Figure 10.
Contribution of autophagy pathway components to OPTN-induced pexophagy. (A–C) Torin-1 induces pexophagy in HeLa cells lacking classical autophagy receptors. Po-mKeima pentaKO HeLa cells—deficient in NDP52, OPTN, TAX1BP1, NBR1, and SQSTM1—were treated overnight with 250 nM Torin-1 or left untreated (CT) and analyzed using fluorescence microscopy or flow cytometry. (A) Representative overlay images showing po-mKeima fluorescence with excitation at 440 nm (false color: green) and 586 nm (false color: red) nm. Scale bar, 10 µm. (B) Representative flow cytometry plots for each condition. Color gradients reflect cell density. (C) Quantification of the percentage of cells within the gated population. Data are presented as mean ± SD from four independent biological replicates. Statistical comparisons were performed (***, P < 0.001). (D and E) Basal pexophagy levels are reduced in cells deficient in FIP200, WIPI2, or ATG8 family proteins. HEK-293 cells lacking FIP200 (ΔFIP200, 293), HEK-293A cells lacking WIPI2 (ΔWIPI2, 293A), and ATG8 hexa-knockout HeLa cells (ΔATG8h HeLa), along with their respective parental control cell lines (CT), were infected with lentiviral particles encoding po-mKeima and cultured in regular DMEM medium. Pexophagy was assessed by flow cytometry, gating for single cells with decreased fluorescence intensity in the neutral (440 nm) channel. (D) Representative flow cytometry plots for each cell line. Color gradients indicate cell density. (E) Quantification of the percentage of cells within the gated population. Data represent the mean ± SD of two independent biological replicates. (F and G) Deficiency of FIP200, WIPI2, or ATG8 family proteins affects OPTN localization to peroxisomes. (F) ΔFIP200 293, ΔWIPI2 293A, and ΔATG8h HeLa cells, along with their respective parental control cell lines (CT), were cultured in standard DMEM medium and analyzed by IF microscopy using antibodies against PEX14 and OPTN. (G) ΔFIP200 HEK-293 cells were additionally treated overnight with 20 nM Baf A1). Nuclei were counterstained with DAPI. Representative images are shown. Scale bars: 10 µm (insets: 50 µm).

Contribution of autophagy pathway components to OPTN-induced pexophagy. (A–C) Torin-1 induces pexophagy in HeLa cells lacking classical autophagy receptors. Po-mKeima pentaKO HeLa cells—deficient in NDP52, OPTN, TAX1BP1, NBR1, and SQSTM1—were treated overnight with 250 nM Torin-1 or left untreated (CT) and analyzed using fluorescence microscopy or flow cytometry. (A) Representative overlay images showing po-mKeima fluorescence with excitation at 440 nm (false color: green) and 586 nm (false color: red) nm. Scale bar, 10 µm. (B) Representative flow cytometry plots for each condition. Color gradients reflect cell density. (C) Quantification of the percentage of cells within the gated population. Data are presented as mean ± SD from four independent biological replicates. Statistical comparisons were performed (***, P < 0.001). (D and E) Basal pexophagy levels are reduced in cells deficient in FIP200, WIPI2, or ATG8 family proteins. HEK-293 cells lacking FIP200 (ΔFIP200, 293), HEK-293A cells lacking WIPI2 (ΔWIPI2, 293A), and ATG8 hexa-knockout HeLa cells (ΔATG8h HeLa), along with their respective parental control cell lines (CT), were infected with lentiviral particles encoding po-mKeima and cultured in regular DMEM medium. Pexophagy was assessed by flow cytometry, gating for single cells with decreased fluorescence intensity in the neutral (440 nm) channel. (D) Representative flow cytometry plots for each cell line. Color gradients indicate cell density. (E) Quantification of the percentage of cells within the gated population. Data represent the mean ± SD of two independent biological replicates. (F and G) Deficiency of FIP200, WIPI2, or ATG8 family proteins affects OPTN localization to peroxisomes. (F) ΔFIP200 293, ΔWIPI2 293A, and ΔATG8h HeLa cells, along with their respective parental control cell lines (CT), were cultured in standard DMEM medium and analyzed by IF microscopy using antibodies against PEX14 and OPTN. (G) ΔFIP200 HEK-293 cells were additionally treated overnight with 20 nM Baf A1). Nuclei were counterstained with DAPI. Representative images are shown. Scale bars: 10 µm (insets: 50 µm).

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal