Figure 6. Activated TGFβ/ActivinB signaling in group 3 MB‐PDXs.

• A
  Immunoblot analysis of phosphorylated Smad2 (P‐Smad2) in Group 3 MB cell lines and PDXs. The level of total Smad2 (Smad2) was assessed, and β‐actin was used as a loading control. Quantification of P‐Smad2 (P‐S2) to β‐actin is shown on right panel.
• B
  Expression of INHBB in Group 3 MB cell lines and PDXs relative to HDMB03 (set at 1) by RT–qPCR.
• C
  Immunoblot of phosphorylated Smad2 (P‐Smad2), total Smad2, and β‐actin upon ActivinB or TGFβ stimulation for 1 h. Quantification of P‐Smad2 (P‐S2) to β‐actin is shown below.
• D
  Conditioned media experiments were performed on HDMB03 MB cell line. Phosphorylation of Smad2 (P‐Smad2) was analyzed by immunoblot upon treatment with either non‐conditioned media (NCM), media conditioned with HDMB03 cells (CM‐HDMB03), or media conditioned on PDX4 cells (CM‐PDX4). Pre‐incubation with blocking antibody against ActivinB (Ab α‐ActB) or with vehicle was performed before HDMB03 cell‐line treatment as indicated. Quantification of P‐Smad2 (P‐S2) to β‐actin is shown below.
• E
  Immunoblots of phosphorylated Smad2 (P‐Smad2), total Smad2, MYC, PMEPA1, OTX2, and β‐actin were performed on extracts from cell cultures of PDX4, PDX3, and PDX7 treated with either DMSO (vehicle), LY364947, SB431542, blocking antibody against ActivinB (Ab α‐ActB), follistatin, PBS, or ActivinB for 24 h. WB quantification is depicted in Appendix Fig S4C.

Data information: P‐Smad2 to total Smad normalization is provided on Appendix Fig S5. The P‐values were determined by unpaired t‐test. *P < 0.05. Detailed statistics are presented in Appendix Table S2 for panel (B). Bars represent the mean ± SD. Number of replicates is n ≥ 3. The exact P‐values and number of replicates are indicated in Appendix Table S5. Source data are available online for this figure.