Extended Data Fig. 3: ROCK-education of mammary fibroblasts yields a tumor-promoting fibroblast phenotype via paracrine Creld2; Related to Fig. 3.
a, Creld2 protein in 4HT-treated K-PyMT and R-PyMT ex vivo-cultured tumor cells. Experiment repeated twice with similar results. b, Creld2 (greyscale and green) in tumors one week following engraftment of tumor cells transfected with non-targeting control (NTC) or Rock1/2 siRNA. Counter-labels: E-cadherin (red); PyMT (blue). Scales: 50 µm. Mean±SEM (n = 5 NTC, 3 siRNA), two-sided unpaired t-test. c, Creld2 (green) in advanced tumor cells treated with fasudil and Y-27632. Counter-labels: Phalloidin (F-actin, red) and DAPI (blue). Scales: 50 µm. Mean±SEM (n = 10/group), one-way ANOVA. FOV = Field of view. d, Creld2 mRNA in R-PyMT cells following Creld2 knockdown (n = 3/group). Mean+SEM, one-way ANOVA. e, Creld2 (greyscale and green) in immortalized PyMT cell line following Creld2-CRISPR, clone E4. Counter-labels: Phalloidin (red); DAPI (blue). Scales: 50 µm. Mean±SEM (n = 13 Parental, 10 E4), two-sided unpaired t-test. f, Mean distance migrated by fibroblasts exposed to CM from PyMT cells with Creld2-CRISPR (n = 3/group) in inverse invasion assay. Mean±SEM, two-sided unpaired t-test. g, Co-engraftment strategies to examine effect of Creld2 on fibroblast function in vivo. h, Fibronectin (n = 5 KD + NTC,6 ROCK + NTC,6 ROCK + siRNA) and periostin (n = 6 KD + NTC,6 ROCK + NTC,5 ROCK + siRNA) (green) in tumors following co-engraftment of tumor cells and fibroblasts exposed to tumor cell CM following Creld2 knockdown. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Mean±SEM, one-way ANOVA. i, Progressive tumor volume following co-engraftment of tumor cells with fibroblasts exposed to CM from K-PyMT cells following Creld2 knockdown (n = 8 tumors/group). Mean&SEM, 2-way ANOVA. j, Final weights of tumors from i, two-sided Mann-Whitney test. Box plot: midline=median; box=25-75th percentile; whisker=minima to maxima. k, Fibronectin and periostin (green) in tumors following co-engraftment of tumor cells and fibroblasts treated with recombinant Creld2. Counter-labels: E-Cadherin (red); DAPI (blue). Scales: 100 µm. Median±IQR (n = 8 tumors untreated, 9 Creld2), two-sided Mann-Whitney. See also Statistics Source Data for Extended Data Fig. 3 and Unprocessed blots for Extended Data Fig. 3.
