Figure 2.
OPTN-mediated pexophagy recruits LC3, is inhibited by Baf A1, and does not alter general autophagy. (A) GFP-LC3 localizes to PEX14/OPTN-positive structures in HEK-293 cells. Cells were transfected with a plasmid encoding GFP-LC3, and after 2 days, treated with 100 nM Baf A1 for 6 h. Cells were then fixed and processed for IF microscopy using a mouse anti-PEX14 antibody and a rabbit anti-OPTN antibody. White arrowheads indicate sites of colocalization between GFP-LC3, OPTN, and PEX14. Enlarged views of boxed areas are shown on the right. The bright blue puncta in the upper panels represent clusters of OPTN. To avoid saturation of these signals due to high expression and to visualize OPTN expression across the cell population, a second field of view (lower panels) with cells expressing lower levels of OPTN was imaged using a longer exposure time in the blue channel, enabling detection of weaker signals. Expr: expression level; Expo, exposure time; ↑, high; ↓ low. Scale bar, 10 µm; insets: 2.5 µm. (B–E) OPTN-mediated pexophagy is sensitive to Baf A1 and does not interfere with general autophagy. (B and C) Lenti-GFP (L-GFP) and lenti-OPTN1–577-GFP (L-OPTN-GFP) Flp-In T-REx 293 cells were transfected with a plasmid encoding non-tagged PEX14. After 48 h, cells were incubated overnight in medium with or without 20 nM Baf A1, followed by IB with antibodies against PEX13, GAPDH, ABCD3, PEX10, and vinculin (VCL). (B) Representative immunoblots. (C) Densitometry quantification of the relative normalized amounts (R-NA) of peroxisomal markers. Expression levels were normalized to GAPDH (for PEX14) or VCL (for ABCD3 and PEX10) and further normalized to the average signal in L-GFP cells. Data represent mean ± SD from ≥3 biological replicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D and E) HEK-293 cells were transiently transfected with plasmids encoding GFP or OPTN-GFP and harvested after 2 days for IB using antibodies against GFP, SQSTM1, LC3B, and GAPDH. (D) Representative immunoblots. (E) Densitometry quantification of the SQSTM1 levels and LC3B-II/I ratios (AU, arbitrary units). Values are presented as means ± SD (n = 3). No statistically significant differences (ns) were observed. Source data are available for this figure: SourceData F2.

OPTN-mediated pexophagy recruits LC3, is inhibited by Baf A1, and does not alter general autophagy. (A) GFP-LC3 localizes to PEX14/OPTN-positive structures in HEK-293 cells. Cells were transfected with a plasmid encoding GFP-LC3, and after 2 days, treated with 100 nM Baf A1 for 6 h. Cells were then fixed and processed for IF microscopy using a mouse anti-PEX14 antibody and a rabbit anti-OPTN antibody. White arrowheads indicate sites of colocalization between GFP-LC3, OPTN, and PEX14. Enlarged views of boxed areas are shown on the right. The bright blue puncta in the upper panels represent clusters of OPTN. To avoid saturation of these signals due to high expression and to visualize OPTN expression across the cell population, a second field of view (lower panels) with cells expressing lower levels of OPTN was imaged using a longer exposure time in the blue channel, enabling detection of weaker signals. Expr: expression level; Expo, exposure time; ↑, high; ↓ low. Scale bar, 10 µm; insets: 2.5 µm. (B–E) OPTN-mediated pexophagy is sensitive to Baf A1 and does not interfere with general autophagy. (B and C) Lenti-GFP (L-GFP) and lenti-OPTN1–577-GFP (L-OPTN-GFP) Flp-In T-REx 293 cells were transfected with a plasmid encoding non-tagged PEX14. After 48 h, cells were incubated overnight in medium with or without 20 nM Baf A1, followed by IB with antibodies against PEX13, GAPDH, ABCD3, PEX10, and vinculin (VCL). (B) Representative immunoblots. (C) Densitometry quantification of the relative normalized amounts (R-NA) of peroxisomal markers. Expression levels were normalized to GAPDH (for PEX14) or VCL (for ABCD3 and PEX10) and further normalized to the average signal in L-GFP cells. Data represent mean ± SD from ≥3 biological replicates (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D and E) HEK-293 cells were transiently transfected with plasmids encoding GFP or OPTN-GFP and harvested after 2 days for IB using antibodies against GFP, SQSTM1, LC3B, and GAPDH. (D) Representative immunoblots. (E) Densitometry quantification of the SQSTM1 levels and LC3B-II/I ratios (AU, arbitrary units). Values are presented as means ± SD (n = 3). No statistically significant differences (ns) were observed. Source data are available for this figure: SourceData F2.

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