Figure 8.
Rab GTPases mediate xenophagy in mammalian cells. (A and B) HeLa WT or Penta KO HeLa cells were transfected with HA-GFP-Rab9, HA-GFP-Rab14, infected WT-RFP-marked S. typhimuriun (Samonella) for 4 h and analyzed by confocal microscopy. Quantified in B (n = 20 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM analyzed with Student’s t test (two-tailed, unpaired). (C and D) Penta KO HeLa cells transiently expressing HA-GFP-Rab9 WT, HA-GFP-Rab9 (C200, 201S), HA-GFP-Rab14 WT, HA-GFP-Rab14 (C213, 215S), were infected WT-RFP-marked S. typhimuriun (Salmonella) for 4 h and analyzed by confocal microscopy. Quantified in D (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (E) HEK 293T cells were co-transfected with HA-GFP-Rab18 and FLAG-NDP52 constructs, cell lysates were collected, coimmunoprecipitation were performed and analyzed by western blot. (F and H) Penta KO HeLa cells transiently expressing HA-GFP-Rab9 and BFP-NDP52 WT or HA-GFP-Rab9 and BFP-NDP52∆(CC+ZN), were infected WT-RFP-marked S. typhimuriun (Salmonella) for 4 h and analyzed by confocal microscopy. Quantified in H (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (G and I) Penta KO HeLa cells were transfected with HA-GFP-Rab14 and BFP-NDP52 WT or HA-GFP-Rab14 and BFP-NDP52∆(CC+ZN), infected WT-RFP-marked S. typhimuriun (Samonella) for 4 h and analyzed by confocal microscopy. Quantified in I (n = 20 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (J and K) Invasion in J and viability in K of WT-RFP-marked S. typhimuriun (Salmonella) in HeLa, Atg7 KO HeLa, NDP52 KO HeLa, NDP52 KO HeLa rescued with NDP52WT, or NDP52 KO HeLa rescued with NDP52∆(CC+ZN). Quantitative data were obtained from six independent replicates. Data are shown as mean ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. (L and M) Invasion in L and viability in M of WT-RFP-marked S. typhimuriun (Salmonella) in Rab14 KD HEK23T rescued with Rab14 WT or Rab14 (C213, 215S). Quantitative data were obtained from six independent replicates. Data are shown as mean ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F8.

Rab GTPases mediate xenophagy in mammalian cells. (A and B) HeLa WT or Penta KO HeLa cells were transfected with HA-GFP-Rab9, HA-GFP-Rab14, infected WT-RFP-marked S. typhimuriun (Samonella) for 4 h and analyzed by confocal microscopy. Quantified in B (n = 20 cells per group). Scale bars, 10 μm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM analyzed with Student’s t test (two-tailed, unpaired). (C and D) Penta KO HeLa cells transiently expressing HA-GFP-Rab9 WT, HA-GFP-Rab9 (C200, 201S), HA-GFP-Rab14 WT, HA-GFP-Rab14 (C213, 215S), were infected WT-RFP-marked S. typhimuriun (Salmonella) for 4 h and analyzed by confocal microscopy. Quantified in D (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (E) HEK 293T cells were co-transfected with HA-GFP-Rab18 and FLAG-NDP52 constructs, cell lysates were collected, coimmunoprecipitation were performed and analyzed by western blot. (F and H) Penta KO HeLa cells transiently expressing HA-GFP-Rab9 and BFP-NDP52 WT or HA-GFP-Rab9 and BFP-NDP52∆(CC+ZN), were infected WT-RFP-marked S. typhimuriun (Salmonella) for 4 h and analyzed by confocal microscopy. Quantified in H (n = 25 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (G and I) Penta KO HeLa cells were transfected with HA-GFP-Rab14 and BFP-NDP52 WT or HA-GFP-Rab14 and BFP-NDP52∆(CC+ZN), infected WT-RFP-marked S. typhimuriun (Samonella) for 4 h and analyzed by confocal microscopy. Quantified in I (n = 20 cells per group). Scale bars, 10 µm. The scale bars in the magnification boxes are 2 µm. Data are shown as mean ± SEM and analyzed with Student’s t test (two-tailed, unpaired). ***P < 0.001. (J and K) Invasion in J and viability in K of WT-RFP-marked S. typhimuriun (Salmonella) in HeLa, Atg7 KO HeLa, NDP52 KO HeLa, NDP52 KO HeLa rescued with NDP52WT, or NDP52 KO HeLa rescued with NDP52∆(CC+ZN). Quantitative data were obtained from six independent replicates. Data are shown as mean ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. (L and M) Invasion in L and viability in M of WT-RFP-marked S. typhimuriun (Salmonella) in Rab14 KD HEK23T rescued with Rab14 WT or Rab14 (C213, 215S). Quantitative data were obtained from six independent replicates. Data are shown as mean ± SEM and analyzed with one-way ANOVA. *P < 0.05, ***P < 0.001. Molecular weight measurements are in kD. Source data are available for this figure: SourceData F8.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal