Fig 2: PCGF1 knockdown inhibits tumour growth in vivo.A, B Tumour size in nude mice after control and shPCGF1 HCT116 CRC cell injection on the day of harvest. C, D Tumour volume was measured every 3 days. Comparisons were performed for the shPCGF1 group versus the control group. E–H The histogram shows the average tumour weight and mouse weight of the two groups. n = 5 per group; error bars represent the SEM; **P < 0.01, *P < 0.05 versus control; one-way ANOVA and t-test. I Tumour sections were subjected to H&E staining and IHC using antibodies against PCGF1. H&E staining and IHC, scale bar, 20 µm. J, K Immunofluorescence staining of Ki-67 (red) and TUNEL (green) was performed to evaluate the proliferation of tumour cells. DAPI was used to stain the nuclei (blue). Ki-67 staining, scale bar, 20 µm; TUNEL staining, scale bar, 50 µm.

Fig 3: PCGF1 enhances colorectal cancer stemness marker transcription by increasing histone methylation at the promoters.A, B Western blot analysis of whole-cell lysates showed the expression of histone methylation and acetylation modification after PCGF1 silencing and overexpression, and the results were semiquantified using ImageJ software. C, D ChIP-qPCR analysis identified the expression of H3K4me3 and H3K27me3 at the promoters of CD133, CD44 and ALDH1A1 after PCGF1 knockdown in HCT116 cells. E, F The mRNA levels of the H3K27me3 methyltransferase EZH2; H3K27me3 demethylases KDM6A and KDM6B; and H3K4me3 methyltransferases KMT2A, KMT2B, and KMT2C in CSCs. G Immunofluorescent staining analysis of KDM6A expression in CSCs. Scale bar, 100 µm. shCtrl, shControl. ***P < 0.001, **P < 0.01, *P < 0.05, t-test. Data are shown as the mean ± SEM of at least three replicates. ß-actin was used as a loading control.

Fig 4: PCGF1 is upregulated in colorectal cancer.A Expression of PCGF1 in different types of tumours in the GEDS database. The red boxes refer to colorectal READ and COAD. B Expression of PCGF1-6 in colorectal cancer in the GEPIA database. COAD (num(T) = 275; num(N) = 41), READ (num(T) = 92; num(N) = 10). T, tumour; N, normal. C PCGF1 expression levels in specimens with various clinical stages. D Kaplan–Meier analyses of the OS rate in CRC patients in the GEPIA database with high or low expression of PCGF1 (n = 135, log-rank test). The hazard ratio (HR) and P value (log-rank test) for each comparison are shown.

Fig 5: PCGF1 is highly expressed in colorectal cancer stem cells.A, B RT-qPCR and western blot analysis of the PCGF1 mRNA and protein levels in HCoEpiCs and a panel of CRC samples, and the relative intensity values (PCGF1/ß-actin) were measured with ImageJ software. C HCT116 and SW620 cells were cultured in a serum-free environment for 9 days. Sphere morphology was photographed using light microscopy. Scale bar, 50 µm. D RT-qPCR analysis of the expression of CD133 and CD44 in colorectal cancer stem-like cells and adherent cells. E The expression levels of the stem cell markers CD133 and LGR5 were detected by immunofluorescence staining. Scale bar, 50 µm. F, G RT-qPCR and western blot analysis of PCGF1 mRNA and protein levels in adherent and CSCs, and the relative intensity values (PCGF1/ß-actin) were measured with ImageJ software. **P < 0.01, *P < 0.05, one-way ANOVA and t-test. Data are shown as the mean ± SEM of at least three replicates. ß-actin was used as a loading control.