Figure 6.
The LC3C autophagic pathway targets PDMBs for lysosomal degradation. (A) Endogenous LC3C colocalized with PDMBs, labeled by MKLP antibody. Split channels and RGB plots are shown. Cells were treated with 30 µM CQ for 24 h. (B) Quantification of the PDMBs colocalized with LC3C compared with the total number of PDMBs in the indicated cell lines. (C) LC3B did not colocalize with PDMBs. Arrowheads point to the PDMBs. (D) Expression of VHL repressed the number of accumulating PDMBs and recovers CQ-dependent accumulation in cell lines without VHL. Ratios of the number of PDMBs to the number of nuclei are shown. Cells were treated 30 µM CQ for 24 h; n is the number of independent experiments in which 10 fields with comparable numbers of cells were counted for PDMBs (labeled for MKLP1) and nuclei (DAPI). (E) Quantification of PDMBs in the UOK 257 cell line without and with FLCN. (F) Quantification of PDMBs in Caki-1 cell line with knockdowns of VHL or FLCN. (G) Knockdown of LC3C, but not LC3B, induced the number of PDMBs and abolished CQ-dependent accumulation of PDMBs in 786-O VHL(+) cells. (H) Knockdown of LC3C, but not LC3B, induced the number of PDMBs and abolished CQ-dependent accumulation of PDMBs in UOK 257 FLCN(+) cells. (I) Knockdowns of LC3C upstream regulators ULK3, PIK3C2A, UVRAG, and RUBCN consistently abolished CQ regulation of PDMBs and increased the number of PDMBs. (J) Colocalization of PDMB (CYK4) with PI3P reporter EGFP-2xFyve in cells with PIK3C2A and PIK3C3 knockdowns. Split channels and RGB profiles are shown. (K) Quantification of PDMBs colocalized with EGFP-2xFyve in cells shown in J. (L) Immunoblot shows CQ-dependent accumulation of PDMB proteins MKLP1, CYK4, and ARF6 in cells with LC3C and CQ-independent accumulation in cells with LC3C knockdown. (M) Quantification of immunoblots shown in L from three independent experiments. (N) Immunofluorescence experiment shows colocalization of PDMB with LAMP1 lysosomal protein. Split channels and RGB profiles are shown. (O) Quantification of colocalization PDMBs with LAMP1 in 786-O VHL(+) cells with and without LC3C. (P) Quantification of PDMBs in the indicated cell lines with TSG101 knockdown. (Q) Endogenous TSG101 coimmunoprecipitated endogenous LC3C but not LC3B. Cells were treated with 100 µM CQ for 1 h. Scale bars are as follows: white = 10 µm; cyan = 5 µm; magenta = 2.5 µm. In all bar graphs, mean ± SEM is shown. **, P < 0.01; ***, P < 0.001 by unpaired two-tailed t test. IB, immunoblot; IP, immunoprecipitation; KD, knockdown; Scr, scramble.

The LC3C autophagic pathway targets PDMBs for lysosomal degradation. (A) Endogenous LC3C colocalized with PDMBs, labeled by MKLP antibody. Split channels and RGB plots are shown. Cells were treated with 30 µM CQ for 24 h. (B) Quantification of the PDMBs colocalized with LC3C compared with the total number of PDMBs in the indicated cell lines. (C) LC3B did not colocalize with PDMBs. Arrowheads point to the PDMBs. (D) Expression of VHL repressed the number of accumulating PDMBs and recovers CQ-dependent accumulation in cell lines without VHL. Ratios of the number of PDMBs to the number of nuclei are shown. Cells were treated 30 µM CQ for 24 h; n is the number of independent experiments in which 10 fields with comparable numbers of cells were counted for PDMBs (labeled for MKLP1) and nuclei (DAPI). (E) Quantification of PDMBs in the UOK 257 cell line without and with FLCN. (F) Quantification of PDMBs in Caki-1 cell line with knockdowns of VHL or FLCN. (G) Knockdown of LC3C, but not LC3B, induced the number of PDMBs and abolished CQ-dependent accumulation of PDMBs in 786-O VHL(+) cells. (H) Knockdown of LC3C, but not LC3B, induced the number of PDMBs and abolished CQ-dependent accumulation of PDMBs in UOK 257 FLCN(+) cells. (I) Knockdowns of LC3C upstream regulators ULK3, PIK3C2A, UVRAG, and RUBCN consistently abolished CQ regulation of PDMBs and increased the number of PDMBs. (J) Colocalization of PDMB (CYK4) with PI3P reporter EGFP-2xFyve in cells with PIK3C2A and PIK3C3 knockdowns. Split channels and RGB profiles are shown. (K) Quantification of PDMBs colocalized with EGFP-2xFyve in cells shown in J. (L) Immunoblot shows CQ-dependent accumulation of PDMB proteins MKLP1, CYK4, and ARF6 in cells with LC3C and CQ-independent accumulation in cells with LC3C knockdown. (M) Quantification of immunoblots shown in L from three independent experiments. (N) Immunofluorescence experiment shows colocalization of PDMB with LAMP1 lysosomal protein. Split channels and RGB profiles are shown. (O) Quantification of colocalization PDMBs with LAMP1 in 786-O VHL(+) cells with and without LC3C. (P) Quantification of PDMBs in the indicated cell lines with TSG101 knockdown. (Q) Endogenous TSG101 coimmunoprecipitated endogenous LC3C but not LC3B. Cells were treated with 100 µM CQ for 1 h. Scale bars are as follows: white = 10 µm; cyan = 5 µm; magenta = 2.5 µm. In all bar graphs, mean ± SEM is shown. **, P < 0.01; ***, P < 0.001 by unpaired two-tailed t test. IB, immunoblot; IP, immunoprecipitation; KD, knockdown; Scr, scramble.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal