG ACC AGU GUA GAG AUG GCAA-3′) or negative control duplex (20 pmol; medium GC; Invitrogen). At 24 hr posttransfection, the cells were detached and replated onto 12-well plates. Next day, the cells were cotransfected with SMARCA3 plasmids (0.2 μg) along with the reporter plasmid mixture (50 ng/0.5 ng), and the luciferase activity was measured 24 hr later. All analyses were performed in triplicate, with each experiment repeated at least twice.

Immunohistochemistry Animals were deeply anesthetized and perfused transcardially with PBS, followed by 4% paraformaldehyde (PFA) in PBS. Brains were postfixed in 4% PFA 12 hr at 4°C, and then saturated with 30% sucrose, followed by freezing in the OCT medium on the dry ice block. A cryostat was used to collect coronal sections of 40 μm thickness along the rostro-caudal axis of the hippocampus. Immunofluorescent staining of the sections was carried out by the free floating method. For immunostaining for p11, SMARCA3, nestin, and Ki-67, the sections were pretreated with citrate buffer (10 mM sodium citrate, pH 6.0, 0.05% Triton X-100) for 30 min at 80°C for antigen retrieval, and then rinsed three times with PBS. For BrdU immunostaining, the sections were pretreated with 1 N HCl for 1 hr at 45°C to denature DNA. After being neutralized, the sections were rinsed twice with PBS, and incubated with the blocking buffer (1 × PBS, 0.2% BSA, 2% normal donkey serum, 0.3% Triton X-100) at room temperature for 1 hr. After blocking, sections were incubated with the primary antibodies diluted in the blocking buffer. The immunohistochemistry was done using the following antibodies: anti-p11 (goat polyclonal, 1:100, R&D systems), anti-eGFP (chicken polyclonal, 1:200, Abcam), anti-eGFP (rabbit polyclonal, 1:200, Abcam), anti-SMARCA3 (rabbit polyclonal, 1:500, Thermo Scientific), anti-calretinin (mouse monoclonal, 1:4000, Swant), anti-parvalbumin (rabbit polyclonal, 1:1000, Swant), anti-parvalbumin (mouse monoclonal, 1:1000, Swant), anti-calbindin-D28K (mouse monoclonal, 1:1000, Swant), anti-neuropeptide Y (rabbit polyclonal, 1:1000, Phoenix Pharmaceutical), anti-GFAP (rabbit polyclonal, 1:1000, Sigma-Aldrich), anti-nestin (mouse monoclonal, 1:200, Abcam), anti-BrdU (rat monoclonal, 1:200, Abcam), anti-Ki-67 (rabbit polyclonal, 1:200, Abcam), anti-doublecortin (goat polyclonal, 1:200, Santa-Cruz Biotech), and anti-NeuN (rabbit polyclonal, 1:1000, Millipore). After incubation for 48-72 hr, sections were washed, incubated with Alexa-fluor-conjugated secondary antibodies (Invitrogen), and counterstained with DraQ5. All the sections were examined under a Zeiss LSM710 confocal microscope or wide-field fluorescence microscope (Zeiss).

Immunohistochemistry of BAC-[p11]-eGFP Mice and Densitometric Analysis Amaral et al., 2007 Amaral D.G.

Scharfman H.E.

Lavenex P. The dentate gyrus: fundamental neuroanatomical organization (dentate gyrus for dummies). Brain tissue (n = 5-6/group) was prepared for immunohistochemistry using a standard protocol. Brains were sectioned at 40 μm, and every twelfth coronal section, along the rostro-caudal axis of hippocampus (−2.5 to −4.0 mm AP) was stained immunohistochemically, as described above. Sections were scanned with a Zeiss LSM710 confocal microscope with a 20 x objective lens. Densitometry on digitized images was carried out using ImageJ software (NIH). Hippocampal subregions () were defined by referencing the well-characterized laminar distribution pattern, as well as well-defined anatomical landmarks visualized with counterstaining with DraQ5, a nuclei-staining dye. The average fluorescent intensity is measured by outlining the region of interest (ROI) for each laminar subregion including the hilus, the inner molecular layer (IML), the granular cell layer (GCL), and the outer molecular layer (OML). Relative fluorescence intensity of each subregion was given after normalization within the same section: the value of individual subregion was divided by the value of the outer molecular layer (OML), which shows background fluorescence with negligible immunostaining and also with no change upon fluoxetine treatment. All imaging and analyses were performed blind to the experimental conditions. For quantification comparisons, we used a two-way ANOVA followed by Bonferroni’s t test for pairwise comparisons using Prism (GraphPad Software).

BrdU Labeling and Neurogenesis Assay Malberg et al., 2000 Malberg J.E.

Eisch A.J.

Nestler E.J.

Duman R.S. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. Malberg et al., 2000 Malberg J.E.

Eisch A.J.

Nestler E.J.

Duman R.S. Chronic antidepressant treatment increases neurogenesis in adult rat hippocampus. 3 . Every sixth section throughout the hippocampus was processed for BrdU immunohistochemistry as described. After the staining procedure, all the slides were coded. An experimenter blinded to the slide code counted all BrdU-labeled cells in the granule cell layer (GCL) and the subgranular zone (SGZ) of the dentate gyrus (DG) in the total 12 sections from the individual mouse. The total number of BrdU-labeled cells per section was determined and multiplied by 6 to obtain the total number of cells per dentate gyrus. Two-way ANOVA and post hoc Bonferroni tests were performed on these totals. The effect of chronic fluoxetine treatment on cell proliferation or cell survival in the WT and KO mice was assessed as described (). For the proliferation assay, the mice were labeled with BrdU solution (200 mg/kg) for 2 hr prior to sacrifice by perfusion. For the survival/neurogenesis assay, the mice were injected with BrdU (75 mg/kg) two times per day for three consecutive days, and were perfused 4 weeks later. Preparation of brain sections and immunostaining were done as described above, and stereological quantitation of BrdU-positive cells were performed as described (). Briefly, serial coronal sections (40 μm) were cut through the entire hippocampus in its rostro-caudal extension on a cryostat and stored in PBS with 0.1% NaN. Every sixth section throughout the hippocampus was processed for BrdU immunohistochemistry as described. After the staining procedure, all the slides were coded. An experimenter blinded to the slide code counted all BrdU-labeled cells in the granule cell layer (GCL) and the subgranular zone (SGZ) of the dentate gyrus (DG) in the total 12 sections from the individual mouse. The total number of BrdU-labeled cells per section was determined and multiplied by 6 to obtain the total number of cells per dentate gyrus. Two-way ANOVA and post hoc Bonferroni tests were performed on these totals.