Fig 2: RAB26 activity is essential for CCV biogenesis. (A) U2OS cells infected with Coxiella NMII were transfected with pLVX-GFP, pLVX-GFP-RAB26, pLVX-GFP-RAB26T77N, pLVX-GFP-RAB26Q123L or pLVX-GFP-RAB26N177I (green) 2 d post-infection. We fixed cells 24 h post-transfection and stained with anti-LC3B (red) and stained DNA using Hoechst 33,258 (blue). White arrows indicate Coxiella colonies. (B) Cells were treated as in A, and the CCV area was measured for at least 100 cells per condition. Values are mean ± SD from 3 independent experiments (n.s. = non-significant, **** = P < 0.0001, one-way ANOVA, Dunnett’s multiple comparison test). (C) Immunoblot of lysates from U2OS Cas9 cells expressing non-targeting guides (Guide CTRL1 and guide CTRL2) or RAB26-targeting guides (RAB26 Guide 3, 5 and 7). Immunoblots were probed with antibodies against RAB26, GAPDH and Cas9. (D) U2OS Cas9 cells expressing the non-targeting guide CTRL2 or the RAB26-targeting guide 5 were challenged for 6 d with Coxiella WT GFP (WT). The normalized CCV area for each condition was determined using the Cell Profiler software. (E) U2OS cells were challenged as in D and Genome Equivalents (GE) were determined by quantitative PCR. Values are mean ± SD from 3 independent experiments (n.s. = non-significant, **** = P < 0.0001, ** = P < 0.001, unpaired t test). Scale bars: 10 μm

Fig 3: CvpF stimulates the recruitment of RAB26 to CCVs. (A) U2OS cells were infected with either Coxiella WT GFP (WT, top panels), cvpF::Tn (middle panels), or the complemented cvpF::Tn strain (cvpF::Tn Comp., bottom panels) and transfected with pLVX-GFP-RAB26 (green) 4 d post-infection. We fixed cells 12 h post-transfection and stained with anti-LAMP1 (red) and anti-Coxiella (blue) antibodies. (B) U2OS cells were infected, as in A, and the presence of RAB26 on CCVs was scored for at least 80 cells per condition. Values are mean ± SD from 3 independent experiments (n.s. = non-significant, **** = P < 0.0001, one-way ANOVA, Bonferroni’s multiple comparison test). Scale bars: 10 μm. (C) Pearson’s correlation coefficient between RAB26 and LAMP1 signals in images acquired in A (**** = P < 0.0001, one-way ANOVA, Sidak’s multiple comparison test)

Fig 4: GOLIM4‐L binds and recruits RAB26 to mediate vesicle transportation. A) Immunofluorescent staining showed the localization of endogenous GM130 (green) and RAB26 (red), as well as colocalizations of exogenous RAB26 with Flag‐tag (red) and endogenous GOLIM4 (green) or exogenous GOLIM4‐L with HA‐tag (green). Scale bar, 100 µm. DNA constructs indicated on top were transiently introduced in 293T cells and whole cell lysis (input) or proteins immunoprecipitated (IP) with B) anti‐HA or C) anti‐FLAG antibody were immunoblotted with antibodies indicated at the left. Immunofluorescent staining of endogenous GM130 (red) and RAB26 (green) in S26 cells transfected with D) GOLIM4‐L siRNAs, or E) RAB26 siRNAs, or control siRNA. Scale bar, 100 µm. F) Electron microscopy showed the ultrastructure of Golgi in S26 cells described in (E). Scale bar, 200 nm. G) Cell proliferation assays were performed using S26 cells transfected with RAB26 siRNAs or control siRNA. H) Colony formation assays were performed with cells described in (E). Quantification of colony numbers was shown at the right. I) Representative images of EdU staining assays (left) for cells described in (E) and corresponding statistics are shown at the right. Scale bar, 100 µm. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Schematic diagram showing a proposed model for the interactions among RBFOX2, GOLIM4, and RAB26 in NPC progression. RBFOX2 mediates the alternative splicing of the exon‐7 of GOLIM4 through binding to GGAA sequence in the exon, which generates the long isoform of GOLIM4‐L. GOLIM4‐L promotes tumorigenesis of NPC, likely through vesicle‐mediated transport pathway involving recruitment of RAB26.