Fig 1: Generation of MCF7-B2 cells. Infected cells with viral vectors expressing GFP (lanes 2 and 3), ephrin-B2-WT (B2-WT) (lanes 4 and 5) and ephrin-B2-5F (B2-5F) (lanes 7 and 8) were treated with two multiplicities of infection (MOI): MOI 25 (lanes 2, 4 and 7) and MOI 1.5 (lanes 3, 5 and 8). Total lysate from SKBR3 cells infected with human ephrin-B2 (EX-M0409-Lv105, Genecopoeia) (~50 kDa) was used as a positive control (C+). Non-infected MCF7 cells (N.I) were used as a negative control. GAPDH was used as a loading control. Ephrin-B2 detected with the anti-pan (A) ephrin B antibody or (B) anti-GFP antibody and visualized as an 80 kDa GFP-fusion protein indicated by arrowheads. Ephrin-B2 activation was verified by (C) immunoprecipitation and column charts, from this representative experiment, and representative immunoblots are shown. Ephrin-B2 expression was visualized by confocal microscopy at ×600 magnification. (D) Cell nuclei are indicated with DAPI staining. Scale bars, 30 µm.
Fig 2: CAFs stimulate expression of RANKL in osteoblasts. (A) mRNA expression of OPG and RANKL in human osteoblasts (HOB) following 24 h culture with conditioned media from CAF and H357 cells. Data represent n=3±s.d. (B) Immunoblot of αSMA protein in NOF treated with TGF-β1 (5 ng ml−1) for 72 h. GAPDH was used as a loading control, and densitometry carried out normalised to GAPDH in the same samples (lower). (C) Conditioned medium was collected from TGF-β1 treated NOF (72 h treatment) and soluble RANKL quantified by ELISA. (D) RANKL was quantified in conditioned medium collected from OSCC-derived CAF (002, 003 and 004). Data represent n=3±s.d. *P⩽0.05; **P⩽0.01; ***P⩽0.001; ****P⩽0.0001.
Fig 3: ERG is downregulated in response to high stiffness and inflammation.A Representative IF images and quantification of ERG (green) in HUVECs seeded for 72 h on 0.5 vs 30 kPa PAA gels and treated for an additional 24 h with IL1b or vehicle (n = 969 cells from 0.5 kPa, n = 744 cells from 0.5 kPa IL1β, n = 1132 cell from 30 kPa and n = 1133 cells from 30 kPa IL1β, from n = 3 independent experiments). B Representative Western Blot in HUVECs seeded for 72 h on 0.5 vs 30 kPa PAA gels and treated for an additional 24 h with IL1b or vehicle. ERG expression was normalized to GAPDH as housekeeping protein (n = 6 independent experiments - Supplementary Fig. 3). C qRT-PCR of ERG gene in HUVECs seeded for 96 h of 0.5 vs 30 kPa (n = 3 independent experiments). D Representative IF images and quantification of ERG in freshly isolated healthy rat LSECs seeded on 0.5 vs 30 kPa PAA gels for 24 h (n = 120 cells from 0.5 kPa and n = 144 cells from 30 kPa were analysed, from n = 4 rats). E Representative IF images and quantification of ERG in freshly isolated cirrhotic rat LSECs seeded on 0.5 vs 30 kPa PAA gels for 24 h (n = 78 cells from 0.5 kPa and n = 178 cells from 30 kPa were analysed, from n = 3 rats). All IF data were normalized to the number of cells. For each experiment, sample distribution was assessed for normality (Kolmogorov-Smirnov test). Normally distributed data were compared with unpaired Student t test, otherwise with Mann-Whitney test. Data for the stiffness + inflammation comparison were normalized to the 0.5 kPa vehicle condition and analyzed with Ordinary One-way ANOVA, * = p < 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001, scale bar HUVEC = 100 µm, scale bar LSEC = 50 µm.
Supplier Page from Abcam for HRP Anti-GAPDH antibody [EPR6256] - Loading Control