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. 2019 Jul 9;9(17):4878–4892. doi: 10.7150/thno.36254

Figure 4.

Figure 4

Brigatinib activates autophagy via ER stress-signaling pathway in CRC cells. A, Immunoblotting of LC3B, Atg5, Atg7, and Beclin 1 in CRC cells treated with the indicated concentrations of brigatinib for 24 hours. B, Interaction among Beclin 1, Bcl-2 and Atg14L was determined by coimmunoprecipitation assay. C-D, Immunofluorescence analysis (C) of LC3B in CRC cells treated with or without 1 µM brigatinib for 12 hours. The number of LC3 puncta (D) was shown. ***, P < 0.001. Scale bar, 10 μm. E, Immunoblotting of LC3B in CRC cells transfected with siATG5 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. F, Immunoblotting of LC3B in CRC cells transfected with siATG7 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. G, Immunoblotting of LC3B in CRC cells transfected with siBECN1 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. H, Immunoblotting of LC3B in CRC cells treated with or without 1 μM brigatinib in the presence or absence of 10 μM chloroquine (CQ) for 24 hours. I-J, Immunofluorescence analysis (I) of colocalized LC3B and LAMP2 in CRC cells treated with or without 1 µM brigatinib for 12 hours. CQ group was negative control. The number of colocalized or non-colocalized LC3B and LAMP2 (J) was quantified. *, P < 0.05. **, P < 0.01. Scale bar, 10 μm. K, Immunoblotting of LC3B, Atg5 and Beclin 1 in CRC cells transfected with siORP8 or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. L, Immunoblotting of LC3B, Atg5 and Beclin 1 in CRC cells treated with or without 1 μM brigatinib in the presence or absence of 2 mM 4-PBA for 24 hours. M, Immunoblotting of LC3B in CRC cells transfected with siIRE1α or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours.