Figure 3. rVP1 induced WIPI1 and WIPI2 puncta formation in a BECN1-independent manner. (A) rVP1 and serum starvation increased WIPI1 and WIPI2 puncta formation. To examine WIPI1 puncta formation, RAW264.7 cells were transfected with DsRed-Wipi1 gene. The cells were then pretreated with or without 2 μM CQ for 30 min and subsequently treated with or without serum starvation for 160 min or 4 μM rVP1 for 4 h as indicated. To examine WIPI2 puncta formation, RAW264.7 cells were immunolabeled with anti-WIPI2 antibody followed by rhodamine-conjugated goat anti-mouse IgG (red). (B) rVP1 but not serum starvation induced DsRed-WIPI1 and WIPI2 puncta formation after knockdown of BECN1. RAW^sh-scrambled, RAW^sh-Benc1 stable cell lines or RAW264.7 cells transfected with DsRed-Wipi1 and scrambled or Becn1 siRNA were treated with serum starvation for 160 min or 4 μM rVP1 for 4 h. Fluorescent images were acquired by confocal microscopy. Scale bar: 2 μm. Data represent means ± SEM of quantitative analyses of DsRed-WIPI1 and WIPI2 puncta per cell in at least 50 cells/experiment in three independent experiments; *p < 0.05, **p < 0.01, ****p < 0.0001, N.S, not significant.