JCS089466 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Results of gene expression analysis in Tiam1-deficient and Tiam1-overexpressing cells. Results of microarray analysis with GenomeStudio software for selected genes. Top panel. Data are shown as fold-change and adjusted p-values for shTiam1-RMF compared with control vector RMF (for Tiam1 silencing) and doxycycline-induced +Tiam-MEF compared with doxycycline-induced vector-MEF (for Tiam1 overexpression). Bottom panel: graphical representation of fold change for genes in top panel.

• Supplemental Figure S2 -

  Fig. S2. Alterations of NFkB pathway components in cells with modulatedTiam1 expression. Fold change in gene expression from cells with Tiam1 deficiency (left panel) and Tiam1 overexpression (right panel) as described in Fig. S1 was analyzed using Ingenuity Pathway Analysis software. Overexpression relative to control is rendered in red; under-expression relative to control is rendered in green; degree of relative change is indicated by color shading.

• Supplemental Figure S3 -

  Fig. S3. OPN transcription is downregulated in Tiam-overexpressing MEF cells. (A) Immunoblots of lysates from indicated MEF cells for Tiam1 and GAPDH as shown. (B) qRT-PCR using OPN-specific primers on cDNA derived from MEF cells as indicated. C-MEF indicates cells with control pBIG vector, +Tiam-MEF indicates cells with pBIG-Tiam1, both under doxycycline-deprivation.

• Supplemental Figure S4 -

  Fig. S4. Expression of Tiam1 in engineered RMF lines. (A,B) Upper panels show immunoblots of lysates from indicated RMF cells for Tiam1 and GAPDH. Numbers below indicate mean quantification of Tiam1 by densitometry for three independent experiments.

• Supplemental Figure S5 -

  Fig. S5. Morphologic changes in RMFs undergoing stress-induced senescence. (A) Stress-associated β-galactosidase (SA-β-gal) staining of RMFs under conditions as indicated. (B) Quantification of numbers of SA-β-gal-positive cells, shown as percentages of total cells. Graph represents mean ± s.d. for three independent experiments. Pre- indicates pre-senescent cells, H2O2 indicates oxidative stress, Bleo indicates bleomycin, XRT indicates radiation.

• Supplemental Figure S6 -

  Fig. S6. Morphologic changes in RMFs with altered Tiam or OPN expression. (A) Stress-associated β-galactosidase (SA-β-gal) staining of indicated RMF lines, with either endogenous protein expression (C-RMF), Tiam deficiency (shTiam), Tiam overexpression (+Tiam), or OPN deficiency (shOPN). Left panels, pre-senescent cells right panels, after oxidative stress. Magnification 4×. (B) Quantification of numbers of SA-β-gal-positive cells, shown as percentages of total cells. Graph represents mean ± s.d. for three independent experiments. Pre- indicates pre-senescent cells, H2O2 indicates oxidative stress.

• Supplemental Figure S7 -

  Fig. S7. Calpain activity is increased in cells undergoing stress-induced senescence. Calpain activity assessed by fluorescent reporter assay is shown for pre-senescent (Pre) and senescent (H2O2) cells, some pretreated with calpain inhibitor (H2O2+inhib). Controls include lysates with no calpain substrate and lysates with exogenous calpain. Results indicate mean ± s.d. for duplicate independent experiments.

• Supplemental Figure S8 -

  Fig. S8. Co-culture with senescent fibroblasts induces increased invasion and migration of HMECs. (A−D) Light micrographs of representative spheroid co-cultures with HMECs and indicated RMFs. (E) Transwell migration of HMECs 2 weeks after isolation out of co-culture with indicated RMFs. Pre indicates control non-senescent RMFs, H2O2 indicates oxidative stress-induced RMFs, Bleo indicates chemical stress-induced RMFs, shTiam indicates Tiam1-deficient RMFs. *P<0.005 compared with pre-senescent cells.

• Supplemental Figure S9 -

  Fig. S9. Isolation of post-co-culture HMECs. (A) Sample flow cytometry analysis of cells 9 days after removal from Matrigel co-culture, showing HMEC (red, y-axis) and RMF (green, x-axis) populations. (B) Percentages of HMECs and RMFs over time in cells isolated after co-culture, assessed by flow cytometry. Results represent mean ± s.d. for triplicate experiments.

• Supplemental Figure S10 -

  Fig. S10. Flow cytometry analysis of post-co-culture cell populations. HMEC (red, y-axis) and RMF (green, x-axis) populations are shown. Top two panels show pure HMEC and RMF populations prior to co-culture. Remaining panels show cell assays 2−3 weeks after isolation from co-cultures, corresponding to post-co-cultured cells used in Figs 5 and 6. C-RMF, control RMF; +Tiam RMF, Tiam overexpression; shOPN, OPN-deficient; H2O2, RMFs rendered senescent by oxidative stress prior to co-culture.