Supplementary Figures Legend:
Fig. S1. Effect of caveolin-1 knockdown on MSC osteogenic differentiation in donor 60f.
MSCs were transfected with a non-targeting control siRNA (Ctrl) or with caveolin-1
siRNA (Cav) then incubated in growth medium (GM) or osteogenic medium (OM). (A)
After 4 days ALP activity was significantly enhanced in cells transfected with caveolin-1
siRNA and incubated in osteogenic medium. Values are the mean ± s.e.m (4 wells). (B)
Alizarin red staining after 10 days. (C) Low magnification images from one well in each
condition in (B); bar = 200 μm. (D) Quantification of Alizarin red stain in each well in (B)
normalized to DNA shows Alizarin red staining was significantly enhanced in osteogenic
cultures transfected with caveolin-1 siRNA. Values are the mean ± s.e.m. of 4 wells.
Fig. S2. Caveolin-1 mRNA expression at the end of osteogenic differentiation in MSCs
from donors 50m and 49f. Caveolin-1 mRNA expression was measured in MSCs
transfected with a non-targeting control siRNA (Ctrl) or caveolin-1 siRNA (Cav) then
incubated in growth medium (GM) or osteogenic medium (OM) for 21 days. (A-B)
Caveolin-1 mRNA expression in cells that were transfected with siRNA once before the
beginning of 21 days osteogenic differentiation. Caveolin-1 knockdown in MSCs
transfected with caveolin-1 siRNA and incubated in osteogenic medium was greater in
MSCs from donor 50m (A) than in MSCs from donor 49f (B). (C-D) Caveolin-1 mRNA
expression in cells that were transfected with siRNA once before the beginning of 21
days osteogenic differentiation, again on day 6 and again on day 13 of osteogenic
differentiation. Caveolin-1 knockdown was much greater in MSCs transfected three times
and caveolin-1 mRNA expression was reduced by caveolin-1 siRNA to the same extent
compared to controls in MSCs from donor 50m (C) and donor 49f (D). Caveolin-1
expression was normalized to the expression GAPDH. Values are the mean of duplicate
samples.
Fig. S3: Transduction of caveolin-1 specific shRNA enhances MSC osteogenesis. MSCs
were left uninfected (Ctrl) or transduced with a lentiviral construct containing shRNA
targeting caveolin-1 (Cav shRNA) and then grown in growth medium (GM) or osteogenic
medium (OM). (A) Caveolin-1 mRNA expression measured by RT-PCR and normalized to
the house keeping gene GAPDH. Values are the mean ± s.e.m (3 wells). Cells
transduced with caveolin-1 shRNA showed reduced caveolin-1 mRNA expression. (B)
After 4 days incubation, ALP activity was significantly enhanced in cells transduced with
caveolin-1 shRNA and incubated in osteogenic medium. Values are the mean ± s.e.m (4
wells). (C) After 21 days incubation, Alizarin red staining was much greater in caveolin-1
shRNA osteogenic cultures compared to uninfected parental cells.
Fig. S4. Expression of caveolin-1 and osteogenic genes in MSCs treated with control or
caveolin-1 siRNA and incubated in growth medium or osteogenic medium for 10 days.
Further data from the samples in Fig. 6 is presented. MSCs from 4 donor populations
(59f, 49f, 50m and 65f) at the same passage were pooled and transfected with either
control siRNA (Ctrl) or caveolin-1 siRNA (Cav). 24 hours after transfection cells were
incubated in growth medium (GM) or osteogenic medium (OM) for 10 days and the RNA
harvested for gene expression analysis. (A) RT-PCR for caveolin-1 expression normalized
to GAPDH expression. Caveolin-1 expression is still significantly reduced in caveolin-1
siRNA transfected samples at day 10 of osteogenesis. Caveolin-1 expression is
upregulated in cells incubated in osteogenic medium compared to cells in growth
medium, in agreement with data in Fig. 2 and Fig. 3. Values are the mean ± s.e.m (3
wells). (B) A qPCR array was performed on the same samples as in (A) for 84 different
osteogenic genes. Plots of some of the fold changes in osteogenic gene expression
(relative to control siRNA transfected cells in growth medium) are presented (scatter
plots for all genes are presented in Fig. 6A). Top panel: Of all genes tested, expression
of COL10A1 was most highly upregulated by osteogenic medium after 10 days.
Caveolin-1 siRNA transfected cells incubated in osteogenic medium showed a two-fold
higher upregulation in COL10A1 expression compared to controls, but this was not
statistically significant. Bottom panel: Other osteogenic genes upregulated by osteogenic
medium on day 10 and showing a trend of further upregulation in caveolin-1 siRNA
transfected cells in osteogenic medium are presented.