Fig 2: Knockdown of PIK3C3 attenuates the inhibitory effect of sh-KDM5B on ESCC cell radio-resistance. ESCC cells transfected with sh-KDM5B were further transfected with sh-PIK3C3 or sh-NC. (A) Detection of mRNA expression of PIK3C3 in cells by RT-qPCR. (B) Cell colony formation was detected by colony formation after 24 h of radiation treatment. (C) Cell apoptosis measured using flow cytometry. (D) Cell cycle distribution examined using flow cytometry. (E) The expression of cell cycle-related proteins Cyclin B1, CDC2 and pCDC2 tested using Western blot analysis. (F) The number of autophagosomes in cells examined using MDC staining. *P < 0.05, **P < 0.01; #P < 0.05 compared with 0 Gy + sh-KDM5B + sh-NC or sh-KDM5B + sh-NC; %%P < 0.01 compared with sh-KDM5B + sh-NC or 8 Gy + sh-KDM5B + sh-NC. The results were measurement data, which were expressed as the mean ± SD. Comparisons between two groups were conducted using unpaired t-test (A), and comparisons between multiple groups analyzed by one-way (C, F) or two-way ANOVA (B, D, E) with Tukey’s post hoc test. The experiment was independently repeated three times.

Fig 3: Circ_0087960 bind to KDM5B and protected from SKP2-induced degradation. (A) Prediction of the interaction between circ_0087960 and KDM5B by RPIseq database. (B) RNA pull-down assay to determine the interaction between circ_0087960 and KDM5B, lncRNA H19 was used as a positive control. (C) Validation of circ_0087960 overexpression in 293T cells and PDLSCs. (D) Determination of KDM5B protein degradation after indicated treatment in 293T cells and PDLSCs. (E) Protein levels of KDM5B, ALP, Runx2 and OCN in PDLSCs after indicated treatment. n = 3, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig 4: Inhibition of KDM5B promotes sensitivity of ESCC cells to radiotherapy and elevates the apoptosis rate. ESCC cells were transfected with sh-KDM5B or sh-NC. (A) mRNA and protein expression of KDM5B in cells by RT-qPCR and Western blot. (B) Cell colony formation was detected by colony formation after 24 h of radiation treatment. (C) Cell apoptosis assessed using flow cytometry. (D–E) Cell cycle distribution examined using flow cytometry. (F) The expression of cell cycle-related proteins Cyclin B1, CDC2 and pCDC2 tested using Western blot analysis. *P < 0.05, **P < 0.01; #P < 0.05 compared with 0 Gy + sh-NC; %P < 0.05, %%P < 0.01 compared with 8 Gy + sh-NC. The results were measurement data, which were expressed as the mean ± SD. Comparisons between multiple groups analyzed by two-way ANOVA with Tukey’s post hoc test. The experiment was independently repeated three times.

Fig 5: Inhibition of KDM5B activates PIK3C3 expression by promoting H3K4me3 methylation modification of the PIK3C3 promoter. (A) UCSC prediction of PIK3C3 histone methylation activation. (B) Detection of PIK3C3 expression in ESCC tissues by RT-qPCR. (C) Detection of mRNA expression of PIK3C3 in cells after transfection by RT-qPCR. (D) The protein expression of H3K4me3 in cells after transfection using Western blot. (E) Detection of PIK3C3 promoter enrichment by Anti-KDM5B and Anti-H3K4me3 examined using ChIP-qPCR. **P < 0.01, ***P < 0.01 compared with adjacent tissues or sh-NC. The results were measurement data, which were expressed as the mean ± SD (n = 30). Comparisons between two groups were conducted using paired t-test (B), and comparisons between multiple groups analyzed by two-way ANOVA (C, D, E) with Tukey’s post hoc test. The experiment was independently repeated three times.