Figure 5.
Interactions between R. parkeri and the ER are dependent on VAPA and VAPB. (A and B) Immunofluorescence detection of endogenous VAPA or VAPB localization around sca2::Tn mutant bacteria (magenta) at 24 hpi. Images are representative of three independent experiments. Scale bar, 10 µm; inset scale bar, 1 µm. (C) Western blot analysis of A549-mCh-Sec61β cell lysates after indicated siRNA treatment at 72 h after transfection. GAPDH, loading control. (D) Percentage of A549-mCh-Sec61β cells displaying BERCs with sca2::Tn mutant bacteria at 24 hpi after the indicated siRNA treatment in C. (E) Western blot analysis of VAPA and VAPB expression in HeLa cell lysates from F. GAPDH, loading control. (F) Percentage of WT or VAPA/B DKO HeLa cells displaying BERCs with sca2::Tn mutant bacteria (28 hpi). For D and F, the mean ± SEM of three independent experiments (large outlined circles) are superimposed over the raw data the percent of cells with BERCs per FOV (small unlined circles) (>100 cells quantified per condition per independent experiment) (one-way ANOVA with Dunnett’s post hoc test). (G) Live-cell imaging snapshots at 24 hpi of Halo-VAPAΔTM-I3-01 or Halo-VAPBΔTM-I3-01 (grey) fusions (WT versus KD/MD FFAT-binding mutant) during infection with sca2::Tn mutant bacteria (magenta). Scale bar, 10 µm; inset scale bar, 1 µm. (H) Percentage of sca2::Tn mutant bacteria in transiently transfected A549 cells showing Halo-VAPΔTM-I3-01 localization. Data are mean ± SD of three independent experiments (≥4 FOV with ≥200 bacteria quantified per condition per independent experiment) (unpaired two-tailed t test). Source data are available for this figure: SourceData F5. Refer to the image caption for details.

Interactions between R. parkeri and the ER are dependent on VAPA and VAPB. (A and B) Immunofluorescence detection of endogenous VAPA or VAPB localization around sca2::Tn mutant bacteria (magenta) at 24 hpi. Images are representative of three independent experiments. Scale bar, 10 µm; inset scale bar, 1 µm. (C) Western blot analysis of A549-mCh-Sec61β cell lysates after indicated siRNA treatment at 72 h after transfection. GAPDH, loading control. (D) Percentage of A549-mCh-Sec61β cells displaying BERCs with sca2::Tn mutant bacteria at 24 hpi after the indicated siRNA treatment in C. (E) Western blot analysis of VAPA and VAPB expression in HeLa cell lysates from F. GAPDH, loading control. (F) Percentage of WT or VAPA/B DKO HeLa cells displaying BERCs with sca2::Tn mutant bacteria (28 hpi). For D and F, the mean ± SEM of three independent experiments (large outlined circles) are superimposed over the raw data the percent of cells with BERCs per FOV (small unlined circles) (>100 cells quantified per condition per independent experiment) (one-way ANOVA with Dunnett’s post hoc test). (G) Live-cell imaging snapshots at 24 hpi of Halo-VAPAΔTM-I3-01 or Halo-VAPBΔTM-I3-01 (grey) fusions (WT versus KD/MD FFAT-binding mutant) during infection with sca2::Tn mutant bacteria (magenta). Scale bar, 10 µm; inset scale bar, 1 µm. (H) Percentage of sca2::Tn mutant bacteria in transiently transfected A549 cells showing Halo-VAPΔTM-I3-01 localization. Data are mean ± SD of three independent experiments (≥4 FOV with ≥200 bacteria quantified per condition per independent experiment) (unpaired two-tailed t test). Source data are available for this figure: SourceData F5.

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