Supplementary Figure 1.
(A) Immunoblotting detection of tyrosine 701 phosphorylated STAT1 in B16 and TC1
cells treated with combination of IFN and TNF for time period as indicated. GAPDH
was used as a loading control. (B) FACS analysis of MHC I expression in B16 and TC-1
cells treated with combination of IFN and TNF for 1-6 days. Non-treated cells were
used as a control. (C) Immunofluorescence detection of p65 subunit of NF-B in B16 and
TC-1 cells treated with combination of IFN and TNF for 30 minutes. Bar 15 m.
Supplementary Figure 2.
(A) Immunoblotting detection of tyrosine 701 phosphorylated STAT1 in HeLa cells
treated with combination of IFN and TNF for time period as indicated. GAPDH was
used as a loading control. (B) Immunofluorescence detection of BrdU incorporation in
HeLa cells treated with combination of IFN and TNF for 14 days. Bar 50 m. (C)
Immunofluorescence detection of p65 subunit of NF-B and (D) PML in HeLa cells
treated with combination of IFN and TNF for 24 h. Bar 10 m. (E)
Immunofluorescence detection of PML in HeLa cells treated with IFN or TNF for 6
days. Bar 10 m. (F) Growth curve of HeLa cells treated with IFN after time period as
indicated. Data represent mean values +/- S.D. from two independent experiments. (G)
Cell cycle profile of HeLa cells treated 48 h with IFN. (H) Senescence-associated galactosidase detection in HeLa cells treated with TNF for 6 days; Bar 50 m.
Supplementary Figure 3.
(A) Senescence-associated -galactosidase activity in BJ cells treated with IFN (500
UI/ml) for 14 days; Bar 50 m. (B) Immunofluorescence detection of 53BP1 and H2AX
in BJ cells treated with IFN (500 UI/ml) for 14 days. Bar 10 m. (C) Immunoblotting
detection of p16INK4a, p21Cip1, p27kip1, total p53 and Chk2, serine 15 phosphorylated p53
and threonine 68 phosphorylated Chk2 in BJ cells treated with IFN (500 UI/ml) for 14
days. GAPDH was used as a loading control. (D) Statistical analysis of BrdU
incorporation in BJ cells treated for 14 days with IFN (500 UI/ml). Data represent mean
values +/- S.D. from two independent experiments. (E) Senescence-associated Page 1
galactosidase activity in MCF7 cells treated with IFN for 14 days; Bar 50 m. (F)
Immunofluorescence detection of 53BP1 and H2AX in MCF7 cells treated with IFN
for 14 days. Bar 10 m. (G) Immunoblotting detection of Nox4 in HeLa cells 24 h after
downregulation of Nox4 by specific siRNA. -tubulin was used as a loading control. (H)
Nox1 and Nox4 mRNA levels detected by PCR in HeLa cells treated for 48 h with IFN
after downregulation of Nox1 or Nox4 by specific siRNA. -actin was used as the
reference gene.
Supplementary Figure 4.
(A) Immunoblotting detection of total STAT1, STAT2, STAT3 and STAT5 in HeLa cells
treated for 48 h with IFN after downregulation of STAT1, STAT2, STAT3 or STAT5 by
specific siRNA, respectively. To assess the effect of transfection, control cells and cells
treated with IFN were transfected with non-targeting siRNA. GAPDH was used as a
loading control. (B) PML mRNA levels quantified by real time qRT-PCR in HeLa cells
treated with IFN for 2 h. -actin was used as the reference gene. (C) IL6-dependent
proliferation assay of B9 mouse hybridoma cells. Estimation of IL6 activity in medium
conditioned by control or IFNtreated HeLa cells three days without or with IL6
depletion using IL6 antibody (IL6 AB; 2 μg/ml). B9 cells treated with human rIL6 (100
pg/ml) were used as a positive control (PC), B9 without rIL6 were used as a negative
control (NC). (D) Immunoblotting detection of SMAD2 phosphorylated on serine
465/467 in HeLa cells treated for 48 h with IFN with presence or absence TGF receptor
inhibitor (TBI, 10 M). GAPDH was used as a loading control. (E) Immunoblotting
detection of SMAD2 phosphorylated on serine 465/467 and STAT1 phosphorylated on
tyrosine 701 in HeLa cells treated for 14 days with IFN alone or in combination with
TNF with presence or absence TGF receptor inhibitor (TBI, 10 M). GAPDH was
used as a loading control. (F) ANT2 mRNA levels quantified by real time qRT-PCR in
HeLa cells treated for 48 h with IFN after downregulation of ANT2 by specific siRNA.
As a control, untreated and IFN-treated cells were transfected with non-targeting siRNA
(siNC). -actin was used as the reference gene. Data in B, C and F represent mean values
+/- S.D. from two independent experiments.
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