Fig. 1: GCLM depletion enhances the chemosensitivity of CRC cells to oxaliplatin.

a Diagram showing the strategy for the CRISPR-Cas9 screen in HCT116 cells under the treatment with PBS or oxaliplatin (10 µM, 7 days). The genomic DNA of control and treated cells was isolated and amplified for comparison of sgRNAs via deep sequencing. b Venn diagram showing the top 100 downregulated genes and the overlapping genes in the three oxaliplatin-treated groups compared with the control group (Supplementary Data 1). c Cell viability of HCT116 cells with or without oxaliplatin treatment (40 µM, 24 h) after each candidate gene (CYP3A5, APEH, ASS1, ALG1, GGH, GFPT1, GSTO2, AGMAT, SHMT2 or GCLM) was knocked out. d IC₅₀ Analysis of oxaliplatin in HCT116 and DLD1 cells treated with different concentrations of oxaliplatin for 48 h. e Annexin V/PI staining analysis was used to evaluate the percentages of apoptotic cells among control and GCLM-knockdown CRC cells treated with PBS or oxaliplatin (40 μM for HCT116 cells and 80 μM for DLD1 cells, 24 h). f Statistical analysis of CDX tumor volumes and weights in nude mice after the implantation of GCLM-knockdown or control HCT116 cells (2 × 10⁶), followed by intraperitoneal injections of PBS or oxaliplatin (5 mg/kg). Statistical analysis of the tumor volumes and weights in nude mice in the PDX #1 (g) and PDX #2 (h) models, followed by intratumoral injections of in vivo-optimized GCLM inhibitor (siGCLM) or the control siRNA (5 nmol per injection), and intraperitoneal injections of PBS or oxaliplatin (5 mg/kg). i Representative images of H&E, IHC staining for Ki67, Tunel staining and Masson’s trichrome staining in PDX #1-based paraffin-embedded subcutaneous tumor sections. The red arrowheads indicate the positive cell of Tunel staining. Scale bar = 50 μm. j Quantification of the proliferation index (Ki67 staining) and apoptotic index (Tunel assay) in the PDX #1 models. n = 3 biologically independent experiments in (c, d) and n = 6 mice in (f–h, j). All the data are presented as the mean ± S.D. The P values were calculated by one-way ANOVA (f–h right and j), and two-way ANOVA (f–h left). Oxa oxaliplatin, CDX cell-derived xenograft, PDX patient-derived xenograft, CRC colorectal cancer, ANOVA analysis of variance.