LEGENDS TO SUPPLEMENTARY FIGURES AND TABLES.
Supplementary Fig S1. (a) Immunofluorescence of endogenous SIX1 and p16 in
ER:Ras IMR90 cells, after 7 days with or without tamoxifen induction of Ras. Note the
absence of nuclear SIX1 staining in Ras-senescent, SAHF-positive and p16-positive
cells. IMR90 with ectopic expression of SIX1 are included as a control (IMR90 SIX1).
(b) Western Blot analysis of SIX1 and p16 in IMR90 fibroblasts expressing the
reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) or Ras. (c), QPCR analysis
of SIX1 transcript levels in replicative-senescent (EP, early passage, lower than PDL
35; LP, late passage, higher than PDL 50) or OSKM-induced senescent fibroblasts. (d)
Validation of senescent phenotype in replicative or OSKM-induced senescent IMR90
fibroblasts used in A and B, using SABGal and Crystal Violet staining. (e), Western Blot
analysis of SIX1 and the indicated senescence markers in human primary dermal
fibroblasts (HDF) expressing an ER:RasV12 cassette at the indicated time-points after
induction with tamoxifen. 7- indicates 7 days without tamoxifen.
Supplementary Fig S2. (a) Expression of SIX family members and the cofactor EYA2
in growing and Ras-senescent fibroblasts. (b) From left to right: QPCR analysis of SIX
family members, quantification of SABGal-postive cells, Western Blot analysis of SIX1
and p16, and QPCR analysis of p16 in IMR90 fibroblasts expressing two different
shSIX1 vectors (c) QPCR analysis of transcript levels of SIX family proteins and
selected targets in IMR90 fibroblasts expressing two different shSIX1 vectors.
Supplementary Fig S3. (a) Immunofluorescence detection of -H2AX and H3K27M in
shSIX1-senescent and control (vector-infected) IMR90 fibroblasts. (b) Western Blot
and QPCR analysis of p21 in IMR90 fibroblasts infected with an shSIX1 vector.
Supplementary Fig S4. (a) Western Blot analysis of the indicated proteins in IMR90
fibroblasts infected with a vector expressing SIX1 followed by a second infection with
RasV12. (b) QPCR analysis of transcript levels of p16 in the same cells. (c)
Percentage of BrdU-positive cells in the same cells. (d) Western Blot analysis of the
indicated proteins in wild-type MEFs immortalized with shp53, and subsequently
infected with a vector expressing SIX1 or empty vector.
Supplementary Fig S5. Expression of a selection of genes differentially regulated in
shSIX1-senescent IMR90 fibroblasts in human tumors. Graphs show log2 mediancentered intensity for the indicated genes in normal tissue (left) and tumor (right) from
datasets of the Oncomine expression database. The datasets used and the tumor type
are indicated at the top of each column.
Supplementary Table 1: Results and GSEA analysis of RNA expression array in shSIX1-senescent IMR90 fibroblast.