Fig. 8: Once PD-L1 penetrates the nucleus, it stimulates the activation of the cancer-promoting factor TM4SF1.

A, H Three shRNAs (sh-PD-L1-1#, sh-PD-L1-2#, and sh-PD-L1-3#) were designed to silence CRC cells (HCT116 and SW480) and analyzed by qRT-PCR and Western blotting for verification. We used sh-PD-L1-3# to downregulate PD-L1. B RNA sequencing was employed to assess the differential expression of mRNAs in the sh-PD-L1 and sh-NC groups. In the Volcano plot, the color red signifies that the gene expression level of the sh-NC group is greater than that of the sh-PD-L1 group, while the color blue suggests a lower expression level. TM4SF1 is denoted by an arrow. C Heatmap displaying the mRNAs that are expressed differently between the sh-PD-L1 and sh-NC groups. Greater intensity of the color red corresponds to a higher degree of expression. D Performing GO analysis on mRNA in CRC cells treated with sh-PD-L1 and sh-NC. E Performing KEGG pathway analysis on mRNA in CRC cells treated with sh-PD-L1 and sh-NC. F, I PD-L1 overexpression in CRC cells (HCT116 and SW480) was confirmed using qRT-PCR and Western blotting techniques. G The expression of TM4SF1 mRNA in cells from each group (Vector, PD-L1, PD-L1+sh-NC, and PD-L1+sh-IPO5) was detected by qRT-PCR. J, K Western blot analysis was performed to examine the expression of TM4SF1 and PD-L1 in each experimental group, including sh-NC, sh-PD-L1, Vector, and PD-L1. L, M Western blot analysis was performed on cells from each group, namely Vector, PD-L1, PD-L1+sh-NC, and PD-L1+sh-IPO5, to detect TM4SF1 protein expression. Following the separation of cells into nuclear and cytoplasmic fractions, the nuclear protein fraction was subjected to PD-L1 Western blot analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.