Journal: International Journal of Molecular Sciences

Article Title: MT1-MMP Cooperates with TGF-β Receptor-Mediated Signaling to Trigger SNAIL and Induce Epithelial-to-Mesenchymal-like Transition in U87 Glioblastoma Cells

doi: 10.3390/ijms222313006

Figure Lengend Snippet: SNAIL induction by concanavalin A involves a TGF-β receptor-mediated signaling axis component. ( A ) Serum starved U87 glioblastoma cells were treated with increasing concentrations of TGF-β in the presence or not of 30 μM EGCG, or 10 μM galunisertib for 24 h. SNAIL, fibronectin and GAPDH protein expression were then assessed by immunoblotting using the respective cell lysates. ( B ) Serum-starved U87 glioblastoma cells were treated with 30 μg/mL of concanavalin A (ConA) for 24 h in the presence or not of 30 μM EGCG or 10 μM galunisertib (Galu) for 24 h. SNAIL and GAPDH protein expression were then assessed by immunoblotting using the respective cell lysates. ( C ) Total RNA was extracted from treated cells, and RT-qPCR was performed to assess SNAIL gene expression as described in the Materials and Methods section. Data are means ± SD from three independent experiments. Probability values of less than 0.05 were considered significant and an asterisk identifies such significance.

Article Snippet: Galunisertib (LY2157299) was from MedChemExpress (Monmouth Junction, NJ, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Gene Expression

Journal: Oncogene

Article Title: Overexpression of PIK3CA in murine head and neck epithelium drives tumor invasion and metastasis through PDK1 and enhanced TGFβ signaling

doi: 10.1038/onc.2016.1

Figure Lengend Snippet: (a) qRT-PCR examination of molecules in the TGFβ signaling pathway. *p<0.05 (n=6 for each group). (b) ELISA quantification of TGFβ1 ligand (n=5) (c) Double IF staining of epithelial marker E-cadherin (green) and Smad3 (red). Note the inverse correlation between Smad3 and E-cadherin in control and PIK3CA- GEMM tumors. (n=4), scale bar: 25 μm. (d) Western blot analysis of phosphorylated Smad3. (e) Western blot analysis of p110α in cells cultured from a 4NQO-induced control tumor (CUCON) and a 4NQO-induced PIK3CA -GEMM tumor (CU110), and CU110 cells with either a PIK3CA knock-down ( sh-PIK3CA ), or a scramble control ( SCR ). (f) qRT-PCR examination of TGFβ1 mRNA levels in CUCON and CU110 cells (left panel), and CU110 cells with PIK3CA knock-down ( sh-PIK3CA ) or scramble control ( SCR ) (right panel). *p<0.05. (g) Western blot analysis of phosphorylation of Smad2 and Smad3 in CU110 cells treated with a TGFβ type I receptor inhibitor, LY2157299, or DMSO (control). (h) Migration assay in CU110 cells treated with a TGFβ type I receptor inhibitor, LY2157299, or DMSO (control). Dotted line defines the edges of cells. All experiments were performed in triplicate. *p<0.05. (i) Invasion assay in CU110 cells treated with a TGFβ type I receptor inhibitor, LY2157299, or DMSO (control). All experiments were performed in triplicate. *p<0.05.

Article Snippet: Cells were seeded into 24-well plates as 1×10 5 cells/well, and were treated with 0–80μM LY2157299 (MedKoo Biosciences) for 48 hr.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Marker, Control, Western Blot, Cell Culture, Knockdown, Phospho-proteomics, Migration, Invasion Assay

Journal: EMBO Molecular Medicine

Article Title: An autocrine ActivinB mechanism drives TGF β/Activin signaling in Group 3 medulloblastoma

doi: 10.15252/emmm.201809830

Figure Lengend Snippet: A Growth curve experiments showing cell proliferation upon TGFβ/Activin signaling inhibition by Galunisertib (LY2157299) in 1603MED (top panel) and D283 (bottom panel) cell lines. B Immunoblots of phosphorylated Smad2 (P‐Smad2), total Smad2, MYC, PMEPA1, OTX2, and β‐actin were performed on extracts from 1603MED and D283 cell lines and PDX3, PDX4, and PDX7 cell cultures treated with Galunisertib for 24 h. C WB quantifications. The relative level to β‐actin is presented for the different proteins. P‐Smad2 to total Smad normalization is provided on Appendix Fig S5 . The level in control conditions was set at 1. D Ki67 and cleaved caspase‐3 staining by IHC of 3 representative tumors per group of mice (Vehicle, Cisplatin, Galunisertib, and Galunisertib + Cisplatin). Scale bars indicate 100 μm. E Boxplots represent quantification of Ki67 and cleaved caspase‐3 (Cc3) staining on tumor (IHC). Data information: Boxplot center lines show data median; box limits indicate the 25 th and 75 th percentiles; lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25 th and 75 th percentiles, respectively. Outliers are omitted. The P ‐values were determined by two‐way ANOVA in (A), unpaired t ‐test in (C) and Mann‐Whitney test (E). * P < 0.05, ** P < 0.01, and *** P < 0.001. 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.

Article Snippet: After 3 days, mice were administrated 300 μl of LY2157299 (Galunisertib, AbMole Bioscience) orally at a dose of 75 mg/kg in 12% DMSO, 30% PEG, and water.

Techniques: Inhibition, Western Blot, Control, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: An autocrine ActivinB mechanism drives TGF β/Activin signaling in Group 3 medulloblastoma

doi: 10.15252/emmm.201809830

Figure Lengend Snippet: A Kaplan–Meier representing survival of mice treated with either vehicle (black) or Galunisertib (LY2157299, red) or Cisplatin (blue) or a combination of Galunisertib and Cisplatin (purple) after orthotopic grafting of PDX4 cells into the cerebellum. The pink rectangle represents Galunisertib treatment duration, while the blue dotted lines represent the 3 Cisplatin administrations. B Boxplot of tumor area after 25 days of treatment. On the right, boxplots represent quantification of P‐Smad2 staining on tumor (IHC). The code color is similar to panel (A). C P‐Smad2 staining by IHC in 3 representative tumors per group after 25 days of treatment. The scale bars represent 500 and 100 μm on the left and right panels, respectively. D Boxplots representing the expression level of INHBB , PMEPA1 , MYC, and NRL in the different MB subtypes, as defined in Cavalli et al . Only P ‐values corresponding to comparisons between Group 3 subtypes are indicated. Detailed statistics are presented in Appendix Table S3 . Patient samples are colored by subtypes as indicated. E Scatter plot of INHBB and PMEPA1 , expression levels in Group 3 patient samples. Colored dots represent each patient sample, and colors represent the group 3 MB subtypes (α in yellow, β in brown, and δ in orange). Note that this panel is identical to that shown in Appendix Fig S3A (yellow) except for the color code. Data information: Center lines show data median; box limits indicate the 25 th and 75 th percentiles; lower and upper whiskers extend 1.5 times the interquartile range (IQR) from the 25 th and 75 th percentiles, respectively (B and D). Squares represents individual tumor (B). Outliers are represented by individual points (D). The P ‐values were determined by log‐rank (Mantel‐Cox) test on panel (A) and unpaired t ‐test on panel (B). Wilcoxon rank‐sum tests were performed for panel (D). Spearman's rank correlation coefficient ρ and P ‐value are indicated on panel E. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. 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 .

Article Snippet: After 3 days, mice were administrated 300 μl of LY2157299 (Galunisertib, AbMole Bioscience) orally at a dose of 75 mg/kg in 12% DMSO, 30% PEG, and water.

Techniques: Staining, Expressing