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. 2022 Sep 22;221(12):e202112032. doi: 10.1083/jcb.202112032

Figure 5.

Figure 5.

ORP1L ER-LE MCSs impact small GTPase switching. (A) Fusion activity of CD63-pHluorin HeLa cells overexpressing Rab27a-wt or Rab27a-DN. Measured over >70 cells from n = 3 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (B and C) Fluorescent analysis of HeLa cells co-expressing Rab27a-GFP and Rab7a-RFP (C) or co-expressing Rab27a-GFP and Arl8b-RFP. Green and red arrowheads indicate (some of the) non co-localizing endosomes, white arrowheads indicate co-recruitment to endosomes. (D) Overlap coefficient of Rab7a-RFP and Arl8b-RFP with Rab27a-GFP expressing HeLa cells. n ≥ 3 images (2≥ cells per image) analyzed from ≥2 independent experiments. Red line indicates median. (E) Fusion activity of CD63-pHluorin HeLa cells treated with siArl8b. siNTC, non-targeting control. Measured over >60 cells from n = 3 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (F) Fusion activity of CD63-pHluorin HeLa cells overexpressing Arl8b-wt or DN. Measured over >40 cells from n = 4 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (G) Fluorescent analysis of Rab27a-GFP localization in HeLa overexpressing Rab5a, Rab7a, or Arl8b. (H) Fluorescent analysis of Rab5a-, Rab7a-, or Arl8b-GFP HeLa cells co-expressing mCherry-ORP1L-wt, -∆ORD, or ∆ORDPHDPHD (red). (I) Co-localization analysis of H. PCC, Pearson’s Correlation Coefficient. Measured over n ≥ 4 images (2≥ cells per image) analyzed from three independent experiments using Student’s two-tailed two-sample t test. Red line indicates median. (J) Fusion activity of CD63-pHluorin HeLa cells treated overexpressing ORP1L-∆ORDPHDPHD without or with Arl8b-DN co-expression. Measured over >70 cells from n = 4 experiments using Student’s two-tailed two-sample t test. Red line indicates median. (K) Co-localization analysis of Rab27a-GFP/CD63-pHuji co-expressing HeLa, treated with siORP1L. To the right: quantification of Rab27a-GFP/CD63-pHuji overlap in ctrl (siNTC) or ORP1L (siORP1L) depleted condition. Measured over n ≥ 5 images (2≥ cells per image) analyzed from three independent experiments using Student’s two-tailed two-sample t test. Red line indicates median. DN, dominant negative. Bars: 5 µm.