Fig 2: Overexpression of LRRC8A rescues the phenotype of CC cells with NSUN2 knockdown. (A) Western blot confirmed the protein expression of NSUN2 and LRRC8A in NSUN2-depleted CC cells with or without LRRC8A re-expression. (B) Overexpression of LRRC8CA rescues the cell proliferation suppressed by NSUN2 knockdown in HeLa and SiHa cells by CCK-8 assays. (C) Overexpression of LRRC8A restores the colony formation of CC cells suppressed by NSNU2 knockdown. (D, E) Cell migration and invasion analysis of NSUN2-depleted CC cells with or without LRRC8A re-expression. Scale bar, 200 µm. (F) Protein expression levels of p-PI3K, p-AKT, PI3K, AKT and Caspase-3 in NSUN2-depleted CC cells with or without LRRC8A re-expression. (G) Spearman's correlation analysis between mRNA levels of NSUN2 and LRRC8A in cervical cancer. (H) Representative images showing protein levels of NSUN2 and LRRC8A in nude mouse xenograft tumors. Scale bar, 500 µm. (I) Statistical analysis of IHC score showing NSUN2 and LRR8CA levels in xenograft tumors. Data are presented as means ± S.D: ***P < 0.001.

Fig 3: LRRC8A maintains cellular homeostasis of CC cells under the hypotonic condition. (A, B) Swelling analysis of CC cells with LRRC8A overexpression or knockdown under the the hypotonic condition. Scale bar, 10 μm. (C, D) CCK-8 assays detecting the survivals of CC cells with LRRC8A overexpression or knockdown upon the hypotonic treatment. (E) Changes of protein levels of p-PI3K p-AKT, PI3K, AKT and Caspase-3 in HeLa and SiHa cells upon LRRC8A knockdown. (F) Protein levels of p-PI3K, p-AKT, PI3K, AKT and in LRRC8A-depleted CC cells with or without SC79 treatment. Data are presented as means ± S.D: **P < 0.01, ***P < 0.001.

Fig 4: NSUN2 promotes m5C modification and expression of LRRC8A mRNA. (A) meRIP-PCR assays detecting the RNA m5C modification of LRRC8A in HeLa and SiHa cells upon NSUN2 knockdown. (B) Dot blot detecting the global m5C modification of mRNAs in CC cells with or without NSUN2 knockdown. Methylene blue (MB) staining was used as a loading control. (C) Heamap of differentially expressed genes from RNA-seq analysis of HeLa and SiHa cells upon NSUN2 knockdown. (D) Gene ontology analysis of differentially expressed genes described in (C). (E) Volcano plot of NSUN2 bound transcripts from RIP-seq analysis in SiHa cells. (F) A Venn diagram shows the potential targets of NSUN2 through the combined analysis of RNA-seq, RIP-seq from the present study and the reported RNA Bis-seq in HeLa cells. (G) Sequence frequency logo for the sequences proximal to mRNA m5C sites on the 30 NSUN2-bound mRNA. (H, I) The RNA and protein levels of LRRC8A in CC cells upon NSUN2 knockdown were measured. (J) RIP-PCR assays detecting the interaction between NSUN2 and LRRC8A mRNAs in HeLa and SiHa cells. IgG was used as an internal control. GAPDH was used as the negative control in western blot assays. Data are presented as means ± S.D: ***P < 0.001.

Fig 5: Effects of VRAC inhibitors and silencing of LRRC8A combined with PTX on pyroptosis-related proteins. (A) Representative western blotting and (C and D) analyzed data was used to detect the effect of PTX combined with DCPIB on the expression of GSDME-N and cl-caspase-3 proteins (n=3; **P<0.01 and ***P<0.001 vs. Control; #P<0.05 and ###P<0.001 vs. the PTX group). (B) Representative western blotting results and (E-G) analyzed data was used to detect the effects of PTX on the expression of LRRC8A, GSDME-N and cl-caspase-3 proteins by LRRC8A siRNA treatment (n=3; **P<0.01 and ***P<0.001 vs. Control; #P<0.05 and ##P<0.01 vs. the PTX group). The experiments were analyzed using Dunnett's t-test. VRAC, volume-regulatory anion channel; LRRC8A, leucine-rich repeat-containing 8a; PTX, paclitaxel; GSDME-N, cleaved-gasdermin E N-terminal fragment; DCPIB, 4-(2-butyl-6,7-dichloro-2-cyclopentyl-indan-1-on5-yl) oxobutyric acid; siRNA or si-, small interfering RNA.