Supplementary Figure S1
Overview of the gene expression analysis of cells treated with IR, PKC 412, or Ro 31-8220
alone, with combinations of IR and PKC 412, or IR and Ro 31-8220. The up- and downregulated genes in each treatment after Venn diagram analyses are shown.
Supplementary Figure S1B
Changes in gene expression in U-1810 cells detected by Affymetrix gene array analysis were
verified using real-time quantitative PCR for a subset of genes, i.e., PPP2R2C, ESR1 and
Rab33A in response to PKC 412, Ro 31-8220 alone, or in combination with IR (8 Gy).
Values are given relative to untreated U-1810 cells. Grey bars: Affymetrix gene expression
array; Black bars: real time quantitative PCR.
Supplementary Figure S2
To confirm the specificity of the Ephrin B3 siRNA, U-1810 cells were treated with
Dharmafect only or transfected with non-target siRNA or Ephrin B3-targeted siRNA with
another target sequence (sequence 2, see Materials & Methods section) for 48 h prior to IR (8
Gy). Silencing of Ephrin B3 was confirmed at the mRNA level (A) using RT-Q-PCR. The
effect of siRNA ablation of Ephrin B3 alone or in combination with IR (8 Gy) was analyzed
48h post-IR and a clear effect on cell morphology similar to Fig. 3C was observed.
Supplementary Figure S3
U-1752 cells were transfected with non-target siRNA or Ephrin B3 siRNA (sequence 1) for
48h prior to IR (8 Gy). Silencing of Ephrin B3 was confirmed at the mRNA level (A) using
RT-Q-PCR as described in Fig.3A. (B) The effects on cell morphology were examined at 48h
post-IR using light microscopy (magnification × 400). Non-target siRNA was used as a
1
negative control. (C) The effect of siRNA against Ephrin B3 on PARP cleavage was
examined using Western blot. Tubulin was used as a loading control.
Supplementary Figure S4
The effect of siRNA against Ephrin B3 on cell proliferation was analyzed using CFSE. Left:
histogram showing the inhibition of proliferation in Ephrin B3 siRNA cells either only
transfected or transfected and incubated for 48 h post-IR. Upper panel: non-target or Ephrin
B3 siRNA alone; lower panel: non-target or Ephrin B3 siRNA in combination with IR at 48h.
Filled grey: non-target siRNA, black line: Ephrin B3 siRNA.
Supplementary Figure S5
Cell cycle distributions after non-target siRNA or siRNA against Ephrin B3 either alone or
combination with IR (8 Gy) were analyzed in ethanol-fixed U-1810 cells using PI staining
(flow cytometry) at 24h post-IR. Data presented are the means of three experiments. Bars
represent SD.
2