Fig 2: IQGAP1 could interact with AEP in gastric cancer cells.Notes: (A and D) IQGAP1 was one of the interacting proteins when used M2-FLAG beads to pull down the proteins. Five percent of the total lysate purified FLAG-tagged proteins were loaded as input. (B and E) IQGAP1 could be detected when AEP monoclonal antibody was used to extract the endogenous interacting proteins. (C and F) IQGAP1 was increased when AEP was knocked out.Abbreviations: AEP, asparaginyl endopeptidase; KO, AEP knockout.

Fig 3: Construction of stable AEP knockout and overexpressive gastric cancer cell lines and the sensitivity to different chemotherapeutic drugs.Notes: (A and B) AEP was suppressed in AEP knockout cells and increased in AEP overexpressive cells by Western blot assay. (C and D) The cell viability was investigated when oxaliplatin, irinotecan, 5-FU, paclitaxel, docetaxel, and T-DM1 treated NC and AEP-KO gastric cancer cells. **P<0.01.Abbreviations: AEP-KO, asparaginyl endopeptidase knockout; AEP-OE, AEP overexpression; NC, negative control; 5-FU, 5-fluorouracil.

Fig 4: AEP and MDR1 were decreased at mRNA level when AEP was knocked out, both in SGC7901 (A) and MKN45 (B) gastric cancer cell lines.Notes: Glyceraldehyde 3-phosphate dehydrogenase was used for an endogenous reference to standardize the mRNA expressive level. **P<0.01.Abbreviations: AEP, asparaginyl endopeptidase; NC, negative control; KO, AEP knockout.

Fig 5: JNK and ERK signaling pathways and MDR1 were inhibited in AEP knockout gastric cancer cell lines, and vice versa.Notes: (A) The expression of MDR-1, p-EGFR, p-JNK, p-ERK and p-Rac1/cdc42 were decreased in AEP-KO cells, but the expression of p-p38 and total EGFR, JNK, ERK, Rac1/cdc42 and p38 did not change. (B) The expressions of MDR1, p-EGFR, p-JNK, p-ERK, and p-Rac1/cdc42 were increased in AEP-OE cells. Vinculin and tubulin were used as an internal control. (C) The AEP-OE cells were treated with varying concentrations of SP600125 or PD98059, and both could inhibit the expressions of MDR1, p-JNK, and p-ERK. (D–F) The IC50 of docetaxel, paclitaxel, and T-DM1 in AEP-OE cells was calculated with the treatment of SP600125 or PD98059 for 24 h. *P<0.05; **P<0.01.Abbreviations: AEP-KO, asparaginyl endopeptidase knockout; AEP-OE, AEP overexpression; IC50, half maximal inhibitory concentration.