Extended Data Fig. 8: PIBF1-induced activation of CRC pathogenesis by suppressing perforin release from NK cells.
a-b. The effect of PIBF1 overexpression (a) and knockdown (b) on their expression level in SW480 cells (top) and HCT116 cells (bottom) was validated by qRT-PCR analysis. n = 9 biologically independent samples for each group. c-d. Western blot assays validated the effect of PIBF1 overexpression (c) and knockdown (d) on the expression level of PIBF1 in SW480 cells (top) and HCT116 cells (bottom). e-f. The effect of PIBF1 overexpression (e) and knockdown (f) on cell proliferation and colony formation ability in HCT116 cells. n = 4 biologically independent samples for cell proliferation and n = 9 biologically independent samples for colony formation ability for each group. g-h. The effect of PIBF1 overexpression (g) and knockdown (h) on tumor cell lysis (%) in HCT116 cells after co-culturing with NK cells at the ratio of 1:1, 2:1 and 5:1 for NK cell: HCT116 cell. n = 9 biologically independent samples per group. i. Representative images and growth curves of MC38 tumors with PIBF1 knockout in C57BL/6 mice. n = 5 per group. j. Representative H&E staining and immunohistochemical analysis (Ki67 and PIBF1). k-l. The effect of PIBF1 overexpression (k) and knockdown (l) on the proportion of intracellular perforin in NK cells (left) and the level of perforin in supernatant solution (right) co-cultured with HCT116 cells. n = 9 biologically independent samples for each group. m. The effect of PIBF1 knockout on the proportion of intracellular perforin in NK cells from the MC38 tumors (left). The effect of PIBF1 knockout on the level of perforin in supernatant solution from the MC38 tumors (right). n = 5 biologically independent samples. n. Immunofluorescent staining shows colocalization between perforin (green) and NK1.1 (red) from MC38 tumors (n = 5 per group). Scale bar, 20μm and 100μm. Results for cell proliferation were shown as the mean ± SEM (e, top, f, left). The results presented colony formation ability relative to control cells (set to 100%) (e, bottom, f, right). Data were shown as the mean ± SD (a-b, e, bottom, f, right, i, k-m). Data were shown as the median, box limits indicate upper and lower quartiles and whiskers indicate the maximum and minimum (g, h). The P values were calculated by unpaired two-sided Student’s t-test (a-b, e-i, k-m).
