Fig. 5: N70 glycosylation of GDF15 relieves its inhibitory effect on EGFR.

A Levels of GDF15, pEGFR (Y1068), EGFR, SRC, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), and AKT proteins were measured by WB in NC, ST-EPI, ST-ENZ, LT-EPI, and LT-ENZ cells. β-tubulin protein was used as loading control. One-way ANOVA, p < 0.05. B Level of GDF15 in culture media was detected by ELISA in NC, ST-EPI, ST-ENZ, LT-EPI, and LT-ENZ cells. One-way ANOVA, p < 0.05. C Effect of GDF15 silencing on cells survival was detected by CCK8 in Parental, ST, and LT cells. D Levels of GDF15, pEGFR (Y1068), EGFR, SRC, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), and AKT proteins in corresponding samples were assessed by WB. E Lectin level was gauged by GDF15 immunoprecipitation-ConA and AAL WB in LT cells transfected with the wild-type GDF15 or N70Q mutant. F Cells survival rate was evaluated by CCK8 in corresponding treatments. G Levels of GDF15, pEGFR (Y1068), EGFR, SRC, pERK1/2 (T202/Y204), ERK1/2, pAKT (S473), and AKT proteins in corresponding samples were measured by WB. β-tubulin protein was utilized as loading control. H 22Rv1-WT (GDF15 overexpression) and 22Rv1-N70Q (GDF15 N70 site mutation) cells suspended in matrigel were injected into the right flank of BALB/c-nude mice. One week after injection, the tumor-bearing mice were castrated and following with tumor size monitoring. After 21 days, the mice were euthanized. The tumors were dissected, and representative images of six mice per group were illustrated (n = 6). Sanger sequencing results showed GDF15 with N70Q site mutation (AAC mutated into CAG) was successfully constructed in 22Rv1 cells. I The corresponding tumor weights were shown in the graph for clarity. Data represents the mean ± std. Student’s t-test, *p < 0.05.