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

Figure 5.

Figure 5

Brigatinib induces ER-phagy in CRC cells. A, Immunoblotting of FAM134B in CRC cells treated with the indicated concentrations of brigatinib for 24 hours. B, Immunoblotting of FAM134B in CRC cells treated with or without 1 μM brigatinib in the presence or absence of 2 mM 4-PBA for 24 hours. C, Immunoblotting of LC3B, Atg5, and Beclin 1 in CRC cells transfected with siFAM134B or siScramble followed by treatment with or without 1 μM brigatinib for 24 hours. D, Interaction between FAM134B and LC3B was determined by coimmunoprecipitation assay. E, CRC cells transfected with either WT or LIRmut HA-FAM134B plasmids for 48 hours, followed by treatment with or without 1 µM brigatinib for 24 hours. Interaction between WT or LIRmut HA-FAM134B and LC3B was determined by coimmunoprecipitation assay. F-H, Immunofluorescence analysis of colocalization of LC3B with WT or LIRmut HA-FAM134B in CRC cells treated with or without 1 µM brigatinib for 12 hours. Scale bar, 10 μm. The number of colocalized LC3 puncta and WT or LIRmut HA-FAM134B (H) was quantified. **, P < 0.01; ***, P < 0.001. I-J, Cells were transfected with GFP-LC3 plasmid for 48 hours, followed by treatment with or without 1 µM brigatinib for 12 hours and staining with ER-Tracker Blue for 30 minutes (I). The number of colocalized GFP-LC3 puncta and ER-Tracker Blue (J) was quantified. Scale bar, 10 μm. ***, P < 0.001.