Fig 2: Progression of ER-localized CD147 correlated with its transfer from CNX to GRP78.(a) The effect of CNX knockdown on the expression of CD147(WT)-EGFP and CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were transfected with control or CNX siRNA for 3 days. Immunoblots of cell lysates were probed with the indicated antibodies. (b) The effect of CNX knockdown on the expression of CD147(N152Q)-EGFP in the ER. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control or CNX siRNA for 3 days. Then, the cells were through fractionating cells into ER using the ER isolation kit. Immunoblots of ER proteins were probed with the indicated antibodies. Relative expression of CD147-EGFP/Tubulin were quantified using Image J software, **P < 0.01. (c) The effect of CNX knockdown on the expression of CD147(N152Q)-EGFP in the presence of proteasome inhibitor (MG-132). K7721 cells stably expressing CD147(N152Q)-CD147 with the control or siRNA against CNX in the presence or absence of MG-132. Immunoblots of cell lysates were probed with the indicated antibodies. (d) Additive effect of CNX and GRP78 co-inactivation on CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with the control or siRNA against CNX, GRP78 or both for 3 days. Lysates prepared from these cells were immunoblotted using the indicated antibodies. (e,f) ATPase activity was not necessary for the degradation of CD147(N152Q)-EGFP. K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were transfected with control or GRP78 siRNA, wild-type GRP78 and mutant G37T-GRP78 for 3 days. The resulting precipitates were examined by immunoblot analysis using the indicated antibodies.

Fig 3: N-glycans at Asn152 are required for CD147 release from the CNX quality control system.(a) The localizations of CD147(WT)-EGFP and CNX. Incubation of K7721 cells stably expressing CD147(WT)-EGFP with 1 mM CAS for the indicated time. CNX was analysed by immunofluorescence staining. Scale bars, 20 µm and 10 µm. (b) The binding of CD147 with CNX. The interaction between CD147(WT)-EGFP and CNX after a 12-h incubation of K7721 cells stably expressing CD147(WT)-EGFP in the presence of 1 mM CAS. **P < 0.01. (c) The localizations of CD147(WT)-EGFP and GRP78. Incubation of K7721 cells stably expressing CD147(WT)-EGFP with 1 mM CAS for the indicated time. GRP78 was analysed by immunofluorescence staining. Scale bars, 20 µm and 10 µm. (d) Localization of CD147-EGFP. K7721 cells transiently expressing mutant CD147(N152/186Q)-EGFP, CD147(N152/186Q)-EGFP, and CD147(N152/186Q)-EGFP (green) were immunostained using an ER-Tracker (red) and DAPI (blue) and observed using a confocal laser scanning microscopy. Scale bars, 20 µm. (e) The localizations of CD147(N44/186Q)-EGFP and CNX. Incubation of K7721 cells transiently expressing CD147(N44/186Q)-EGFP with 1 mM CAS for the indicated time, CNX was analysed by immunofluorescence staining. Scale bars, 20 µm and 10 µm. (f,g) CD147(WT)-EGFP or CD147(N152Q)-EGFP was immunoprecipitated before or after the incubation of K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP with 20 µg/mL cycloheximide for the indicated periods of time.

Fig 4: CD147(N152Q)-EGFP was more associated with the heat shock proteins than with the ER lectin chaperones.(a) The localizations of CD147-EGFP and its binding partners, CNX, CRT, GRP78, and GRP94, were determined by immunofluorescence staining in K7721 cells stably expressing CD147(WT)-EGFP and CD147(N152Q)-EGFP. Scale bars, 20 μm and 10 μm. (b) The interaction between CD147 and the chaperones. Extracts prepared from K7721 cells stably expressing CD147(WT)-EGFP or CD147(N152Q)-EGFP were immunoprecipitated using the indicated antibodies. The resulting precipitates were examined by immunoblot analysis using the indicated antibodies. The signal intensities of each chaperone and CD147-EGFP were quantified using Image J software and are presented as the ratio of chaperone/CD147-EGFP signal. *P < 0.05, **P < 0.01, and **P < 0.001. (c) The effect of CNX knockdown on the binding ability among CD147(N152Q)-EGFP, GRP78, and GRP94. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control or CNX siRNA for 3 days, followed by immunoprecipitated with the indicated antibodies, **P < 0.01. (d) The effect of GRP78 knockdown on the binding ability among CD147(N152Q)-EGFP, CNX, and GRP94. K7721 cells stably expressing CD147(N152Q)-EGFP were transfected with control or CNX siRNAs for 3 days, followed by immunoprecipitated with the indicated antibodies, **P < 0.01. (e) Expression of CNX, CRT, GRP78, and GRP94. Lysates of K7721 cells stably expressing the CD147(WT)-EGFP and CD147(N152Q)-EGFP mutants were analysed by Western blotting with the indicated antibodies.

Fig 5: Endoplasmic reticular stress is involved in the activation of autophagy of INS-1 cells induced by LRG. INS-1 cells were treated with normal saline (CON), FFA, LRG and FFA + LRG for 24 h. (A) CHOP and GRP78 proteins were detected using immunoblotting, the results were normalized to that of GAPDH. Quantification of the expression levels of (B) CHOP and (C) GRP78. LRG, liraglutide; CON, control; FFA, free fatty acids.