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. 2023 Nov 8;20(5):994–1014. doi: 10.1080/15548627.2023.2281128

Figure 3.

Figure 3.

TRIM2, TRIM3 and TRIM71 inhibit autophagy initiation. (A) Schematic depiction of the domain organization of the FLN-NHL TRIMs (TRIM2, TRIM3, TRIM71) and closely related TRIM proteins. TRIM, tripartite motif. (B) Quantification of autophagosome levels in HEK293T GL cells transiently expressing the indicated FLAG-tagged TRIMs at 48 h post transfection by flow cytometry. Lines represent mean ± SEM, n = 3. (right panel) Representative immunoblot of whole cell lysates. (C) Representative confocal laser scanning fluorescence microscopy images of HeLa GL cells transiently expressing indicated TRIMs (48 h) or treated with chloroquine (CQ, 10 µM, overnight). TRIMs (FLAG, red), eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (D) Quantification of eGFP-puncta area per cell in the images from (C). Lines represent mean ± SEM, n = 61–139 (individual cells). (E) Representative immunoblots of whole cell lysates of HEK293T cells transiently expressing indicated TRIMs (48 h post transfection). Blots were stained with anti-SQSTM1 and anti-GAPDH. Torin-1 (5 µM) and bafilomycin A1 (BafA1, 250 nM) treatment were used as positive controls. (F) Quantification of the immunoblotting data of (E). Lines represent mean ± SEM, n = 3. (G) Representative confocal laser scanning fluorescence microscopy images of HeLa GL cells (48 h post transfection) depleted of the indicated TRIMs by siRNA or treated with chloroquine (CQ, 10 µM, overnight). eGFP-LC3B (green), nuclei (DAPI, blue). Scale bar: 10 µm. (H) Quantification of the eGFP-puncta area per cell in the images from (F), Lines represent mean ± SEM, n = 61–117 (individual cells). (I) Representative immunoblots of whole cell lysates of HDF cells depleted of the indicated TRIMs by siRNA at 40 h post transfection. Blots were stained with anti-p-ULK1 (S757), anti-ULK1, anti-p-BECN1 (Ser15), anti-BECN1 and anti-ACTB. (J) quantification of the band intensities of p-ULK1 (Ser757) to total ULK1 of the immunoblotting data of (I). Bars represent mean ± SEM, n = 3. (K) Representative immunoblots of whole cell lysates of HEK293T cells that transiently expressed empty vector or TRIM3 at 40 h post transfection. Blots were stained with anti-p-ULK1 (Ser757), anti-ULK1, anti-p-BECN1 (Ser15), anti-BECN1, anti-FLAG and anti-ACTB. (L) Quantification of the band intensities of p-ULK1 (Ser757):ULK1 levels of the immunoblotting data of (K) at highest TRIM3 transfection levels. Bars represent mean ± SEM, n = 5. Student’s t-test with Welch correction. *, p < 0.05; **, p < 0.01; ***, p < 0.001.