Fig. 4: RAB33A induces nondegradative autophagy to stabilize RhoC.

a–d LC3-II protein levels in wild-type or RAB33A-overexpressing HeLa and SiHa cell lines (a) and wild-type or RAB33A-knockdown HeLa and SiHa cell lines (c) were measured by western blotting. These results are presented from three independent experiments. The protein levels of LC3-II/LC3-I were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). e–h Increased LC3-II expression in cells treated with Baf A1 combined with CQ (Baf A1: 200 nM, 6 h; CQ: 40 μM, 6 h). Wild-type or RAB33A-overexpressing HeLa (e) and SiHa (g) cell lines were treated with the late autophagosome inhibitor Baf A1 (200 nM) or CQ (40 μM) for 6 h. The protein levels of LC3-II were determined by western blotting. These results are presented from three independent experiments. The protein levels of LC3-II/LC3-I were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). i Electron microscopic images; autophagosomes are indicated by red arrows. j, k Immunofluorescence image of increased numbers of autophagosomes after RAB33A overexpression (P < 0.0001). Localization of endogenous LC3 in HeLa cells (j), and the number of LC3 puncta per cell was calculated (k, n = 20 cells). The data are presented as the mean ± SD. P values are shown; two-tailed Student’s t-test. Vector, vector-only control. l, m The colocalization of GFP and RFP puncta in live cells was quantified. The numbers of puncta, either yellow puncta with both green and red fluorescence (autolysosome) or red puncta with only red fluorescence (autophagosome), were quantified (right panel). P values were calculated by Student’s t-test. n–s Effects of ATG5 (n) and ATG7 (q) knockout combined with RAB33A overexpression on RhoC and LC3-II levels and migratory capabilities. RhoC and LC3-II protein levels in HeLa cells with ATG5 or ATG7 knockout combined with RAB33A overexpression. These results are presented from three independent experiments. The protein levels of RhoC and LC3-II/LC3-I were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). t Migration and invasion of HeLa cells with ATG5 knockout combined with RAB33A overexpression. u, v An in vivo model of cervical cancer lymph node metastasis was established using the indicated stable cell lines; the model was established in 7 biologically independent mice. Dissected popliteal and inguinal LNs (u) and their volumes (v) from mice with tumors derived from wild-type or RhoC/ATG5-knockout SiHa cells. The data are presented as the mean ± SD. P values are shown; Student’s t-test.