Skip to main content
. 2019 Jan 16;39(3):e00264-18. doi: 10.1128/MCB.00264-18

FIG 1.

FIG 1

Rab35 activation alters TMV shedding. (A) TMVs from an equal number of control LOX cells, along with cells expressing Rab35-Q67L or Rab35-S22N, were isolated and analyzed by NTA. Data are presented as means ± the standard errors of the mean (SEM) of replicate measures at each particle diameter. (B) TMV concentrations in conditioned media from cells expressing constitutively active (Q67L) and dominant-negative (S22N) Rab35 were examined relative to the control. Data are presented as means ± the SEM (*, P < 0.001). (C) LOX melanoma cells were transfected with constitutively active myc–Rab35-Q67L or dominant-negative myc–Rab35-S22N, and the impact on TMV shedding was examined by Western blotting. (D) Clonal cell lines generated after the expression of Cas9 with guide RNA targeting Rab35 were lysed, and Rab35 protein levels were examined by Western blotting. (E) A375-P, A375-MA2, and LOX cells were infected with Cas9/Rab35gRNA lentivirus to deplete endogenous Rab35. After pharmacological selection, Rab35 levels were examined by Western blotting. (F) Isolated TMVs released from equal numbers of LOX or LOXRab35KO cells were quantified by NTA. Data are presented as means ± the SEM of replicate measures at each particle diameter. (G) TMV concentrations in conditioned media conditioned by cells described for panel E were determined by NTA. Data are presented as means ± the SEM (*, P < 0.001; **, P < 0.005). (H) TMVs isolated from equal numbers of LOX or LOXRab35KO cells were overlaid onto poly-l-lysine-coated coverslips. After 16 h, the TMV were fixed, stained as indicated and analyzed by confocal microscopy. Scale bars, 10 µm. (I) OvCar3 cells were infected with retrovirus to express constitutively active Rab35-Q67L, dominant-negative Rab35-S22N, or empty vector (Control). At 60 h postinfection, the cells were switched to EV-free media and cultured for an additional 24 h. TMVs were then isolated from the media and examined by NTA. (J) OvCar3 cells were infected with Cas9/Rab35gRNA lentivirus to deplete endogenous Rab35. The Rab35 levels were then examined by Western blotting, and TMV concentrations in conditioned media were measured by NTA. Data are presented as means ± the SEM (*, P < 0.001). (K) OvCar3 cells were infected with lentiCRISPRv2-Rab35 virus, and TMVs were examined as described previously (D). Data are presented as means ± the SEM of replicate measures at each particle diameter.