Fig 1: CCN2 promotes colony formation and 3D growth of transformed keratinocytes expressing α3β1.(A) WB of CCN2 and integrin α3-expression of selected CCN2 KO and control clones. (B) Representative image (top) and quantification (bottom) of colony-formation assay of MSCC Itga3 WT and Itga3 KO cells and MSCC CCN2 KO G1, KO G2, control G1, and control G2 clones. Deletion of α3β1 results in a strong reduction of colony formation and colony size. Moderate reduction of colony formation can be seen upon CCN2 deletion. Quantification of colony size can be found in Fig S6A. Average values of technical triplicates of five independent experiments are presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005, ****P < 0.0001). (C) Representative image (top) and quantification (bottom) of colony-formation assay of MSCC CCN2 KO G1 and KO G2 clones, grown in control conditions or in the presence of 45 ng/ml CCN2, as well as CCN2 WT control G1 and control G2 clones. Treatment with exogenous CCN2 significantly increases colony formation of CCN2 KO clones. Quantification of colony size can be found in Fig S6B. Average values of technical triplicates of four independent experiments are presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005). (D) No differences in the number of colonies can be observed upon CCN2 treatment of Itga3 KO–transformed keratinocytes. Quantification of colony size can be found in Fig S6B. Average values of technical triplicates of four independent experiments are presented (mean ± SD, Fisher’s LSD test, ****P < 0.0001). (E) Quantification (left) and IF as maximum intensity projection (right) of CCN2 expression of MSCC Itga3 WT and KO spheroids, grown in 3D Matrigel matrix for 1 or 7 d (scale bar: 20 μm). The expression of CCN2 is α3β1 dependent, which is particularly prominent at the beginning of spheroid growth. The percentage of CCN2-positive area was quantified from 17 MSCC Itga3 KO and 30 MSCC Itga3 WT spheroids 1 d after seeding and from 15 MSCC Itga3 KO and 27 MSSC WT spheroids 7 d after seeding (mean ± SEM, unpaired t test, *P < 0.05). (F) Spheroid growth in 3D Matrigel is α3β1 dependent and moderately reduced upon CCN2 deletion. Top: bright-filed images of representative spheroids (scale bar: 50 μm). Bottom: size quantification of 60–80 spheroids measured over three to four independent experiments (mean ± SD, Fisher’s LSD test, ****P < 0.0001). (G) 3D growth of CCN2 KO MSCC spheroids shows small but significant increase when cells are seeded with 45 ng/ml of CCN2. Left: bright-filed images of representative spheroids (scale bar: 50 μm). Right: size quantification of 85–90 spheroids measured over three independent experiments (mean ± SD, Fisher’s LSD test, **P < 0.005, ***P < 0.0005 ****P < 0.0001). (H) Seeding Itga3 KO MSCCs with 45 or 180 ng/ml of CCN2 does not impact the 3D growth pf spheroids. Left: bright-filed images of representative spheroids (scale bar: 50 μm). Right: size quantification of 70 spheroids measured over two independent experiments (mean ± SD, one-way ANOVA, P = 0.9491).Source data are available for this figure.
Fig 2: CCN2 expression in transformed keratinocytes is a3ß1-dependent.(A) GAPDH-normalized relative mRNA expression of CCN2 is significantly decreased in non-stimulated as well as IL-6 and TPA-treated a3ß1-depleted keratinocytes. The average of up to four independent measurements of technical duplicates of four RNA samples per group (dots) is presented (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). (B) IF (left) and quantification of the mean intensity (right) of CCN2 in non-stimulated, IL-6, and TPA-treated MSCC Itga3 KO and WT keratinocytes. Expression of CCN2 is a3ß1 dependent and increases upon IL-6 and TPA treatment (scale bar: 50 µm). 90 cells imaged over three independent experiments were quantified (mean ± SD, Fisher’s LSD test, P < 0.0001). (C) Representative WB confirming a3ß1-dependent and IL-6– and TPA-mediated CCN2 expression. Quantification can be found in Fig S5A.Source data are available for this figure.
Fig 3: HB-originating a3ß1-depleted keratinocytes show decreased expression of CCN2 during the initiation stage of tumorigenesis.(A) A heat map showing expression of selected epidermal markers, selected from gene list, in GFP-positive HB-originating keratinocytes, isolated from the skin of short-term DMBA/TPA–treated K19 Itga3 KO and WT mice. (B) IHC staining for GFP and CCN2 in the skin of short-term DMBA/TPA–treated K19 Itga3 KO and WT mice (scale bar: 100 µm). (C) Sparse CCN2-positive cells can be observed in epithelia and stroma of all tumors of K19 Itga3 WT mice (arrow heads). Top: quantification of the CCN2-positive area in cross-section of 127 tumors, isolated from six K19 Itga3 WT mice. CCN2 represents less than 1% of total tumor surface in most tumors. Bottom: representative IHC staining for CCN2 (scale bar: 500 µm). (D) Consecutive section of papilloma, isolated from K19 Itga3 WT mouse, stained for GFP and CCN2. No overlap of the two markers can be observed (scale bar: 500 µm).
Fig 4: CCN2 expression in transformed keratinocytes is α3β1-dependent.(A) Quantification of WB from Fig 7C. Bars represent the mean of five independent experiments (mean ± SD, Fisher’s LSD test, *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). (B) IF images showing co-localization of excreted CCN2 and laminin-332 in the culture of non-treated MSCC Itga3 KO and WT keratinocytes.
Fig 5: a3ß1-depleted keratinocytes show an increased differentiation signature and decreased expression of CCN2 during the initiation stage of tumorigenesis.(A) GFP-positive Cre-induced HB SCs localize to growing hair follicles (HFs) and, in some cases, to isthmus, infundibulum, and IFE (black arrows) after short-term DMBA/TPA treatment in K19 Itga3 KO and WT mice. Left: IHC staining for GFP (scale bar: 100 µm). Right: quantification of the number of HFs, where GFP-positive cells were observed in the upper parts of HFs and in adjacent IFE. Each dot represents a mouse (mean ± SD, unpaired t test). (B) Heat map of row-scaled significantly differentially expressed protein-coding genes of GFP-positive keratinocytes, isolated from three K19 Itga3 KO and three K10 Itga3 WT mice after short-term DMBA/TPA treatment. Protein-coding genes have an adjusted P < 0.05 and an average normalized expression across all samples >4 (as calculated with Voom) and a logFC > 0.6 between K19 Itga3 WT an KO mice. (C) IF staining (left) and quantification of mean intensity of the signal (right) for CCN2 in GFP-positive HFs after short-term DMBA/TPA treatment. Each dot represents a GFP-positive HF. HFs of five K19 Itga3 KO and six K19 Itga3 WT mice were quantified (mean ± SD, unpaired t test, P < 0.0001).
from BioVendor Laboratory Medicine, Inc. for Connective Tissue Growth Factor Mouse HEK293