Fig 2: Membrane contact sites and ceramide uptake are maintained during inhibition of ERES.Cells were infected with C. trachomatis, then transfected with a plasmid expressing pmScarlet-CERT (A, purple in merge) or pmClover3-PDI (B, green in merge). At 20 hpi, DMSO, 10 μM FLI-06, 5 mM 4PBA, or 3 μg/mL BFA were added to cells. At 24 hpi, cells were fixed and stained with anti-Sec31A (A, green in merge) or anti-IncA (B, purple in merge). DNA was stained with DAPI (blue in merge). Images are single planes from deconvolved z-series. Scale = 16 μm. (B) Inset shows regions along the inclusion membrane where PDI was closely apposed or overlapping IncA. (C) Average inclusion fluorescence after incubation with NBD-C6-Ceramide. Representative images shown on right, NBD-ceramide in green, DAPI in blue. Significance determined by one-way ANOVA with Tukey’s multiple comparison test. Any comparisons not shown were not significant. **, p < 0.01; ***, p < 0.001.

Fig 3: ERES proteins Sec16A and Sec31A are recruited to the C. trachomatis inclusion.(A) Immunofluorescence microscopy of cells showing cellular distribution of Sec16A (anti-Sec16A, first column, green in merge), Chlamydia IncA (anti-IncA, second column, magenta in merge), and DNA (DAPI, third column, blue in merge). (B) Distribution of the COPII outer coat protein Sec31A (anti-Sec31A, first column, green in merge), IncA, and nuclei. Single channel images are displayed in inverted grayscale. Merged panels display all three-color channels. Protein distribution in uninfected HeLa cells are shown in the top row. Deconvolved images from cells infected with C. trachomatis L2 at 24 hpi are shown as a summation of z-series images (merged planes) or a single xy plane. Enlargements (far right) represent the regions marked with white boxes. Scale bars = 16 μm.

Fig 4: Mea6 regulates OL differentiation in vitro(A) Representative images of CG4 cell morphology and dead cells during differentiation. Arrowheads indicate dead cells. Scale bars: 100 μm.(B and C) Immunoblotting (B) and quantification (C) of the indicated protein levels in cultured CG4 cells 6 days after differentiation. Five KO samples originated from distinct single clones.(D) Immunoblotting and quantification of indicated proteins in different WT and Mea6 KO CG4 clones.(E) Immunostaining for SEC31A in WT and Mea6 KO CG4 cells. Scale bar: 10 μm.(F) Reduced signal and puncta number of SEC31A in Mea6-KO cells. Data represent the mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, Two-tailed unpaired Student’s t test.

Fig 5: Inhibition of ERES cargo loading abrogates ERES recruitment to the inclusion.(A) Treatment of C. trachomatis infected cells with FLI-06 disrupted the recruitment of COPII coat protein Sec31A to inclusion membranes. Immunofluorescence microscopy of cells showing cellular distribution of Sec31A (anti-Sec31A, first column, green in merges), inclusion membrane protein IncA (anti-IncA, second column, magenta in merges), and DNA (DAPI, third column, blue in merges). Representative deconvolved merged z-series images of uninfected cells are shown in upper panels, and cells infected with C. trachomatis L2 at 24 hpi are shown in lower panels. 10 μM FLI-06 treatment for 4 h, from 20–24 hpi, resulted in Sec31A distribution similar to that of uninfected cells, and away from inclusion membranes. Scale bars = 16 μm. (B) Quantification of Sec31 in cells infected and treated with FLI-06 as described in A. Sec31 punctae that were touching or overlapping with IncA in untreated or FLI-06 treated cells were counted using Volocity to assess the number of overlapping spots per inclusion. At least 20 inclusions were analyzed per condition, for two independent experiments. Each dot represents an inclusion, lines represent mean (SD). Significance determined by unpaired t-test with Welch’s correction, ****, p < 0.0001.