Loss of GABARAP abrogates CRT exposure during ICD. (A) Correlation between CRT exposure and GABARAP protein expression in a panel of 10 MM cell lines. The surface exposure of CRT was determined by flow cytometry on viable cells after 16 hours of treatment of different cell lines, according to their BTZ sensitivity. Fold change of CRT increase was correlated with abundance of GABARAP protein (as shown in supplemental Figure 2A). (B) Analysis of surface CRT exposure in KMS11 WT and GABARAP^OE after treatment with BTZ (7.5 nM; 16 hours) by flow cytometry of viable cells. (C-D) Effect of BTZ treatment (16 hours) on the exposure of surface CRT in AMO1 (5 nM) (C) and H929 (2.5 nM) cells (D) both with WT and GABARAP^KO as assessed by flow cytometry of viable cells (left). Representative overlay histogram (right) of surface CRT fluorescence (MFI) in AMO1 (C) and H929 (D). (E) Representative images of immunofluorescence staining of surface CRT (red) in nonpermeabilized AMO1 WT and GABARAP^KO before and after treatment with BTZ. DAPI was used to stain nuclei; scale bars, 10 μm. Enlargement pictures of the squared area show CRT exposure on dying cells only in WT condition; scale bars, 2 μm. (F) Analysis of surface CRT exposure in AMO1 WT, GABARAP^KO, and GABARAP^KO in which GABARAP was re-expressed (GABARAP^KO + add-back) after treatment with BTZ (5 nM; 16 hours) by flow cytometry of viable cells. For panels B-D,F, ∗P < .05; ∗∗P < .01. ns, not significant (unpaired Student t test).