Fig. 2: RhoB induces LC3 lipidation and upregulates Beclin1 level.

a Protein levels of LC3 and P62 in HEK293 cells transfected with RhoB or vector. Signal densities of RhoB and P62 were normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: 0 vs. 0.1 P = 0.1768, 0 vs. 0.5 P = 0.0306, 0 vs. 1.0 P = 0.0001; P62/β-Actin: 0 vs. 0.1 P = 0.2468, 0 vs. 0.5 P = 0.0103, 0 vs. 1.0 P = 0.0031. b TEM images of RhoB overexpressing cells. Quantification on the right shows the number of autophagic vacuoles per cell (n = 12 cells per group from three independent experiments). Arrows show autophagic vacuoles. Scale bars, 2 or 0.5 μm. P = 7.4e−7. c Representative images of HEK 293 cells transfected with mCherry-GFP-LC3 in the presence of RhoB or vector. Autophagosomes, yellow puncta; autolysosomes, red-only puncta. Bar graph shows the quantification of LC3 puncta per cell (n = 10 random areas per group from three independent experiments). Scale bar, 10 μm. Yellow P = 1.1e−5, Red P = 3.2e−8. d, e Western blotting showing the levels of autophagic proteins in RhoB knockdown 5637 bladder epithelial cells with LPS (d) or with CFT073 infection (e). 5637 bladder epithelial cells were transfected with RhoB siRNAs or scramble siRNA for 48 h followed by infection of CFT073 at MOI of 50 for 2 h. Signal densities of LC3-II, P62, and Beclin 1 were normalized to that of β-Actin. The relative density of siScr-transfected cells was set to 100%. n = 3 independent experiments. d LC3-II/β-Actin: siScr vs. siRhoB #1 P = 0.0133, siScr vs. siRhoB #2 P = 0.0011, siScr vs. siRhoB #3 P = 0.0013; P62/β-Actin: siScr vs. siRhoB #1 P = 0.5419, siScr vs. siRhoB #2 P = 0.2053, siScr vs. siRhoB #3 P = 0.0290; Beclin 1/β-Actin: siScr vs. siRhoB #1 P = 0.3483, siScr vs. siRhoB #2 P = 0.0400, siScr vs. siRhoB #3 P = 0.0390. e LC3-II/β-Actin: siScr vs. siRhoB #1 P = 0.1356, siScr vs. siRhoB #2 P = 0.0225, siScr vs. siRhoB #3 P = 0.0030; Beclin 1/β-Actin: siScr vs. siRhoB #1 P = 0.0131, siScr vs. siRhoB #2 P = 0.0004, siScr vs. siRhoB #3 P < 1.0e−15. f Knockdown of Beclin 1 restores LC3 lipidation in RhoB-overexpressing HEK 293 cells. Three independent experiments. g Knockdown of Beclin 1 restores bacterial survival in RhoB-overexpressing 5637 bladder epithelial cells. 5637 cells were co-transfected with RhoB and Beclin 1 siRNAs for 48 h followed by bacterial invasion assay. Bacterial survival of vector and siScr-transfected cells was set to 100%. n = 3 independent experiments. RhoB siScr vs. Vector siScr P = 0.0062, RhoB siScr vs. RhoB siBeclin 1 #1 P = 0.0003, RhoB siScr vs. siBeclin 1 #2 P = 0.0004, RhoB siScr vs. siBeclin 1 #3 P = 0.0002. h LC3 lipidation in HEK293 cells transfected with plasmids encoding RhoB-WT, Q63E, Q63L, and T19N. The LC3-II density was normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: Vector vs. RhoB P = 0.0069, Vector vs. Q63E P = 0.0249, Vector vs. Q63L P = 0.0480, Vector vs. T19N P = 0.0002. i LC3 lipidation and Beclin 1 level in HEK293 cells transfected with plasmids encoding RhoB-WT, S185A, and C193S for 48 h. Signal densities of LC3-II and Beclin 1 were normalized to that of β-Actin. The relative density of vector-transfected cells was set to 100%. n = 3 independent experiments. LC3-II/β-Actin: Vector vs. RhoB P = 0.0459, Vector vs. S185A P = 0.0398, Vector vs. C193S P = 0.7997; Beclin 1/β-Actin: Vector vs. RhoB P = 0.0136, Vector vs. S185A P = 0.0092, Vector vs. C193S P = 0.9421. Data are the mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired Student’s t test (b), one-way ANOVA (a, d–e, g–i), or two-way ANOVA (c).