Fig 2: Schematic model of ATG9A's role in trastuzumab resistant cellsATG9A promotes c-Cbl recruitment to the receptor kinase Her2 on T1112, a known c-Cbl docking site, leading to an increase in Her2 poly-ubiquitination. This ultimately results in a decrease in Her2 protein levels, Her2 transcriptional activity and Her2 downstream PI3K/AKT signaling.

Fig 3: ATG9A induces Her2 protein degradation via c-Cbl E3 ubiquitin ligase(A) Immunoprecipitation of ATG9A and (B) Her2 followed by western blotting of indicated antibodies in BT474-TR and BT474 cells. IgG antibodies were used as controls. (C) Immunoprecipitation of Her2 in BT474-TR and (D) BT474 cells transfected with vector and ATG9A-encoding plasmid in the presence of trastuzumab (20 µg/ml) for 48 hours, followed by western blotting of indicated antibodies. (E) Western blotting for Her2, ATG9A, pAKT and c-Cbl upon sic-Cbl with or without ATG9A overexpression in the presence or absence of trastuzumab (20 µg/ml) in BT474-TR. Protein levels were analysed and quantified using Image J software. Her2 bands were normalised to a-tubulin. (F) BT474 and BT474-TR cell proliferation was analysed after siRNA silencing against c-Cbl (or a non-targeting siRNA (siCTL)), followed by overexpression of ATG9A (or control vector).

Fig 4: Candidalysin is necessary and sufficient to activate Gab1, Grb2, Shc, Shp2, and c-Cbl adaptors in human oral epithelial cells.A, candidalysin (Clys) activates Gab1, Grb2, Shc, Shp2, and c-Cbl adaptors in human TR146 oral epithelial cells (OECs). Immunoblot showing phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl in response to 2 h infection of TR146 OECs with the indicated strains of C. albicans. B, Clys is sufficient to activate Gab1, Grb2, Shc, Shp2, and c-Cbl. Immunoblot showing phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl following stimulation with Clys at 30 min and 2 h in OECs. Blots are representative of three independent experiments.

Fig 5: Candidalysin-induced adaptor activation requires EGFR and MMP activity. Inhibition of EGFR activity suppresses (A) C. albicans- and (B) candidalysin (Clys)-induced adaptor activation. Use of Gefitinib or PD153035 EGFR TK inhibitors suppresses WT C. albicans–induced and Clys-induced activation of Gab1, Grb2, Shc, Shp2, and c-Cbl. C, MMP inhibition suppresses candidalysin-induced adaptor activity. Pretreatment of TR146 cells with Marimastat (10 or 1 µM, designated M10 or M1) suppressed Clys-induced phosphorylation of Gab1, Grb2, Shc, Shp2, and c-Cbl. Protein lysates were isolated 2 h post stimulation. Blots are representative of two (Grb2) or three (Gab1, Shc, Shp2, and c-Cbl) independent experiments. (Note: because data from other experiments presented in this article were originally run on the same gel, the a-actin loading control is reused in Fig. 2, A–C). MMP, matrix metalloproteinase.