TRIMs regulate IFN-γ–induced autophagy. (A) THP-1 cells were subjected to TRIM knockdown and treated with 1,000 U/ml IFN-γ for 4 h, and high content (HC) analysis was performed using a Cellomics HCS scanner (epifluorescence) and iDEV software. HC (magenta, endogenous LC3B immunofluorescence; blue, nuclei stained with Hoechst). Mask overlay, software-defined objects (primary objects, cell outlines; internal secondary objects, LC3 puncta). (B) Average count of LC3 puncta per cell from cells treated as in A (data from two 96-well plates with identical siRNA arrangements; the corresponding data are shown in Fig. S1 C). Encircled are IFN-γ–treated wells (right) and wells with vehicle controls (bottom left). TRIM knockdowns that reduced LC3 puncta readout in both of the two experiments by 3 SDs (horizontal dot lines) from the average of IFN-γ–treated controls (horizontal solid line) are indicated by corresponding TRIM numbers (open circle). TRIMs that were chosen in follow up experiments in Fig. 1 C are also indicated with number. (C) Similar to B, except that THP-1 cells were subjected to specific TRIM or scrambled (Scr; control) knockdowns and were analyzed in quadruplicates or more repeats. (D) Model of TRIMs-mediated IFN-γ–induced autophagy based on the results obtained in Fig. 1 and Fig. S1 thus far. (E) THP-1 cells were treated with TRIM20 or scrambled siRNAs, incubated with or without IFN-γ for 4 h in the presence of bafilomycin A1 (Baf A₁), and LC3-II conversion was determined by immunoblots. RI, relative intensity. (F) HeLa cells were transfected with GFP or GFP-TRIM20, and HC analysis was performed. Data, means ± SE; n ≥ 3. *, P < 0.05 (ANOVA, C and E, or t test in F). Bars, 5 µm.