Fig 2: Effects of siWHSC1 on cell growth, migration, and invasion of C33A cells. (A) Effects of WHSC1 inhibition on C33A cell growth were detected by Cell Counting Kit-8 assay: (a) C33A-siWHSC1 in 6 days (×10); (b) C33A-NC in 6 days (×10); (c) growth of C33A cells was inhibited by siWHSC1; *P<0.05, compared to that of siNC-treated cells. (B) Effects of WHSC1 inhibition on the migration of C33A cells were detected by Transwell assay: (a) migration of C33A-siWHSC1 cells (×10); (b) migration of C33A-NC cells (×10); (c) inhibition of migration of C33A cells by siWHSC1; *P<0.05, compared to that of siNC-treated cells. (C) Effects of WHSC1 inhibition on the invasion of C33A cells were detected by Transwell assay: (a) invasion of C33A-siWHSC1 cells (×5); (b) invasion of C33A-NC cells (×5); (c) inhibition of C33A cell invasion by siWHSC1; *P<0.05, compared to that of siNC-treated cells.Abbreviations: NC, normal control; si, small interfering; WHSC1, Wolf–Hirschhorn syndrome candidate 1.

Fig 3: NSD2 upregulation leads to cisplatin resistance in ESCC cells(A) Western blot analysis of NSD2 and H3K36me2 protein levels in ESCC cells transfected with empty vector or NSD2-expressing plasmids. (B) Bar graphs show the cisplatin IC50 values in ESCC cells transfected with empty vector or NSD2-expressing plasmids. (C) Cell proliferation was determined by EdU incorporation assay after treatment with or without cisplatin for 72 h. Left, representative merged images of EdU incorporation (red) and nuclear staining (blue). Scale bar, 50 µm. Right, quantification of EdU-positive cells. *p < 0.05. ns indicates no significance. (D) Measurement of apoptosis by Annexin V/PI staining in the transfected cells after 10 µM cisplatin treatment. (E) Colony formation assay. NSD2 overexpression enhanced long-term survival of ESCC cells after treatment with 10 µM cisplatin. Right, quantitative analysis of colonies. *p < 0.05.

Fig 4: NSD2 depletion increases cisplatin sensitivity in ESCC cells in vivoESCC cells stably transfected with shCtrl or shNSD2 were injected into nude mice. In the drug treatment groups, 3 mg/kg cisplatin was injected. (A and E) Representative images of subcutaneous xenograft tumors from 2 mice of each group. (B and F) Tumor growth curves were plotted. (C and G) Quantitative analysis of the weight of xenograft tumors. (D and H) TUNEL staining showed that NSD2 depletion increased cisplatin-induced apoptosis in xenograft tumors. *p < 0.05.

Fig 5: Clinical significance of NSD2 and MACC1-AS1 in ESCC(A and B) Quantitative real-time PCR analysis of (A) MACC1-AS1 and (B) NSD2 in 70 pairs of ESCC and normal esophageal tissue samples. (C) Immunohistochemistry for NSD2 in 70 ESCC cases and normal tissues. Left, representative images of NSD2 staining. Scale bar, 40 µm. Right, quantitative evaluation of NSD2 staining results. (D) Survival analysis of ESCC patients from TCGA dataset indicated that ESCC patients with high MACC1-AS1 expression in tumors had worse overall survival compared to those with low MACC1-AS1 expression. (E) Analysis of the correlation between MACC1-AS1 and NSD2 expression in ESCC tissues (n = 70). (F) Schematic model showing that NSD2 promotes the expression of MACC1-AS1 and FOXD2-AS1, enhancing ESCC cell survival and cisplatin resistance.