a, Shear moduli of soft substrates during liquid-to-solid gelation over the course of 60 min, shown as G′ and G′′. Time = 0 min indicates the start of the time sweep. The experiment was repeated seven times over two separate days; each experiment is shown in a different colour. b, Shear moduli of preformed soft substrates over the course of an additional 60 min, shown as G′ and G′′. Time = 0 min indicates the completion of 60 min of incubation at 37 °C for gelation and the start of a second 60-min time sweep. The experiment was repeated twice; each repeat is shown in a different colour. The shear modulus increased rapidly and reached a plateau within 5 min, with a sixfold difference between G′ and G′′. Although the substrates are soft, this ratio indicates the formation of stable elastic hydrogels39. c, Left, representative images of calcein AM (green) and ethidium homodimer (red) staining of HBEC76 cells on stiff and soft substrates. Scale bar, 50 μm. Right, fraction of calcein-AM-positive cells relative to the combined count of calcein-AM-positive and ethidium-homodimer-positive cells on stiff (n = 50 images) and soft (n = 50 images) substrates from a single imaging experiment. Individual data points indicate fractions per image. d, Metabolomic profiling of HEBC76 cells on stiff and soft substrates (n = 3 independent cultures per substrate stiffness). Metabolic profiling was performed once. e, Volcano plot of metabolic shifts between substrates derived from d. Each dot shows the average ratio in metabolite concentration between stiff and soft substrates versus P values, based on three independent cultures. Vertical dotted line, log 2 -transformed fold change ±0.5; horizontal dotted line, P value = 0.05). f, Glycolytic rates, normalized to cell number, of HBEC30 and HBEC34 cells on stiff and soft substrates. g, Fraction enrichment of 13C-labelled lactate synthesized from uniformly labelled d-13C-glucose. M, no 13C labelling; M + 1, one 13C label; M + 2, two 13C labels; M + 3, three 13C labels. Data are shown as mean ± s.e.m. of three independent experiments. h, Glycolytic pathway illustrating enzymes tested in Fig. 1d. PFKP, PFKL and PFKM are highlighted in red. i, Verification of the specificity of PFK-isoform-targeting antibodies. GFP-tagged PFK was expressed in HEK293 cells for each isoform, and immunoblotting was performed. The experiment was performed once. j, PFK activity normalized by protein abundance on stiff and soft substrates. Data are shown as mean ± s.e.m. The experiment was performed once, with three technical repeats per substrate stiffness. k, Abundance of PFKFB2 and PFKFB3 in HBEC76 cells, per substrate stiffness. Representative data from three independent experiments. l, Normalized expression of PFKP, PFKL and PFKM in human bronchial epithelium. Data of two replicates are shown as the mean obtained from BioGPS: PFKP (no. 201037), PFKL (no. 201102) and PFKM (no. 201102). m, Abundance of PFKP on stiff and soft substrates in the head and neck epithelial cell line HHN2. The experiment was performed once. n, Normalized expression of glycolytic genes, ranked according to oncogenic enrichment. PFKP is highlighted in red; and GLUT1 (also known as SLC2A1), PFKL and PFKM shown in black. o, Effect of oncogenic transformation of HBEC3 cells on oxidative phosphorylation rates on stiff and soft substrates. Data are normalized to cell number. p, Glycolytic rates, normalized to cell number, of NSCLC cells (H2009, H1819 and HCC827 lines) on stiff and soft substrates. q, Abundance of PFKP in H2009, H1819 and HCC827 cells on stiff and soft substrates. The experiment was performed once. Data in f, o, p are from three independent experiments, shown as mean ± s.e.m. Statistical significance was assessed using two-tailed Mann–Whitney test (c), unpaired multiple t-test (e) or two-tailed Student’s t-test (f, g, j, o, p). Protein abundance was normalized to the abundance of GAPDH (k, m, q). Source data