Journal: Cellular Oncology

Article Title: Upregulation of limb-bud and heart (LBH) drives liver cancer progression by interacting with the oncoprotein Mortalin

doi: 10.1007/s13402-026-01228-z

Figure Lengend Snippet: LBH is transcriptionally upregulated by TGF-β in liver cancer cells. (A) Heatmap displaying the common 474 DEGs whose expressions were altered upon treatment with 100 pM of TGF-β1 for 4 h in three different HCC cell lines, i.e. MHCC97H, MHCC97L and HCCLM3. (B) Heatmap classification displaying some typical upregulated genes in response to TGF-β1 administration in HCC cell lines. (C-H) After being treated with 100 pM of TGF-β1 (C-E) or 10 µM of SB431542 (F-H) for the indicated periods, HCCLM3, HLE and SMMC-7721 cells were harvested for RNA purification and gene expression analyses through qPCR. (I-J) HCCLM3, HLE cells transfected with a control siRNA (NS) or those targeting Smad2/3 were treated with 100 pM of TGF-β1 for 6 h. Then the expression levels of Smad2, Smad3 and LBH were analyzed by qPCR. (K) Gene track view of Smad2/3, H3K4me3, and H3K27ac at the LBH promoter from the Cistrome Data database (top), and schematic diagram of ChIP primers designed within and near the LBH promoter region (bottom). HCCLM3 cells were treated with 2.5 ng/ml TGF-β for 24 h. ChIP assays were then performed to examine the binding of Smad2/3 to the LBH promoter region, and the enriched DNA fragments were quantitatively analyzed by qPCR. qPCR experiments described above were analyzed by one-way ANOVA followed by Tukey’s or Dunnett’s post hoc test. Statistical significance: * p < 0.05, ** p < 0.01 and *** p < 0.001; ns, no significance. Data were shown as mean ± SD

Article Snippet: Recombinant human TGF-β1 protein was obtained from R&D Systems Inc., and the small molecule inhibitor SB431542 was purchased from MedChemExpress (MCE, USA).

Techniques: Purification, Gene Expression, Transfection, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: APOE4 affects basal and NMDAR mediated protein synthesis in neurons by perturbing calcium homeostasis

doi: 10.1101/2020.12.10.418772

Figure Lengend Snippet: A – Experimental workflow. Primary rat/mouse cortical neurons (DIV15) were treated with APOE from different sources - stem cell secreted APOE, astrocyte secreted APOE and recombinant APOE protein. The APOE treated neurons were probed for readouts of global protein synthesis. B - Rat primary cortical neurons (DIV15) were treated with APOE (10-15nM) from iPSC conditioned media for 20 minutes and probed for phosphorylation of eEF2. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=5, One-way ANOVA (p=0.0001) followed by Tukey’s multiple comparison test. C - Rat primary cortical neurons (DIV15) were treated with recombinant APOE protein (15nM) for 20 minutes and probed for phosphorylation of eEF2. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=4, One-way ANOVA (p=0.0002) followed by Tukey’s multiple comparison test. D – Rat primary cortical neurons (DIV15) were treated with recombinant APOE protein (15nM) for 20 minutes and subjected to fluorescent non-canonical amino acid tagging (FUNCAT) along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different APOE treatment conditions are shown (Scale bar - 10μM). E – The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different APOE treatment conditions. Data is represented as mean +/- SEM. N = 20-40 neurons from 4 independent experiments, One-way ANOVA (p=0.0001) followed by Tukey’s multiple comparison test. F - Mouse primary cortical neurons (DIV15) were treated with APOE from mouse primary astrocyte conditioned media for 20 minutes and probed for phosphorylation of eEF2. Top - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Bottom - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=7, One-way ANOVA (p<0.0001) followed by Tukey’s multiple comparison test. G – Synaptoneurosomes prepared from P30 rat cortices were treated with recombinant APOE protein (15nM) for 20 minutes and probed for phosphorylation of eEF2. Top - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Bottom - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=4, Unpaired Student’s t-test. H - Rat primary cortical neurons (DIV15) were treated with APOE receptor antagonist RAP (200nM) along with APOE (10-15nM) from iPSC conditioned media for 20 minutes and probed for phosphorylation of eEF2. Top - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Bottom - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=5, One-way ANOVA (p=0.003) followed by Tukey’s multiple comparison test.

Article Snippet: The iPSCs were subjected to monolayer Dual SMAD inhibition by changing the media to Neural Induction Media (NIM) which is composed of NBM supplemented with small molecules SB431542 (10μM, an inhibitor of TGFβ pathway) (72232, Stem Cell Technologies) and LDN193189 (0.1μM, an inhibitor of BMP pathway) (72142, Stem Cell Technologies).

Techniques: Recombinant, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: APOE4 affects basal and NMDAR mediated protein synthesis in neurons by perturbing calcium homeostasis

doi: 10.1101/2020.12.10.418772

Figure Lengend Snippet: A – Experimental workflow. Rat primary cortical neurons (DIV15) were treated with APOE from iPSC conditioned media/recombinant protein for 20 minutes. During the last 5 minutes of the APOE treatment, the neurons were subjected to stimulation of NMDA receptors (20μM NMDA, 5 minutes). The NMDAR-mediated translation response in APOE treated neurons was probed by using the following readouts - phosphorylation eEF2, FUNCAT and increased levels of PTEN and PSD95 proteins. B - Rat primary cortical neurons (DIV15) were treated with APOE3/APOE4 (10-15nM) conditioned media for 20 minutes along with NMDAR stimulation for 5 minutes (20μM NMDA). The cell lysates were probed for phosphorylation of eEF2. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=4-5, One-way ANOVA (p=0.0047) followed by Dunnett’s multiple comparison test. C - Rat primary cortical neurons (DIV15) were treated with recombinant APOE3 or APOE4 protein (15nM) for 20 minutes along with NMDAR stimulation for 5 minutes (20μM NMDA). They were subjected to fluorescent non-canonical amino acid tagging (FUNCAT) along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different treatment conditions are shown (Scale bar - 10μM). D – The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different treatment conditions. Data is represented as mean +/- SEM. N = 20-40 neurons from 2 independent experiments, One-way ANOVA (p<0.0001) followed by Tukey’s multiple comparison test. E - Rat primary cortical neurons (DIV15) were treated with APOE3/APOE4 (10-15nM) conditioned media for 20 minutes along with NMDAR stimulation for 5 minutes (20μM NMDA). The cell lysates were probed for PTEN protein. Left - representative immunoblots indicating levels of PTEN and Tuj1; Right - graph indicating PTEN levels normalized to Tuj1. Data is represented as mean +/- SEM. N=4, One-way ANOVA (p=0.002) followed by Dunnett’s multiple comparison test. F - Rat primary cortical neurons (DIV15) were treated with APOE3/APOE4 (10-15nM) conditioned media for 20 minutes along with NMDAR stimulation for 5 minutes (20μM NMDA). The cell lysates were probed for PSD95 protein. Left - representative immunoblots indicating levels of PSD95 and Tuj1; Right - graph indicating PSD95 levels normalized to Tuj1. Data is represented as mean +/- SEM. N=4, One-way ANOVA (p=0.0216) followed by Dunnett’s multiple comparison test. G - Synaptoneurosomes prepared from P30 rat cortices were treated with recombinant APOE3 protein (15nM) for 20 minutes along with NMDAR stimulation for 5 minutes (40μM NMDA). The lysates were probed for phosphorylation of eEF2. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=3, Unpaired Student’s t-test. H - Synaptoneurosomes prepared from P30 rat cortices were treated with recombinant APOE4 protein (15nM) for 20 minutes along with NMDAR stimulation for 5 minutes (40μM NMDA). The lysates were probed for phosphorylation of eEF2. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=5, Unpaired Student’s t-test.

Article Snippet: The iPSCs were subjected to monolayer Dual SMAD inhibition by changing the media to Neural Induction Media (NIM) which is composed of NBM supplemented with small molecules SB431542 (10μM, an inhibitor of TGFβ pathway) (72232, Stem Cell Technologies) and LDN193189 (0.1μM, an inhibitor of BMP pathway) (72142, Stem Cell Technologies).

Techniques: Recombinant, Western Blot, Immunostaining

Journal: bioRxiv

Article Title: APOE4 affects basal and NMDAR mediated protein synthesis in neurons by perturbing calcium homeostasis

doi: 10.1101/2020.12.10.418772

Figure Lengend Snippet: A - Rat primary cortical neurons (DIV15) were treated with NMDA (20μM) for 1 minute, 5 minutes, 20 minutes and probed for phosphorylation of eEF2. Top - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Bottom - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. N=3, One-way ANOVA (p=0.0015) followed by Tukey’s multiple comparison test. B - Rat primary cortical neurons (DIV15) were subjected to NMDAR stimulation for 1 minute and 20 minutes (20μM NMDA). The cell lysates were subjected to polysome profiling and probed for the distribution of ribosomal protein RPLP0. Top – line graph showing the percentage distribution of RPLP0 under different conditions. Bottom - bar graph representing the ratio of RPLP0 protein distribution in fractions 7-11 (translating pool) to fractions 1-6 (non-translating pool). Data is represented as mean +/- SEM. N=3. One-way ANOVA (p=0.0248) followed by Tukey’s multiple comparison test. C - Rat primary cortical neurons (DIV15) were treated with APOE3/APOE4 (10-15nM) conditioned media for 1 minute and 20 minutes and probed for phosphorylation of eEF2. Top - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Bottom - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. Dotted line indicates the untreated condition. All the APOE treatments were normalized to untreated condition. N=4. One-way ANOVA (p<0.0001) followed by Dunnett’s multiple comparison test. D – Rat primary cortical neurons (DIV15) were treated with recombinant APOE protein (15nM) for 1 minute and subjected to fluorescent non-canonical amino acid tagging (FUNCAT) along with immunostaining for MAP2. The representative images for MAP2 and FUNCAT fluorescent signal under different APOE treatment conditions are shown (Scale bar - 10μM). E – The graph represents the quantification of the FUNCAT fluorescent intensity normalized to MAP2 fluorescent intensity under different APOE treatment conditions. Data is represented as mean +/- SEM. N = 20-40 neurons from 4 independent experiments, One-way ANOVA (p=0.0058) followed by Tukey’s multiple comparison test. F - Rat primary cortical neurons (DIV15) were treated with APOE3/APOE4 (10-15nM) conditioned media for 1 minute and subjected to recovery for 5 minutes/ 10 minutes / 20 minutes using pre-conditioned neurobasal media. The samples were probed for the phosphorylation of eEF2. The data from each recovery time point is normalized to its corresponding 1-minute APOE treated set. Left - representative immunoblots indicating levels of phospho-eEF2, eEF2 and Tuj1; Right - graph indicating ratio of phospho-eEF2 to eEF2 normalized to Tuj1. Data is represented as mean +/- SEM. For APOE3 1’ and APOE4 1’, N=8, Unpaired Student’s t-test. For APOE 1’ + 5’R, N=4, Unpaired Student’s t-test. For APOE 1’ + 10’R N=5. For APOE 1’ + 20’R, N=3.

Article Snippet: The iPSCs were subjected to monolayer Dual SMAD inhibition by changing the media to Neural Induction Media (NIM) which is composed of NBM supplemented with small molecules SB431542 (10μM, an inhibitor of TGFβ pathway) (72232, Stem Cell Technologies) and LDN193189 (0.1μM, an inhibitor of BMP pathway) (72142, Stem Cell Technologies).

Techniques: Western Blot, Recombinant, Immunostaining

Journal: Journal of cell science

Article Title: FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-β signaling.

doi: 10.1242/jcs.176248

Figure Lengend Snippet: Figure 1. TGFβ1 induces Endothelial-Mesenchymal Transition in an ALK5-Smad2/3-

Article Snippet: Small molecule inhibitors to ALK5 (SB431542, 5μM), p38 MAPK (SB203580, 1μM, both Sigma Aldrich, St. Louis, MO), Ras (Farnesyl Thiosalicylic Acid (FTS), 5μM, Cayman Chemical, Ann Arbor, MI), PI3Kinase (LY294002, 10μM, SelleckChem, Munich, Germany), Erk1/2 (U0126, 5μM, Promega, Madison, WI), or JNK (SP600125, 1μM, EMD Millipore, Darmstadt, Germany) were used where indicated for 72h.

Techniques:

Journal: Journal of cell science

Article Title: FGF2 inhibits endothelial-mesenchymal transition through microRNA-20a-mediated repression of canonical TGF-β signaling.

doi: 10.1242/jcs.176248

Figure Lengend Snippet: Figure 3. microRNA-20a Gain-of-Function inhibits TGFβ1-ALK5-Smad2/3 Signaling.

Article Snippet: Small molecule inhibitors to ALK5 (SB431542, 5μM), p38 MAPK (SB203580, 1μM, both Sigma Aldrich, St. Louis, MO), Ras (Farnesyl Thiosalicylic Acid (FTS), 5μM, Cayman Chemical, Ann Arbor, MI), PI3Kinase (LY294002, 10μM, SelleckChem, Munich, Germany), Erk1/2 (U0126, 5μM, Promega, Madison, WI), or JNK (SP600125, 1μM, EMD Millipore, Darmstadt, Germany) were used where indicated for 72h.

Techniques: