Fig 2: TECPR1 recruits LC3C vesicles to lysosomes.a Quantitative analysis of the colocalization of GFP-LC3C and GFP-TECPR1 with immunostained LAMP2 and STX17 in starved HeLa cells (n = 20 cells pooled from three independent experiments). Data are represented as mean ± SD. b Structured-illumination microscopy of starved HeLa cells expressing GFP-TECPR1 or RFP-TECPR1 together with GFP-LC3C as indicated. Cells were fixed and immunostained for LAMP2 or STX17. LC3C puncta are in the immediate vicinity of TECPR1/LAMP2 compartments. Scale bars, 10 µm, insets, 2.5 µm. c Time-lapse video microscopy of TECPR1KO cells coexpressing RFP-TECPR1 and GFP-LC3C. Confocal images were captured every 10 s and every other frame is shown. Arrows indicate moving structures that transiently interact. Scale bar, 10 µm, insets, 2.5 µm. See also Supplementary Movies 1, 2, 3 and 4. Source data are provided as a Source Data file.

Fig 3: U18666A and bafilomycin A1 impairs Syntaxin17 trafficking to autophagosomes. A RPE1 cells were treated with U18666A (2 µg), chloroquine (5 µM), or bafilomycin A1 (25 nM) for 24 h in a serum-free medium. Cells were then subjected to lysotracker staining. Scale bar, 10 µm. B Intensities from 50 cells were acquired for each experimental group using ImageJ software. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. C Cells were transfected with GFP-LC3 for 12 h in serum-supplemented media. Cells were then treated with U18666A (2 µg), chloroquine (5 µM), or bafilomycin A1 (25 nM) for 4 h in a serum-starvation medium followed by 4% paraformaldehyde fixation and immunostaining with STX17 antibody. Scale bar, 10 µm. Arrowheads represent STX17 trafficking to autophagic structures (GFP-LC3). D Quantification of GFP-LC3 from 50 transfected cells was analyzed for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. E Determination of the number of STX17 positive GFP-LC3 puncta. Fifty transfected cells were analyzed for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. F Percentage of STX17 positive GFP-LC3 puncta were calculated from D and E. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test. G Cells were treated with bafilomycin A1 (25 nM) for different time points in serum-starvation medium. Cells were then subjected to Lysotracker staining. Scale bar, 10 µm. H Lysotracker intensities from 50 cells were acquired using the ImageJ software for each experimental group. The bar graph represents the mean ± SD (n = 3 experiments). *P < 0.05, Student's t-test