Journal: JCI Insight

Article Title: A G protein–coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23

doi: 10.1172/jci.insight.125007

Figure Lengend Snippet: (A) FGF23 mRNA expression in YFP- and XLαs-overexpressing Ocy454 cells. **P < 0.01, and ***P < 0.001, determined by Welch’s t test (2 tailed) followed by Bonferroni’s correction. (B) FGF23 mRNA expression in control or XLαs adenovirus–infected Ocy454 cells treated with PKC inhibitors, bisindolylmaleimide I (Bisindol), Ro-31-8220, or Ro-32-0432. Data are shown as mean ± SEM of 3 independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. XLαs-overexpressing Ocy454 cells. (C and D) Ocy454 cells were treated with PMA, with PKC inhibitors. qRT-PCR analysis of (C) FGF23 and (D) IL-6 mRNA after 6 hours of treatment. Data are shown as mean ± SEM of 4 independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. PMA-treated group. (B–D) Significance was calculated by 1-way ANOVA followed by Tukey’s multiple-comparisons test. (E) Representative Western blot of phosphorylated ERK1/2 (p-ERK) and total (phosphorylated and nonphosphorylated) ERK1/2 (total ERK) in femur bone lysate samples from P10 XLKO (KO) and WT mice. Loading control: tubulin. (F) Densitometric analysis of the ratio of p-ERK to total ERK in KO and WT femurs. Data are shown as mean ± SEM of 7 mice from each group. (G) Western blot analysis of p-ERK, ERK, and tubulin levels in control and XLKO Ocy454 cells. (H) Densitometric analysis of p-ERK/total ERK ratio represented in G. Data are shown as mean ± SEM from 3 independent experiments. (F and H) *P < 0.05, **P < 0.01 by 2-tailed Student’s t test. (I) Western blot analysis of p-ERK, ERK, and tubulin in XLKO and control cells treated with PMA or vehicle. (J) Densitometric analysis of p-ERK/ERK ratio represented in I. aP < 0.05 vs. vehicle-treated control cells; bP < 0.05 vs. vehicle-treated KO cells; cP < 0.05 vs. PMA-treated control cells. (K) FGF23 mRNA levels in Ocy454 cells treated with PMA, with or without MEK inhibitor U0126. Data are shown as mean ± SEM of 3 independent experiments. *P < 0.05 vs. control group; #P < 0.05 vs. PMA-treated group. Statistical differences in J and K were assessed with 1-way ANOVA followed by Tukey’s test.

Article Snippet: Antibodies against phospho-PKC substrates (catalog 2261S), total ERK (catalog 9102S), PKCα (catalog 9960S), and PKCδ (catalog 9960S) were from Cell Signaling Technology.

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot

Journal: JCI Insight

Article Title: A G protein–coupled, IP3/protein kinase C pathway controlling the synthesis of phosphaturic hormone FGF23

doi: 10.1172/jci.insight.125007

Figure Lengend Snippet: (A) Western blot analysis of phosphorylated PKC (p-PKC) substrates, p-ERK, and total ERK in WT and Hyp mice. (B) Densitometric analysis of the relative abundance of p-PKC substrates. (C) Densitometric analysis of p-ERK/total ERK ratio represented in A. Data are shown as mean ± SEM of 7 or 8 mice from each group. *P < 0.05, calculated by unpaired, 2-tailed Student’s t test. (D) Serum C-terminal FGF23, (E) intact FGF23, and (F) phosphate levels in Hyp and XLKO intercross littermates. *P < 0.05, and ***P < 0.001, determined by Welch’s t test (2 tailed) followed by Bonferroni’s correction.

Article Snippet: Antibodies against phospho-PKC substrates (catalog 2261S), total ERK (catalog 9102S), PKCα (catalog 9960S), and PKCδ (catalog 9960S) were from Cell Signaling Technology.

Techniques: Western Blot

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: β-HB treatment reverses sorafenib resistance by shifting glycolysis-lactate metabolism in HCC.

doi: 10.1016/j.biopha.2023.115293

Figure Lengend Snippet: Fig. 5. β-HB treatment reversed sorafenib resistance and enhanced regorafenib sensitivity by inhibiting the B-raf/MAPK pathway in Huh7-SR and Sk-Hep-1-SR cells. (A) Huh7-SR and SK-Hep-1-SR cells were treated with sorafenib (0 and 10 μM) and β-HB (0, 2.5, and 5 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (B) Huh7-SR and SK-Hep-1-SR cells were treated with regorafenib (0 and 5 μM) and β-HB (0, 2.5, 5, and 10 mM) for 48 h, and cell viability was then analyzed using an MTT assay. (C) Western blotting was conducted to analyze the protein expression of phosphorylated (p)- and total (t)-MAPK-related signaling cascades; α-tubulin was used as an internal control. (D) Western blot images from Fig. 4 C were quantified using ImageJ software. (E) Representative contour plots of apoptosis were detected by flow cytometry stained with Annexin V-FITC and PI. (F) Apoptosis rates were assessed in dose-dependent regorafenib with or without β-HB treatment in Huh7-SR and Sk-Hep-1-SR with Annexin V/PI staining. * p < 0.05; * * p < 0.01; * ** p < 0.001 vs. 0 mM β-HB cells. Data are presented as mean ± SD.

Article Snippet: The following antibodies were used in the experiments: HMGCS2 (1:1000, #ab137043, Abcam, Cambridge, UK), hexokinase I (1:1000, #2024, Cell Signaling, Danvers, MA, USA), hexokinase II (1:1000, #2867, Cell Signaling), phosphofructokinase (1:1000, #8164, Cell Signaling), LDHA (1:1000, #3582, Cell Signaling), PDH (1:1000, #3205, Cell Signaling), IDH (1:1000, #8137, Cell Signaling), phosphorylated (p)- and total (t)-B-raf (1:1000, #2696 and #9433, Cell Signaling), p- and t-MAPK kinase (MEK; 1:1000, #2338 and #9122, Cell Signaling), ERK (1:1000, #9101 and #4695, Cell Signaling), β-catenin (1:1000, #8480, Cell Signaling), ZO-1 (1:1000, #8193, Cell Signaling), Vimentin (1:1000, #5741, Cell Signaling), N-cadherin (1:1000, #13116, Cell Signaling) and antiα-tubulin (1:5000 dilution, #T9026, Sigma-Aldrich).

Techniques: MTT Assay, Western Blot, Expressing, Control, Software, Flow Cytometry, Staining

Journal: Journal of Virology

Article Title: ERK Is a Critical Regulator of JC Polyomavirus Infection

doi: 10.1128/JVI.01529-17

Figure Lengend Snippet: JCPyV induces multiphasic ERK activation upon infection. (A) SVG-A cells were infected with JCPyV (MOI = 1 FFU/cell) for the specified duration and were analyzed via Western blotting using antibodies specific for phosphorylated ERK (pERK) and total ERK (protein control). The percentage of pERK was determined using phosphorylated ERK and total ERK band intensities, determined by using the equation [pERK/(pERK + total ERK)] × 100. The percentage of pERK for JCPyV-infected samples was normalized to the value for mock-infected samples at each time point (100%; dashed line). Percentages of phosphorylated ERK for each time point were plotted via box-and-whisker plot and are representative of results from three independent experiments. (B) SVG-A cells were either pretreated (PT) for 1 h prior to JCPyV infection with U0126 (10 μM) or DMSO (1:1,000) or incubated in complete medium. Cells were then infected with JCPyV (MOI = 0.1 FFU/cell) at 4°C for 1 h. Following infection, cells were washed with medium and then incubated in media with inhibitors for the indicated times. At the specified time point, medium was removed, cells were washed, complete medium was added, and cells were incubated at 37°C for 72 h. Cells were fixed and stained by indirect immunofluorescence using a VP1-specific antibody, and infectivity was scored by quantitating nuclear VP1 expression. Data are representative of the average number of infected cells per visual field for five 20× fields of view for triplicate samples. Data are representative of results from three independent experiments. Error bars indicate SDs. Student's t test was used to determine statistical significance. *, P < 0.05.

Article Snippet: Antibodies used for pERK Western blot analysis were a rabbit-specific phosphorylated ERK antibody (Cell Signaling Technology; no. 9101) and mouse-specific total ERK antibody (Cell Signaling Technology; no. 4696) and corresponding secondary anti-mouse 680 antibody (LICOR) and goat anti-rabbit 800 antibody (LICOR).

Techniques: Activation Assay, Infection, Western Blot, Whisker Assay, Incubation, Staining, Immunofluorescence, Expressing