Fig 2: The morphological alterations of RER in the mDAergic neurons of VMP1cKO mice.A The representative TEM images of the observed RER. Scale bar, 0.5 μm. B The mean width of RER tubules was shown (N = 487 RER collectively counted from 3 VMP1cWT mice, N = 500 RER collectively counted from 3 VMP1cKO mice). C The proportion of RER tubules (>100 nm) was quantified from (B). D Double-label immunofluorescence of SEC31A (green) and TH (red) in the mDAergic neurons. The nuclei were labeled with DAPI (blue). Scale bar, 10 μm. E Quantification of the number of SEC31A+ puncta (>0.1 μm2) per TH+ neuron (N = 3 mice per genotype). Data were analyzed by using Student’s t-test and were represented as mean ± SEM. **p < 0.01, ****p < 0.0001. RER rough endoplasmic reticulum, NM nuclear membrane, RER rough ER.

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: 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.

Fig 5: 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.