DEV028423 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. ILK expression is retained in myoepithelial cells in Ilk^−/− mammary glands. (A) Immunohistochemical analysis of Ilk^fx/fx;WapiCre^Tg/• mammary gland wax sections demonstrate ILK-positive brown staining in cells at edges of the alveoli. (B-D) This is also evident by immunofluorescence staining with anti-ILK antibody (red), which colocalises with smooth muscle actin (SMA), a marker for myoepithelial cells (green). Scale bar: 30 µm.

• Supplemental Figure S2 -

  Fig S2. Ad-Cre infection does not affect either ILK or β-casein expression in wild-type MEC. Wild-type primary mammary epithelial cells were uninfected or infected with adenovirus encoding β-Gal (Ad-LacZ) or Cre (Ad-Cre) and then plated onto BM matrix in growth medium for 3 days. Differentiation was induced by prolactin stimulation 24 hours before harvest. The levels of β-casein and ILK are not affected by viral infection. The faint band below β-gal (red asterisk) is non-specific.

• Supplemental Figure S3 -

  Fig. S3. PKB and ERK activation in the absence of ILK. (A) Ilk^fx/fx primary mammary epithelial cells were infected with Ad-LacZ or Ad-Cre and plated onto BM-matrix in growth medium for 3 days. Differentiation was induced by prolactin stimulation 24 hours before harvest. Cell lysates were analysed by immunoblotting. Ad-Cre infected cells show similar steady-state levels of phosphorylated GSK3β-S9 as control or mock-infected cells. (B,C) Acute stimulation of the PKB and ERK pathways in the absence of ILK was measured by culturing infected cells on BM-matrix either without insulin for 3 days and then treating with insulin for 30 minutes, or by serum starvation for 4 hours before a 30-minute EGF treatment, respectively. (B) In the Ad-Cre-infected cells, PKB is still phosphorylated on both Ser473 and Thr308 after insulin stimulation. (C) Phosphorylation of ERK takes place in response to EGF stimulation in both control cells and those following Ad-Cre infection.