Supplementary Figure legends
Supplementary Figure 1: p63-regulated microRNAs target the TGFβ pathway
(a) Significantly regulated miRs after p63 knock-down in JHU-029 and their potential targets
in the TGFβ pathway. (b) Evaluation of TAp63α regulation of miR-527 and miR-665, by realtime quantitative RT-PCR (QRT-PCR) at 1 and 2 hours post tretracyclin treatment using a
tetracyclin inducible JHU-029 human SCC cells. (c) Validation of p73 activation and its
direct target PUMA, by real-time quantitative RT-PCR (QRT-PCR) at 1 and 2 hours post
tretracyclin treatment using a tetracyclin inducible TAp73β JHU-029 human SCC cells. (d). A
reporter construct containing the SMAD4 biding motif of PAI-1 promoter fused to luciferase
has been co-transfection with a shp63 construct. Results show relative luciferase units (RLU)
normalized to the control reporter lacking the SMAD4 biding motif. All error bars show
s.e.m. for triplicate measurements from representative experiments. * p< 0.05, ** p< 0.01,
*** p< 0.001. Two-way ANOVA followed by Tukey’s multiple comparison tests were used.
Supplementary Figure 2: miR-527 and miR-665 repress Smad4 and TβRII, respectively
(a) Schematic of the Smad 4 and TGFβR2 3’UTR reporter constructs showing predicted miR527 and miR-665 seed-binding sequences respectively. Wild type seed-binding sequences
(WT seed BS) and nucleotide changes introduced in the mutant reporter (mutant seed BS) are
shown. (b) Predicted alignment of the seed-binding sequences of miR-527 and miR-665
within the Smad 4 and TGFβR2 3’UTRs respectively and accordingly to TargetScan
software(c, d) JHU-029 cells were cotransfected with the indicated miR mimics/antimiRs or
ct (control) miR/antimiR, together with the UTR-reporter, or control reporter. Results are
shown as relative luciferase units (RLU) normalized to the control miR/antimiR.(e) qRT-PCR
for Smad 4 and TGFβR2 RNA levels in JHU-029 cell line transfected with the indicated
antagomiRs (antimiRs) compared with the control antimiR. (f) Immunoblotting for SMAD4,
TGFRII, P-SMAD 3 and SMAD 3 from MNNG-HOS osteosarcoma cells transfected with
pre/anti miR ct (controls) or pre/anti miRs candidates. Cells were treated or not with 5ng/mL
TGFβ during 15 minutes. * p< 0.05, ** p< 0.01, *** p< 0.001. Two-way ANOVA followed
by Tukey’s multiple comparison tests were used.
Supplementary Figure 3: p63 and p73 regulate SMAD4 and TβRII
(a) Evaluation of mRNA levels of key proteins of the TGFβ pathway in JHU-029 cell line
after 48h transfection with shRNA control (ct) or shRNA p63 or (b) after overexpression of
p63 in MNNG-HOS osteosarcoma cell line . (c) Immunoblotting for Smad 3 and P-Smad 3 in
MNNG-HOS osteosarcoma cell line after 48h of transfection with ΔNp63α or pcDNA3. Cells
were treated or not with 5ng/mL TGFβ for 15 minutes to show activation of the TGFβ
pathway. SMAD 4 and TβRII protein levels were analysed in MNNG-HOS cells after 48h of
transient overexpression of ΔNp63α. * p< 0.05, ** p< 0.01, *** p< 0.001. Two-way
ANOVA followed by Tukey’s multiple comparison tests were used.
Supplemental Figure 4: The p63 and p73-controlled miR network regulates TGFβ
induced migration
(a) Viability of JHU-029 cell line transfected with miR control (ct), miR-527 or miR-665 after
72h transfection. Colorimetric WST-1 assay was used to measure cell viability. (b) Western
Blot performed in MNNG-HOS osteosarcoma cells (c) JHU-029 cells were transfected with
indicated antimiRs or with control plus TAp73β or PCDNA3 (control). (d) JHU-029 cells
were transfected with indicated PremiR or with control plus TβR2 or PCDNA3 (control). 24h
later cells were treated or not with TGFβ (5ng/mL) and were re-treated before the migration
test. Migrated cells were counted after 24h of migration in boyden chambers. Migrated cells
were counted after 24h of migration in boyden chambers. * p< 0.05, ** p< 0.01, *** p<
0.001. Two-way ANOVA followed by Tukey’s multiple comparison tests were used. All
error bars show s.e.m. for triplicate measurements from representative experiments.
Supplemental Figure 5: p63 induces TGFβ-mediated metastasis in vivo
(a) qPCR for miR-527 and 665 in HOS cell line stably transfected with plasmid control (ct) or
ΔNp63α. (b) HOS cell line stably transfected with plasmid control (ct) or ΔNp63α and
treated or not with 5ng/mL TGFβ after 5h starvation. Secretion of matrix metalloproteinase
(MMP)-2 and MMP-9 was measured by gelatin zymography. (c) Immunohistochemistry
staining for SMAD4 and P-SMAD3 in primary tumors that gave rise to metastasis or not from
the p63 overexpressing mice group (d) qPCR of miR-527 and miR-665 and their targets
(Smad4 and TGFβR2 respectively) in primary tumors (PT) and their matching metastasis
(MET) from 6 osteosarcoma patients. * p< 0.05, ** p< 0.01, *** p< 0.001. Two-way
ANOVA followed by Tukey’s multiple comparison tests were used.
Supplemental Figure 6: miR-527 and miR-665 regulate metastasis dissemination in vivo
(a) miR-527 , miR-665, TβRII and Smad 4 expression levels were analysed by qRT-PCR.
Protein levels of SMAD4 and P-SMAD3 were performed by immunohistochemistry (b)
TβRII and Smad 4 expression levels were analysed by qRT-PCR. Protein levels of SMAD4
and P-SMAD3 were performed by immunohistochemistry. All error bars show s.e.m. for
triplicate measurements from representative experiments. (c) Inverse correlation between p63
and miR-527 in human primary breast tumor specimens, assessed by RT-qPCR. N number
of samples. (d) ) Inverse correlation between miR-527 and Smad4 in human primary breast
tumor specimens analysed from GEO. For the same specimens mRNA and microRNA
microarray analysis have been performed. GSE22216 – miRNA expression database and
GSE22220 – mRNA expression database. N, number of samples. . * p< 0.05, ** p< 0.01, ***
p< 0.001. Two-way ANOVA followed by Tukey’s multiple comparison tests were used.