Journal: BMC Cancer

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

doi: 10.1186/s12885-024-12316-4

Figure Lengend Snippet: Oxaliplatin treatment triggers YAP and TAZ nuclear accumulation. A Immunofluorescence experiments performed on HCT116 cells treated, or not, with oxaliplatin (0.5 µM) monitoring TEAD4 (top panels), YAP (middle panels), and TAZ (bottom panels) nuclear localization (red). DAPI (blue) was used to stain DNA and the nuclei. Quantification of YAP and TAZ stainings are shown on the right side of the figures and are represented as the corrected nuclear fluorescence. Data are represented as the mean ± SEM ( n = 3). Unpaired two-tailed Student’s t-test; **** p < 0.0001. B Western blot analysis showing protein expression and/or activation of Hippo pathway components in HCT116 cells treated (Oxa), or not (NT) with oxaliplatin (0.5 µM). GAPDH was used as a loading control ( n = 3). B’ Quantification of the ratio Oxa/NT of the western-blots shown in B after normalization by GAPDH intensity. Average is shown with standard deviation. Unpaired two-tailed t-test; ** p < 0.01, *** p < 0.001, ns non-significant. C Western blot analysis after subcellular fractionation showing the relative amount of YAP and TAZ protein in the nuclear fraction of HCT116 cells treated (Oxa), or not (NT) with oxaliplatin (0.5 µM). Histone H3 was used as a nuclear loading control for the fractionation ( n = 3). C’ Quantification of the ratio Oxa/NT of the western-blots shown in B after normalization by Histone H3 intensity. Average is shown with standard deviation. Unpaired two-tailed t-test; ** p < 0.01, *** p < 0.001, ns non-significant

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05–636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques: Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Western Blot, Expressing, Activation Assay, Standard Deviation, Fractionation

Journal: BMC Cancer

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

doi: 10.1186/s12885-024-12316-4

Figure Lengend Snippet: YAP and TAZ accelerate oxaliplatin-mediated cell death. A HCT116- shLuc, HCT116- shYAP , and HCT116 transfected with siTAZ were treated with increasing doses of oxaliplatin for 96 h. Cell viability analysis was then assessed using SRB assay and the IC50 of oxaliplatin was calculated as the concentration needed to kill 50% of the cells (shown in the inset). One-way ANOVA statistical test, * p < 0.05. B HCT116- shYAP-siTAZ and HCT116- shLuc-siCtl (control) cell lines were treated with increasing doses of oxaliplatin for 96 h. Cell viability analysis was then assessed using SRB assay and the IC50 of oxaliplatin was calculated as the concentration needed to kill 50% of the cells (shown in the inset). Paired two-tailed Student’s t-test, ** p < 0.01. C Western blot analysis showing protein expression of TAZ and YAP in HCT116- shLuc , -siTAZ , -shYAP and both -shYAP-siTAZ used in panel A and B. Tubulin was used as a loading control ( n = 3). D HCT116- shYAP-siScramb (control), HCT116- shYAP-siTAZ, and HCT116- shYAP-siTAZ transfected with a Flag tagged murine Taz (pEFmTaz) cell lines were treated with increasing doses of oxaliplatin for 96 h. Cell viability analysis was then assessed using SRB assay and the IC50 of oxaliplatin was calculated as the concentration needed to kill 50% of the cells. Paired two-tailed Student’s t-test, ** p < 0.01, ns non-significant. E Western blot analysis showing protein expression of TAZ and Flag in the different cell lines used in panel D. GAPDH was used as a loading control. F HCT116 cells were incubated with increasing concentrations of oxaliplatin and either Verteporfin or CA3. Cell viability was assessed with the SRB assay in 2D to obtain the viability matrix. Drug concentrations were as follows: Verteporfin (from 0.437 to 7 µM), CA3 (from 0.004 to 0.75 µM) and Oxaliplatin (from 0.0185 to 1.2 µM). The synergy matrices were calculated as described in “ ” section

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05–636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques: Transfection, Sulforhodamine B Assay, Concentration Assay, Two Tailed Test, Western Blot, Expressing, Incubation

Journal: BMC Cancer

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

doi: 10.1186/s12885-024-12316-4

Figure Lengend Snippet: Oxaliplatin and YAP/TAZ drugs interactions in p53 mutant CRC cell lines. A-D HCT116 p53mut ( A ), HT-29 ( B ), SW480 ( C ), and Caco-2 ( D ) cells were incubated with increasing concentrations of oxaliplatin and either Verteporfin or CA3. Cell viability was assessed with the SRB assay in 2D to obtain the viability matrix. The synergy matrices were calculated as described in “ ” section

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05–636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques: Mutagenesis, Incubation, Sulforhodamine B Assay

Journal: BMC Cancer

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

doi: 10.1186/s12885-024-12316-4

Figure Lengend Snippet: Src inhibition by Dasatinib reduces HCT116 cells sensitivity to oxaliplatin. A Western blot analysis showing protein expression of YAP and TAZ in HCT116 treated, or not, with oxaliplatin (0.5 µM) and/or Dasatinib (50 nM and 100 nM). Phopsho-SRC blotting was used to evaluate the inhibition of SRC activity using Dasatinib. GAPDH was used as a loading control ( n = 3). Quantification of the blots (performed using Image J software) is shown on the right side of the figure. B Immunofluorescence experiments performed in HCT116 cells treated, or not, with oxaliplatin (0.5 µM) and/or Dasatinib (50 nM) monitoring YAP (top panels) and TAZ (bottom panels) nuclear localization (red). DAPI (blue) was used to stain DNA and the nuclei. Quantification of both stainings are shown on the right side of the figures and are represented as the corrected nuclear fluorescence. Data are represented as the mean ± SEM ( n = 3). Unpaired two-tailed Student’s t-test; **** p < 0.0001. C HCT116 colorectal cancer cell lines were incubated with increasing concentrations of oxaliplatin and Dasatinib. Cell viability was assessed with the SRB assay in 2D to obtain the viability matrix. Drug concentrations were as follows: Dasatinib (from 1 to 16 µM) and oxaliplatin (from 0.0185 to 1.2 µM). The synergy matrix was calculated as described in “ ” section

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05–636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques: Inhibition, Western Blot, Expressing, Activity Assay, Software, Immunofluorescence, Staining, Fluorescence, Two Tailed Test, Incubation, Sulforhodamine B Assay

Journal: BMC Cancer

Article Title: The Hippo pathway terminal effector TAZ/WWTR1 mediates oxaliplatin sensitivity in p53 proficient colon cancer cells

doi: 10.1186/s12885-024-12316-4

Figure Lengend Snippet:

Article Snippet: Antibodies used were rabbit anti-53BP1 (1/100; CST #4937), mouse anti-phospho-Histone H2AX clone JBW301 (1/200; Millipore #05–636), anti-TAZ (1/100; CST #4883), mouse anti-TEAD4 (1/50; Santa Cruz #sc101184), and rabbit anti-YAP (1/100; CST #14074).

Techniques:

Journal: Physiological Reports

Article Title: Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine

doi: 10.14814/phy2.12782

Figure Lengend Snippet: Characterization of the antibody used for the proUgn western blot assay. (A) Experiments with recombinant propeptides demonstrate the selectivity of anti‐proUgn antibody 6910. The two left lanes were loaded with identical samples of purified recombinant proGn that were run side‐by‐side on the same gel, then separated for incubation with different primary antibodies, as indicated. Anti‐proGn antibody 2538 recognizes proGn (the minor immunopositive band of lower molecular weight presumably represents small amounts of a proGn degradation product), but antibody 6910 does not. In contrast, antibody 6910 selectively recognizes purified recombinant proUgn (right lane). (B) Rat plasma (50 μ L) was fractionated by C‐18 reverse‐phase HPLC , and then 500 μ L of each 1 mL fraction was dried, resuspended in electrophoresis sample buffer, and analyzed by immunoblotting. Antibody 6910 should not recognize Ugn or proGn, and the blot confirms a complete lack of crossreactivity at retention times where Ugn or proGn would elute from the column. Here, and in all other relevant figures, retention times for recombinant proUgn, proGn, and Ugn standards were established in parallel column runs, and are indicated by the arrows. The left‐most lane of the gel was loaded with a rat proUgn standard. (C) Western blot analysis of urine samples obtained from three normal rats (control) and three animals previously subjected to surgical ablation of five‐sixths of their renal mass (renal failure). Each urine sample (100 μ L) was prefractionated on a Superdex column, as described in the Methods, and then fractions 31–34 (where proUgn elutes from this column) were pooled, dried, resuspended in electrophoresis sample buffer, and half of this final sample was analyzed by immunoblotting. The left‐hand lanes of the gel were loaded with a dilution series of recombinant rat proUgn, as a standard curve.

Article Snippet: Samples were loaded on 4–12% polyacrylamide gels (Invitrogen Corp., Carlsbad, CA), and a standard curve was constructed with known amounts of recombinant rat proUgn (Ironwood Pharmaceuticals, Cambridge, MA).

Techniques: Western Blot, Recombinant, Purification, Incubation, Molecular Weight, Clinical Proteomics, Electrophoresis, Control

Journal: Physiological Reports

Article Title: Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine

doi: 10.14814/phy2.12782

Figure Lengend Snippet: Validation of a novel Ugn binding assay. (A) Ability of synthetic proUgn, Ugn, and Gn to displace radioligand in the competitive binding assay. (B) The analyte responsible for the binding displacement activity of urine coelutes with authentic Ugn from a high‐resolution C‐18 HPLC column. A 200 μ L sample of urine (collected at 3 am on day 2 of a high‐salt diet) was loaded on the column; this is four times the volume that is loaded in a typical assay. Retention times of Gn, Ugn, proGn, and proUgn standards are indicated by the arrows. (C) Displacements of radioligand by urine samples from a Ugn knockout mouse and a wild‐type mouse are interpolated in a standard curve generated with a rat Ugn standard. (D) Endogenous Ugn is not detected in rat plasma (black symbols), whereas a strong peak is readily observed when 2 nmol exogenous Ugn are added to a plasma sample prior to chromatography (white symbols – retention times of Gn, Ugn, proGn, and proUgn standards are indicated by the arrows).

Article Snippet: Samples were loaded on 4–12% polyacrylamide gels (Invitrogen Corp., Carlsbad, CA), and a standard curve was constructed with known amounts of recombinant rat proUgn (Ironwood Pharmaceuticals, Cambridge, MA).

Techniques: Biomarker Discovery, Binding Assay, Competitive Binding Assay, Activity Assay, Knock-Out, Generated, Clinical Proteomics, Chromatography

Journal: Physiological Reports

Article Title: Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine

doi: 10.14814/phy2.12782

Figure Lengend Snippet: Western blot analysis of plasma proUgn. The quantitative western blot assay is described in the Methods and in Figure . The number of replicates ( n ) is given in parentheses for each measurement. (A) Time course of plasma levels of proUgn (black circles) and inulin (white circles) after ligation of splanchnic or renal arteries, as indicated. (B) Circadian fluctuations in circulating plasma levels of proUgn are illustrated for a subset of the animals included in the diet study. Data were acquired from rats fed normal chow ( N ), after 5 days on low‐salt chow (L5), and after 5 days on high‐salt chow (H5). Plasma samples (100 μ L) were taken every 6 h via an indwelling carotid cannula, and assayed for proUgn content using the quantitative western blot assay. Data points are plotted against the time of sample acquisition. The dashed line indicates 12 am (midnight). Asterisk indicates P < 0.05. (C) Diurnal (white bars) and nocturnal (black bars) plasma levels of proUgn in animals fed ad libitum (left panel) or animals fasted (right panel) for 12 h (diurnal fasting values) or 24 h (nocturnal fasting values). (D) Representative western blots, showing a subset of the raw data used to generate the graphs in panels e and f. For quantitation, the infrared signal intensity of each sample was interpolated in a standard curve derived from known amounts of recombinant rat proUgn, as shown at the right for gel 2 (black symbols = standards, white symbols = plasma samples). Values from multiple gels could be combined to generate the final graphs in panels e and f, because every gel was loaded with an identical standard curve. (E) Circulating levels of proUgn at 3 am under defined dietary conditions. (F) Average rates of nocturnal urinary Ugn excretion ( U U gn V) plotted as a function of average nocturnal plasma proUgn concentration for animals consuming normal salt ( NS ), low salt for 5 days ( LS 5), or high salt for 1 ( HS 1), 2 ( HS 2) or 5 ( HS 5) days ( R ² = 0.009 for a linear fit to the data).

Article Snippet: Samples were loaded on 4–12% polyacrylamide gels (Invitrogen Corp., Carlsbad, CA), and a standard curve was constructed with known amounts of recombinant rat proUgn (Ironwood Pharmaceuticals, Cambridge, MA).

Techniques: Western Blot, Clinical Proteomics, Ligation, Quantitation Assay, Derivative Assay, Recombinant, Concentration Assay

Journal: Physiological Reports

Article Title: Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine

doi: 10.14814/phy2.12782

Figure Lengend Snippet: Primers and probes used for qRT‐PCR

Article Snippet: Samples were loaded on 4–12% polyacrylamide gels (Invitrogen Corp., Carlsbad, CA), and a standard curve was constructed with known amounts of recombinant rat proUgn (Ironwood Pharmaceuticals, Cambridge, MA).

Techniques:

Journal: Physiological Reports

Article Title: Dietary salt regulates uroguanylin expression and signaling activity in the kidney, but not in the intestine

doi: 10.14814/phy2.12782

Figure Lengend Snippet: Characteristics of the Ugn binding assay

Article Snippet: Samples were loaded on 4–12% polyacrylamide gels (Invitrogen Corp., Carlsbad, CA), and a standard curve was constructed with known amounts of recombinant rat proUgn (Ironwood Pharmaceuticals, Cambridge, MA).

Techniques: Binding Assay

Journal: Antioxidants

Article Title: MicroRNA-4732-3p Is Dysregulated in Breast Cancer Patients with Cardiotoxicity, and Its Therapeutic Delivery Protects the Heart from Doxorubicin-Induced Oxidative Stress in Rats

doi: 10.3390/antiox11101955

Figure Lengend Snippet: Identification and validation of miR-4732-3p target genes. ( A ) GO terms among predicted targets of miR-4732-3p and their intersections. ( B ) Interactome of combined target genes of miR-4732-3p, miR-145-5p, miR-125b-5p, miR-29b-3p, miR-155-5p. ( C ) Representative western blots of p-YAP, YAP and Smad2 in HCF non-transfected or transfected with either NC-Mimic or 4732-Mimic. Relative levels were quantified by densitometry relative to the levels of HCF without transfection. GAPDH was used as a loading control. ( D ) Representative western blots of p-YAP, YAP and Smad2 in hiPSC-CMs non-transfected or transfected with either NC-Mimic or 4732-Mimic. Relative levels were quantified by densitometry relative to the levels of hiPSC-CMs without transfection. GAPDH was used as a loading control. Data are represented as mean ± standard deviation. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies used for western blotting were: anti-Smad2 (1/500, Abcam, Cambridge, UK, ab228765), anti-YAP (1/1000, Cell Signaling Technology, Danvers, MA, USA, D8H1X), anti-phospho-YAP (1/1000, Cell Signaling Technology, D9W2I) and anti-GAPDH (1/1000, Cell Signaling Technology, D16H11).

Techniques: Western Blot, Transfection, Standard Deviation