Figure 2

CD109 protein, but not mRNA, expression is increased in systemic sclerosis skin fibroblasts. (A) Cell lysates prepared from systemic sclerosis (SSc) (in = 6, Patients S1 to S6) and normal (n = 4, controls N1 to N4) skin fibroblasts were analyzed by western blot using an anti-CD109 antibody, as described in Materials and methods. The membrane was reprobed with an anti-β-actin antibody to confirm that equal amounts of protein were loaded in each lane. (B) Densitometric analysis of CD109 protein levels (180 kDa band) in the cell lysates of SSc and normal skin fibroblasts was performed using β-actin as a loading control. Results from two independent experiments corresponding to skin fibroblasts from two different randomly selected groups of SSC patients versus normal subjects are shown: Experiment 1, normal (n = 4, controls N1 to N4) and SSc (n = 7, Patients S1 to S7); Experiment 2, normal (n = 5, controls N5 to N9) and SSc (n = 9, Patients S8 to S16). P values denote statistical differences of the means between normal and SSc fibroblasts using Student's t test. (C) Data presented in (B) obtained from 16 SSc patients (nine with limited SSc and seven with diffuse SSc) and nine normal healthy controls were reorganized into normal control, limited SSc and diffuse SSc groups and were reanalyzed. One data point (outlier) from the control group (value = 1.04) was removed. Fold-change in the ratio of CD109/β actin in the limited SSc and diffuse SSc groups compared with normal controls is shown. One-way analysis of variance analysis showed a statistically significant difference (P = 0.0127); Holm-Sidak analysis overall significance level = 0.05. Significant differences observed are indicated. (D) Total RNA extracted from SSc (n = 7, Patients S1, S6, S8, S14, S7, S12 and S5) and normal (n = 7, controls N1 to N7) skin fibroblasts was analyzed by RT-PCR using human CD109-specific (top panel) or GAPDH-specific (bottom panel) oligonucleotide primers. PCR products were resolved by agarose gel (1.5%) electrophoresis and visualized by ethidium bromide staining. (E) Densitometric analysis of data presented in (D) expressed as the ratio of CD109 PCR product to GAPDH internal control (CD109/GAPDH) is shown (P = 0.717).