Fig 2: The inhibitory effect of fasudil hydrochloride on penicillin-induced upregulation of ROCK1, p-MYPT1 and p-MLC2 expression, F-actin reorganization and endothelial monolayer hyperpermeability in HUVECs.(a) ROCK1, p-MYPT1 and p-MLC2 expression in HUVECs incubated with 5 kU/mL penicillin with or without pretreatment with 10 µM fasudil hydrochloride (n = 6, per group). (b–g) F-actin formation and assembly in confluent endothelial monolayer or un-confluent HUVECs ((b,e) untreated cells; (c,f) 20 kU/mL penicillin; (d,g) 10 µM fasudil hydrochloride +20 kU/mL penicillin). (h) Fluorescence apparent permeability coefficients of FITC-dextran through the HUVEC monolayer (n = 3, per group). Blank: untreated cells; P: 20 kU/mL penicillin; F + P: 10 µM fasudil hydrochloride +20 kU/mL penicillin. *p < 0.05, **p < 0.01 compared with the penicillin-treated group; #p < 0.05, ##p < 0.01 compared with untreated cells; one-way ANOVA. Error bars represent the s.e.m.

Fig 3: The effect of penicillin on endothelial monolayer permeability, F-actin distribution and GTP-RhoA, ROCK1, p-MYPT1 and p-MLC2 expression in HUVECs.(a) Fluorescence apparent permeability coefficients of FITC-dextran through the HUVEC monolayer (n = 3, per group). (b–i) F-actin formation and assembly in confluent endothelial monolayer or un-confluent HUVECs ((b,f) untreated cells; (c,g) 1.25 kU/mL penicillin; (d,h) 5 kU/mL penicillin; (e,i) 20 kU/mL penicillin). (j) GTP-RhoA, ROCK1, p-MYPT1 and p-MLC2 expression in HUVECs incubated with 5 kU/mL penicillin for different time courses (n = 6, per group). Blank: untreated cells. *p < 0.05, **p < 0.01 compared with untreated cells; one-way ANOVA. Error bars represent the s.e.m.

Fig 4: Targeted ablation reveals unique roles for PSC-derived CAFs in regulation of the ECM and mechanosignaling. A, IHC staining and quantification of GFP+ cells in normal pancreas tissue from Fabp4-Cre;Rosa26mTmG mice and from Rosa26mTmG/iDTR mice 7 days after intraductal injection with AAVKP1-Fabp4-Cre (n = 5). Data are presented as mean ± SEM. Scale bar, 50 µm. B, Schematic of tumor modeling using intraductal injection of AAVKP1-Fabp4-Cre and orthotopic transplantation of KPC PDAC cells into Rosa26mTmG/iDTR hosts. C, IHC staining for GFP, PDPN, and a-SMA of KPC FC1199 PDAC in Rosa26mTmG/iDTR hosts with intraductal injection of AAVKP1-Fabp4-Cre, enrolled when tumors reached 5 to 6 mm in diameter and treated with PBS or DT for 5 days (n = 4). Scale bar, 20 µm. D, IHC staining for TNC of KPC FC1199 PDAC in AAVKP1-Fabp4-Cre–injected Rosa26mTmG/iDTR hosts, enrolled at 5 to 6 mm in tumor diameter and treated with PBS or DT for 5 days (n = 3). Scale bar, 50 µm. Data are presented as mean ± SEM. E, IHC staining for p-MLC2 of PDAC samples as described in D. Scale bar, 50 µm. Data are presented as mean ± SEM. Quantification (F) and images (G) of fibrillar collagen content analyzed by second harmonic generation with normalized intensity as a quantification of concentration in control and PSC-depleted PDAC (n = 3 per group). Scale bar, 42.5 µm. H, Force maps generated by atomic force microscopy (AFM) on KPC FC1199 PDAC in AAVKP1-Fabp4-Cre–injected Rosa26mTmG/iDTR hosts (n = 3 per treatment group, control: 1,063 data points, depleted: 717 data points), excised after 5 days of treatment with PBS or DT. I, Quantification of Young's modulus per AFM measurements on control and PSC-depleted PDAC as described in F. The dashed line on the graph to the right denotes the approximate stiffness of normal murine pancreas tissue. J, IHC staining for p-FAK (Y397) of control and PSC-depleted PDAC harvested after 5 days of depletion (n = 3). Scale bar, 50 µm. Data are presented as mean ± SEM. K, Kaplan–Meier plot depicting overall survival of patients with PDAC with high versus low expression of a PSC-derived CAF ECM gene signature comprising 99 genes (see Methods), plotting the upper versus lower quartile (n = 73 per arm). *, P < 0.05; ****, P < 0.0001 by unpaired t test; ns, not significant.

Fig 5: DOCK3 inhibits WAVE2 expressiona. Quantification of RhoA (left panel) and Rac1 (right panel) activation in HuH-7 cells transfected with siDOCK3 or siCtrl at 48 h after transfection. ns p > 0.05 by student's t-test. b. Co-immunoprecipitation of endogenous RhoA (left panel) or Rac1 (right panel) with DOCK3. c. HuH-7 cells transfected with siDOCK3 or siCtrl were analyzed for p-mlc2 and DOCK3 by Western blot at 48 h after transfection. d. HuH-7 cells transfected with siDOCK3 or siCtrl were analyzed for WAVE2 by relative quantitative real-time RT-PCR (left panel) and Western blot (right panel) at 48 h after transfection. ***p < 0.001 by student's t-test. e. Relative quantitative real-time RT-PCR analysis of DOCK3 (left panel) and WAVE2 (right panel) in A549 cells transfected with siDOCK3 or siCtrl at 48 h after transfection. ***p < 0.001, **p < 0.01 by student's t-test. f.-h. Confocal microscopy images of p-mlc2 expression (f), WAVE2 expression (g) and actin cytoskeleton rearrangement (h) in A549 cells transfected with siDOCK3 or siCtrl at 48 h after transfection. i. DIC microscopy images of A549 cells transfected with siDOCK3 or siCtrl at 48 h after transfection. Arrows indicate lamellipodium dynamics. **p < 0.01 by student's t-test. j. Quantitative analysis of relative migration distance. ***p < 0.001, **p < 0.01 by ANOVA. Error bars indicate the standard deviations (SD) from at least triplicate determinations (n > 3)