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. Author manuscript; available in PMC: 2021 Mar 19.
Published in final edited form as: Cell. 2020 Mar 10;180(6):1160–1177.e20. doi: 10.1016/j.cell.2020.02.017

Figure 2. Intact OXPHOS Promotes ER-phagy.

Figure 2.

(A) Genes that are components of the OXPHOS pathway were top hits in screen (highlighted in blue). Additional mitochondria-related genes are indicated in black and all other targeting sgRNAs are indicated in gray.

(B) Knockdown of NDUFB4 and NDUFB2 significantly inhibit ER-phagy. HCT116 CRISPRi EATR cells were transduced with sgRNAs targeting ULK1, NDUFB4, NDUFB2, or ATP5O and starved for 16 h before FACS measurement for ER-phagy. Data are presented as mean ± SD of eight biological replicates.

(C) Re-expression of cognate cDNA of each OXPHOS gene rescues ER-phagy. HCT116 CRISPRi EATR cells were transduced with NDUFB4 or NDUFB2 cDNA constructs and then transduced with the indicated sgRNAs. Cells were starved for 16 h before FACS measurement for ER-phagy. Data are presented as mean ± SD of three biological replicates.

(D) Small-molecule inhibitors of the different OXPHOS compartments phenocopy the effect of genetic inhibitions. HCT116 EATR cells were treated with rotenone, antimycin A, or oligomycin A and starved for 16 h before FACS measurement of ER-phagy. Data are presented as mean ± SD of three biological replicates.

(E) General autophagy proceeds in cells where NDUFB2, NDUFB4, or ATP5O are knocked down. HCT116 CRISPRi cells expressing mCherryeGFP-LC3B were transduced with the indicated sgRNAs. Cells were starved for 4 h before FACS measurement for general autophagy. Data represent mean ± SD of four biological replicates.

See also Figures S2 and S3.