Fig. 4: Platinum drugs promote GCLM nuclear localization via GCLM binding to importin a5.

a Schematic illustration of the potential NLS sequence of the GCLM protein predicted by the cNLS Mapper tool. The strategy for mutating the NLS of the GCLM is also shown. b HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS mutant (Mut) with or without importazole treatment (40 μM, 24 h). The localization of GCLM was detected by IF staining. Scale bar = 10 μm. c, d HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS Mut with oxaliplatin or IPZ treatment (40 μM, 24 h). IB detected the nuclear and total GCLM expression (c) and Co-IP analysis showed the level of interaction between NKRF and GCLM (d). e–g GCLM-knockdown HCT116 or DLD1 cells overexpressed rGCLM WT or rGCLM-NLS Mut with or without oxaliplatin treatment (40 μM, 24 h). The transcriptional activity of NF-κB/p65 (e), cell viability (f) and apoptotic cells (g) were detected. h Co-localization of endogenous GCLM and importin α5 in HCT116 cells was detected by IF. Red arrowheads showed the co-localization. Scale bar = 10 μm. i HCT116 cells overexpressed Flag-tagged GCLM WT or GCLM-NLS Mut with or without oxaliplatin treatment (40 μM, 24 h). Co-IP analysis showing the GCLM-importin α5 interaction. IF staining of the localization of GCLM (j) and IB detection of nuclear and total GCLM expression (k) in HCT116 cells with control or importin α5 silencing in presence of oxaliplatin treatment (40 μM, 24 h). Scale bar = 10 μm. l, m GCLM-knockdown HCT116 cells overexpressed vector or rGCLM WT with control or importin α5 silencing under oxaliplatin treatment (40 μM, 24 h). The transcriptional activity of NF-κB/p65 (l), cell viability and apoptotic cells (m) were detected. IB experiments were repeated three times and n = 3 biologically independent experiments in (e–g, l, m). All the data are presented as the mean ± S.D. The P values were calculated by one-way ANOVA (e, l, m) and two-way ANOVA (f, g). IPZ importazole, Imp α5 importin α5.