|
|
Blood, Vol. 110, Issue 12, 4111-4119, December 1, 2007
Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling
Blood Wang et al.
110: 4111
Supplemental materials for: Wang et al
Files in this Data Supplement:
- Figure S1. Key for Phospho-RTK array (JPG, 159 KB)
-
- Figure S2. Characteristic morphology of undifferentiated and differentiated hESC colonies (JPG, 179 KB)
-
- Figure S3. Phosphorylation status of RTKs in hESCs following stimulation by insulin (JPG, 48.4 KB)
-
H1 cells starved overnight with DMEM/F12 with 0.5% BSA were stimulated with 10 µg/ml insulin for 15 minutes. Cell lysates were harvested and applied to phospho-RTK arrays. RTK profiles show that insulin induces not only phosphorylation of insulin receptor, but also strong phosphorylation of IGF1R. Faint phosphorylation of ERBB2 and FGFR2 was also observed following insulin stimulation.
- Figure S4. Full lane gel pictures for Figure 1C (JPG, 115 KB)
-
- Figure S5. IGF1R/ERBB2 coexpression with the pluripotency markers (SSEA-3, SSEA-4 and OCT4) in hESCs (JPG, 78.1 KB)
-
Double staining H1 cells with IGF1R or ERBB2 and the pluripotency markers SSEA-3, SSEA-4, or OCT4 demonstrates that all SSEA-3, SSEA-4, OCT-4 positive cells express IGF1R/ERBB2. In a small percentage of spontaneously differentiating cells (<2% SSEA-4 or OCT4 negative cells), the intensity of IGF1R and ERBB2 expression remains constant.
- Figure S6. AG825 inhibits ERBB2 Y1248 phosphorylation in hESCs (JPG, 50.9 KB)
-
BG01 hESCs grown in DC-HAIF were starved of growth factors overnight, or starved in the presence of 50 µM AG825. The cultures were washed and then treated as indicated with a 15-minute pulse of fresh medium (no growth factors), medium containing10 ng/ml HRG-1 , or medium containing 10 ng/ml HRG-1/50 µM AG825. The parental BG01 DC-HAIF culture was used as a steady-state positive control. Cell lysates were prepared as per RTK blotting, separated by 4-20% gradient SDS-PAGE, blotted and detected with an immunoaffinity purified anti-ERBB2 (phosphor-Y1248) antibody (Upstate/Millipore Cat#06-229). Y1248 is a major site of ERBB2 auto-phosphorylation, and treatment with AG825 inhibited the increase of phospho-Y1248 observed when a starved culture was pulsed with HRG-1. The level of phospho-1248 was normalized with pan-phosphotyrosine control spots on RTK blots of the same samples. The stead-state sample was not normalized.
- Figure S7. Phosphorylation status of RTKs in hESCs following stimulation by IGF1 and IGF2 (JPG, 75.7 KB)
-
H1 cells starved overnight with DMEM/F12 with 0.5% BSA were stimulated with either 100 ng/ml IGF1 or 100 ng/ml IGF2 for 15 minutes. Cell lysates were harvested and applied to phospho-RTK arrays. RTK profiles show that the RTKs activated by IGF1 or IGF2 are very similar with predominant phophorylation of IR and IG1R, moderate phosphorylation of ERBB2, VEGFR2, FGFR2, and faint phosphorylation of EGFR, ERBB3, FGFR3, AXL, MER, TIE-2, EPHA7, EPHA2, EPHA1, c-RET, VEGFR1.
- Figure S8. Expression of the pluripotency markers (OCT4, SSEA-4, TRA-1-60 and TRA-1-81) in hESCs after long-term passage in DC-HAIF medium (JPG, 230 KB)
-
BG02 cells maintained for 34 passages in DC-HAIF medium (76 total passages after derivation) exhibited expression of OCT4, SSEA-4, TRA-1-60, and TRA-1-81. Antigen expression was detected using unlabeled primary monoclonal antibodies and secondary isotype-specific antibodies conjugated with Alexa-488. No staining was observed using unlabeled isotype controls with the appropriate secondary antibody, or with secondary antibody alone.
|
|
