Fig 1: E-Syt1 is required in PKCd secretion in liver cancer cell lines. (A) Immunoblot analysis of lysates or media from starved control or E-Syt1 KO HepG2 cells for 24 h; n = 3 independent experiments. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and AFP were used as a loading control. (B) Immunoblot analysis of lysates and media (24 h) from starved HuH7 cells treated with scrambled (Scr) or E-Syt1 siRNA; n = 3 independent experiments. AFP was used as a loading control. (C) PKCd secretion measured by HiBiT extracellular assay in doxycycline-inducible HepG2 cells treated with scrambled (Scr), E-Syt1, SEC22B, or STX3 siRNA for 24 h in 10% FBS condition; n = 4 independent experiments. Luminescence was measured after cells were re-cultured in a medium containing 0.5 µg/mL doxycycline for 24 h. Data are shown as the mean ± s.d. One-way ANOVA, p = 0.002 with post-test Bonferroni’s test with pairwise comparison with siScr (siE-Syt1, * p < 0.0001; siSEC22B, ** p = 0.0007; and siSTX3, *** p = 0.0041). (D) Confocal micrographs to detect the interaction between SEC22B and STX3 (a SNARE on the PM) in control or E-Syt1 KO HepG2 cells. Each cell was fixed, reacted with a combination of mouse anti-SEC22B and rabbit anti-STX3 antibodies (SEC22B × STX3), and subjected to Duolink in situ PLA. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Data are shown as the mean ± s.d., * p < 0.0001 (two-tailed Student’s t-test) (n = 49 for control HepG2 cells and n = 52 for E-Syt1 KO HepG2 cells). Images represent three independent experiments. Scale bars, 10 µm.