Fig. 4: The GAPDH switch allows cells to survive the loss of anchorage.
From: The GAPDH redox switch safeguards reductive capacity and enables survival of stressed tumour cells
a, Colonies formed after seeding of 100 WT or mutant (Y314F) HAP1 cells into adherent cell culture plates. Before seeding, singularized cells were kept in ultralow-attachment plates for either 0.25 h (left), or 6 h (middle and right), in the absence (left and middle) or presence (right) of 10 mM NAC. Data are presented as mean ± standard deviation (s.d.), based on n = 3 biological replicates with n = 3 technical replicates each. NS, non-significant; **P < 0.01; P = 0.8860, 0.0059 and 0.4174, based on a two-tailed unpaired t-test. b, Relative intracellular oxidant levels as indicated by DCF fluorescence, corresponding to a. Data are presented as mean ± s.d., based on n = 3 biological replicates with n = 3 technical replicates each. NS, non-significant; *P < 0.05; **P < 0.01; P = 0.4696, 0.0057 and 0.0418, based on a two-tailed unpaired t-test. c, The roGFP2-Orp1 fluorescence ratio in WT and mutant (Y314F) HAP1 cells grown in adherent monolayer cell culture. Left: representative histogram. Right: quantitation presented as mean ± s.d. based on n = 3 biological replicates. NS, non-significant; P = 0.1583, based on a two-tailed unpaired t-test. d, Visualization of roGFP2-Orp1 in WT (top) and mutant (bottom) HAP1 cells grown in adherent monolayer culture. Fluorescence was recorded following sequential excitation at 405 nm (left) and 488 nm (middle). The fluorescence ratio image (right) was calculated pixel-by-pixel and colour-coded. Representative of n = 3 biological replicates. e, The roGFP2-Orp1 fluorescence ratio in WT and mutant (Y314F) HAP1 cells grown in non-adherent spheroid culture. Left: representative histogram. Right: quantitation presented as mean ± s.d., based on n = 9 biological replicates. ****P < 0.0001, based on a two-tailed unpaired t-test. f, Visualization of roGFP2-Orp1 in WT (top) and mutant (bottom) HAP1 cells grown in non-adherent spheroid culture. Fluorescence was recorded following sequential excitation at 405 (left) and 488 nm (middle). The fluorescence ratio image (right) was calculated pixel-by-pixel and colour-coded. Representative of n = 6 (WT) and n = 7 (Y314F) biological replicates. g, Staining of WT (top) and mutant (bottom) HAP1 spheroids with Hoechst dye (left) or PI (middle). Representative of n = 3 biological replicates. h, Staining of WT (top) and mutant (bottom) EF spheroids with Hoechst dye (left) or PI (middle). Representative of n = 3 biological replicates.
