p-ERK

β-tubulin

V S Shh + cyclopamine

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).

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.

Guldal et. al.

Vehicle

Shh

DAPI/BrdU/p38αp38α

Vehicle

Shh

DAPI/BrdU/p-p38αp-p38α

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.

Guldal et. al.

IRS1

-Shh:

-

48h

-

48h

48hSANT-2:

48h

24h

β-tubulin

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.

ASK1

p38α

V S V S V S

β-tubulin

p-p38α

cyclin D2

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.

Guldal et. al.

Subtype p-p38 low p-p38 high Total Comments

WNT 1 4 5Not enough samples to provide statistical power

SHH 3 28 31 High, χ2 = 9.343, p<0.05

C 9 18 27 No over-representation, χ2 = 0.077

D 25 21 46 Low, χ2 = 7.753, p<0.05

All Subtypes

38 71 109 65% are p-p38-high

Supplementary Table ESM 4 Summary 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.

Supplementary Table ESM 3 Immunofluorescence 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.

tumor

non-tumor

V

Shh

Shh+

p38α shRNA

Shh+

p38α ShRNA

DAPI/GFP DAPI/GFP/Ki67

Shh+

GFP shRNA

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).

Guldal et. al.

cyclin D2

β-tubulin

p38

p-ATF-2

Shh

20uM SB-

Supplementary Figure ESM 6 CGNPs 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.

c. PARP

β-tubulin

-DMSO

+DMSO

+5

+10

+20

Shh:

SB (μM):

β-tubulin

c. PARP

DMSOSB

5 μMSB

20 μM-

Wild type CGNPs Pzp53med cells

Supplementary Figure ESM 7 Comparison 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.