Fig 2: Suppression of phosphorylated (p-)SMAD family member 2 (Smad2) signaling by LY364947 reverses the inductive effect of jumonji AT-rich interactive domain 1B (JARID1B) on the expression of glioma cancer stem cell-related markers. The expression of transforming growth factor-ß1 (TGF-ß1), p-Smad2, Smad2, CD133, octamer-binding transcription factor 4 (Oct4), nestin and BMI1 proto-oncogene, polycomb ring finger (Bmi-1) protein levels in LY364947-exposed U251-pBabe-JARID1B and its control cells were measured by western blot analysis. ß-actin was used as the internal control for western blot analysis.

Fig 3: TGIF1 inactivates the TGFß signaling pathway to enhance ESCC cell malignant phenotypes. A Expression of the TGFß signaling pathway-related proteins (Smad2, Smad4 and TGF-ßRII) in ESCC and adjacent tissues normalized to GAPDH. n = 79. * p < 0.05 vs. adjacent tissues. B Expression of the TGFß signaling pathway-related proteins (Smad2, Smad4 and TGF-ßRII) in EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381 normalized to GAPDH. C Proliferation of EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381. D Apoptosis of EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381. E Migration of EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381. F Invasion of EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381. G Protein levels of cleaved caspase-3, total caspase-3, MMP-2 and MMP-9 normalized to GAPDH in EC9706 and Eca109 cells treated with oe-TGIF1 or in combination with SRI-011381. In Panel B-G, *p < 0.05 vs. EC9706 and Eca109 cells treated with oe-TGIF1 + DMSO. #p < 0.05 vs. EC9706 and Eca109 cells treated with oe-TGIF1 + SRI-011381. Measurement data were expressed as mean ± standard deviation derived from at least 3 independent experiments. Data between ESCC and adjacent tissues were compared by paired t test while data comparison between other two groups was analyzed by unpaired t test

Fig 4: TGF-ß Signaling Enhances the Expression Level of Ankyrin-G(A) Confocal image of a neuron immunostained for ankyrin-G and TGF-ßRI or TGF-ßRII in the axon initial segment (AIS) and dendrite. Scale bars, 20 µm (left); 5 µm (right).(B) Comparing intensities in the AIS (white bars) and dendrite (black bars) (n = 6 for each group). *p < 0.05; ***p < 0.001 by two-tailed unpaired t test. All data are presented as mean ± SEM.(C) Representative images of western blot. Time-dependent stimulation of expression of ankyrin-G after treatment with TGF-ß (20 ng/mL) in cultured neurons. Phosphorylation of Smad2 (p-Smad2) was used for confirmation of TGF-ß signaling activity. ß-Actin was the internal loading control.(D) Confocal images of neurons transfected with mCherry, and subsequently treated with vehicle (top) or TGF-ß (20 ng/mL) (bottom) for 1 h. Scale bar, 2 µm.(E and F) Spine head area (E) and density (F) with vehicle (plain pattern) or TGF-ß (comb pattern) (n = 9 neurons for each group). *p < 0.05; ***p < 0.001 by two-tailed unpaired t test. All data are presented as mean ± SEM.

Fig 5: TGF-ß-Mediated Phosphorylation of Usp9X Enhances the Interaction with Ankyrin-G(A) Usp9X homology model (gray) was generated using crystal structure (PDB: 5WCH) as template. S1,593, S1,600, and S1,609 (orange) are positioned on a loop absent from crystal structure 5WCH. Energy minimization of this loop allowed an energetically favorable conformation to be predicted (blue) and suggests that all serine sites lie distal to the catalytic triad (magenta).(B) Representative western blot of His-Usp9X1,547–1,962, expressed and purified from E. coli, was used to pull down HEK293T-expressed hemagglutinin (HA)-AnkG1–808 by nickel affinity purification. A GFP-transfected sample was used as a negative control.(C) Representative western blot of HA-AnkG1–807 with co-expressing FLAG-Usp9X1,555–1,958 (FLAG-Usp9XWt) or FLAG-Usp9X1,555–1,958 (S1,593;1,600;1,609A) (FLAG-Usp9XS3A) construct from HEK293T cells. Immunoprecipitation (IP) of phosphoserine (pSer) and detection of phosphorylated Smad2 (p-Smad2) were performed to confirm TGF-ß-mediated signaling.(D) In situ PLA measurement of the interaction between HA-AnkG1–807 and FLAG-Usp9XWt or FLAG-Usp9XS3A in HEK293T cells. Scale bar, 5 µm.(E) Quantification of PLA signal from (D) (n = 9–16 cells). HA-AnkG1–807 and FLAG-Usp9XWt (black bars) or FLAG-Usp9XS3A (red bars) with vehicle (plain pattern) or TGF-ß (comb pattern). ***p < 0.001; †††p < 0.001 by two-way ANOVA followed by Bonferroni post-tests. All data are presented as mean ± SEM. TGF-ß (-), vehicle; TGF-ß (+), 20 ng/mL TGF-ß for 1 h.See also Figure S1.