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Guldal et. al. V. S. Shh + cyclopamine. p - ERK.

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slide1

Guldal et. al.

V

S

Shh + cyclopamine

p-ERK

Supplementary Figure ESM 1a ERK MAP Kinase activity is not affected by Shh pathway activity. Western blot analysis of PN 5 CGNPs cultured for 48 hours with (S) or without (V) Shh and/or cyclopamine (1 mg/mL) for the last 24 hours. Protein lysates were probed for phospho-ERK (p-ERK) and -tubulin was used as a loading control. Three independent cultures with Shh and cyclopamine were used (last three lanes).

β-tubulin

Supplementary Figure ESM 1b p38α/MAPK14 is not transcriptionally affected by Shh in CGNPs. CGNPs from PN 5 mice were cultured with (S) or without (V, vehicle) Shh for 3, 6, 12, 24, and 48 hours and total RNA was probed for MAPK14 mRNA levels by quantitative PCR analysis. Fold expression was compared to the internal control mRNA, HPRT1. None of the time points yielded a statistically significant difference between CGNPs treated with or without Shh in two-tailed t-tests.

slide2

Guldal et. al.

p38α

DAPI/BrdU/p38α

Vehicle

Shh

p-p38α

DAPI/BrdU/p-p38α

Vehicle

Shh

Supplementary Figure ESM 1c Proliferating CGNPs have higher levels of p38 activity. Higher magnification images of the immunofluorescence staining performed in Figure 1c. CGNPs positive for BrdU also exhibit high levels of p38α and phospho-p38α, even in CGNP cultures without Shh.

slide3

Supplementary Figure ESM 1d Western blot of CGNP lysates as described in Figure 1e. IRS1 protein levels confirm the activity level of the Shh pathway, which reduces with SANT-2 treatment in the presence of Shh, as expected.

V

S

V

S

V

S

ASK1

p38α

p-p38α

cyclin D2

β-tubulin

Supplementary Figure ESM 2 p38 pathway activation in proliferating CGNPs in the C57BL/6 mouse strain background is the same as in the Swiss-Webster strain. Western blot analysis of CGNPs from PN 5 C57BL/6 mice cultured for 48 hours with (S) or without (V) Shh. Protein lysates were probed for ASK1, total and phospho-p38α, and cyclin D2, and -tubulin was used as a loading control.

-

48h

48h

48h

Shh:

-

-

48h

24h

SANT-2:

IRS1

β-tubulin

slide4

Guldal et. al.

non-tumor

tumor

Supplementary Table ESM 3Immunofluorescence staining of SmoA1 tumor and adjacent non-tumor tissue with Ki67 to delineate the tumor region for Figure 3b. Ki67 (red) marks the tumor, also indicated by the yellow dashed line. DAPI was used to stain nuclei. The non-tumor region in Figure 3b is marked by the white square and the tumor region by the yellow rectangle.

Supplementary Table ESM 4Summary of classification of 109 human pediatric medulloblastoma samples on tissue microarray (TMA) by subtype and p-p38 immunohistochemistry staining. Staining, classification, and statistical analysis was done as described in Materials and Methods section.

slide5

Guldal et. al.

DAPI/GFP

DAPI/GFP/Ki67

V

Shh

Shh

+

GFP shRNA

Shh

+

p38αshRNA

Supplementary Figure ESM 5 Knocking down p38 reduces CGNP proliferation in the presence of Shh. Immunofluorescence of CGNPs cultured without Shh (V), with Shh (Shh) and either with lentiviruses expressing the control shRNA targeting GFP (GFP shRNA) or targeting p38 (p38-1 shRNA). Cells were immunostained for Ki67 (red) and GFP (green). DAPI staining was used to label all nuclei. Since CGNPs do not express any GFP and the GFP shRNA construct does not express GFP, only the cultures infected with p38 shRNA are positive for GFP staining, as this construct expresses eGFP. Magnified view of the p38 shRNA-infected cultures shows mutually exclusive GFP and Ki67 staining (bottom panels).

Shh

+

p38αShRNA

slide6

Shh

-

20uM SB

p38

p-ATF-2

cyclin D2

β-tubulin

-

+

+

+

+

Shh:

Supplementary Figure ESM 6CGNPs were cultured with Shh for 48 hours with or without 20 M of SB203580 (SB) for the last 24 hours of culture. Protein lysates were probed for p38α and phospho-ATF-2, a known target of p38 activity, in western blot analysis. -tubulin was used as a loading control. Cyclin D2 indicates CGNP proliferation levels.

SB (μM):

5

10

20

DMSO

DMSO

c. PARP

β-tubulin

SB

5 μM

SB

20 μM

-

DMSO

Wild type CGNPs

Pzp53med cells

c. PARP

β-tubulin

Supplementary Figure ESM 7Comparison of the apoptotic effect of SB203580 (SB) on wild type CGNPs (left panel) and Pzp53med cells (right panel) indicates that the latter were more sensitive to p38 inhibition at lower doses of SB203580. CGNPs were cultured with Shh for 48 hours with or without indicated concentrations of SB for the last 24 hours of culture. Pzp53med cells were cultured as described in Materials and Methods section with SB for 24 hours. Cultures without SB were incubated with DMSO as control. Protein lysates were probed for cleaved PARP in western blot analysis. -tubulin was used as a loading control.

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