Extended Data Fig. 4: Pde5 perturbation restores DC migration through cGMP-regulated myosin-II activity.
From: Rescuing dendritic cell interstitial motility sustains antitumour immunity
a, pMLC signal visualization using DNA-PAINT imaging at nanoscale resolution in whole cell (overlay) or y-z slice view (x = 200 nm) of BMDCs treated with vehicle or 8-Br-cGMP. pMLC signal quantifications are presented on the right (n = 75 steps per group, 1 step = 50 nm). White arrows indicate enhanced pMLC signals in peri-nuclear regions. Colour bar denotes depth of z axis. Scale bars, 1 μm. b, pMLC signal visualization in mature BMDCs treated with vehicle or 500 μM 8-Br-cGMP. Colour bar denotes depth. Scale bar, 1 μm. c, Quantifications of the pMLC signal ratio between the back and front of DCs treated with vehicle or 500 μM 8-Br-cGMP (n = 20 cells per group) (see Methods). d, Western blot analysis of pMLC expression in mature BMDCs treated with 500 μM 8-Br-cGMP for varying durations (0, 5, and 15 min). GAPDH was used as control. e-f, Western blot analysis of pMLC and pROCK levels in mature BMDCs treated with 500 μM 8-Br-cGMP plus 10 μM Y27632 (e) or 50 μM Rhosin hydrochloride (f) for 5 min. g, Quantifications of RhoA activity in BMDCs treated with vehicle (n = 26 cells) or 500 μM 8-Br-cGMP (n = 50 cells) using FRET sensor, with FRET ratios plotted. h, Mean velocities of BMDCs treated with vehicle (n = 125 cells), 500 μM 8-Br-cGMP (n = 158 cells), 8-Br-cGMP plus 50 μM Rhosin (n = 100 cells) or 10 μM Y27632 (n = 132 cells). i,j, Transcript analysis of PRKG1 and PRKG2 in different cell populations of Pan-cancer (i) or colorectal cancer (CRC) (j) patients (see Methods). k, Mean velocities of mature BMDCs treated with vehicle (n = 132 cells), 500 μM 8-Br-cGMP (n = 115 cells), 30 μM sildenafil (n = 137 cells), or each plus 86 μM PKG inhibitor (n = 121, 130, 123 cells correspondingly) in 3D collagen gel assay. Data are representative (a-f) or merged (g, h, k) of at least two to three independent experiments and present as mean ± s.e.m. Statistical analysis was performed using two-tailed Mann–Whitney U-tests (g) or Kruskal–Wallis test with multiple comparisons (h, k).
