(A) A549 cells infected with lentiviruses expressing either FLAG-tagged WT Bach1 or Bach1(Y11F) under the control of a doxycycline-inducible promoter were treated (where indicated) with doxycycline for 24 hours. Doxycycline was then washed out to stop transcription and allow protein decay, and cells were collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM. w-o, wash-out.

(B) H2009 cells infected with lentiviruses expressing either FLAG-tagged WT Bach1 or Bach1(Y11F) under the control of a doxycycline-inducible promoter were treated (where indicated) with doxycycline for 24 hours. Doxycycline was then washed out to stop transcription and allow protein decay, and cells were collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM. l.ex., long exposure; s.ex., short exposure; w-o, wash-out.

(C) A549 cells infected with lentiviruses expressing either FLAG-tagged WT Bach1 or Bach1(Y11F) under the control of a doxycycline-inducible promoter were treated (where indicated) with doxycycline for 24 hours. Doxycycline was then washed out and, after 4 hours, cells were treated with hemin (10 μM), collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM. l.ex., long exposure; s.ex., short exposure; w-o, wash-out.

(D) H2009 cells infected with lentiviruses expressing either FLAG-tagged WT Bach1 or Bach1(Y11F) under the control of a doxycycline-inducible promoter were treated (where indicated) with doxycycline for 24 hours. Doxycycline was then washed out and, after 4 hours, cells were treated with hemin (10 μM), collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM. w-o, wash-out.

(E) A549 cells infected with lentiviruses expressing either FLAG-tagged WT Bach1 or Bach1(Y11F) under the control of a doxycycline-inducible promoter were transfected with either an EV or HA-tagged Fbxo22 as indicated. Cells were treated with doxycycline for 24 hours. Doxycycline was then washed out and, after 4 hours, cells were treated with CHX, collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM. l.ex., long exposure; s.ex., short exposure.

(F) Schematic representation of the Bach1 genomic locus and 2 different gRNA target locations. Exon 2 refers to the mouse Bach1 gene ( GRCm38/mm10; chr16:87,698,954-87,733,346 ).

(G) The indicated proteins in KP-sgTom and KP-sgBach1 clones were analyzed by immunoblot.

(H) KP-sgBach1 clone #4 was infected with lentiviruses expressing either an EV, FLAG-tagged WT Bach1, or Bach1(Y11F) under the control of a doxycycline-inducible promoter in combination with lentiviruses expressing either an EV or HA-tagged Fbxo22. Where indicated, cells were treated with doxycycline for 24 hours. Protein expression was analyzed by immunoblot with antibodies to the indicated proteins.

(I) Cells used in (H) were treated with doxycycline for 24 hours. Doxycycline was then washed out and, after 4 hours, cells were treated with CHX, collected at the indicated times, lysed, and immunoblotted as indicated. The graph shows the quantification of protein levels. Values are presented as means ± SEM.