Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: Analysis of fatty acids in mouse liver following PHx. ( A ) Immunohistochemistry analysis of FAT/CD36 (brown) in the liver of chow- and MCD-diet fed mice during liver regeneration. Bar = 20 μm; 40× objective lenses were used. ( B ) qPCR analysis of FAT/CD36 expression in mouse liver after PHx. ( C , D ) Levels of fatty acids in liver and serum during liver regeneration were assessed using enzymatic assays. Data represent the mean ± SEM for 6–8 mice per group. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: The sections were then reacted with the following primary antibodies for 16–17 hr: anti-proliferating cell nuclear antigen (PCNA) (Dako, M0879), anti-FAT/CD36 (Novus Biologicals, NB400-144), anti-S1PR2 (Novus Biologicals, NBP2-26691), anti-HNF-4α (Abcam, ab181614), anti-F4/80 (Abcam, ab6640), and anti-Ly6G (Biolegend, #127605).

Techniques: Immunohistochemistry, Expressing

Journal: Pharmaceutics

Article Title: CAVPENET Peptide Inhibits Prostate Cancer Cells Proliferation and Migration through PP1γ-Dependent Inhibition of AKT Signaling

doi: 10.3390/pharmaceutics16091199

Figure Lengend Snippet: Protein expression levels of fatty acid synthase (FASN) ( A ), CD36 ( B ) and carnitine palmitoyltransferase (CPT1) ( C ) after incubation with CAVPENET. Cell viability of PC-3 cells after 48 h incubation with 50 μM of etomoxir (CPT1 inhibitor), 10 μM of CAVPENET and their combination ( D ). The PC-3 cells were incubated with 10 µM of CAVPENET for 48 h, collected and lysed using RIPA1x lysis buffer. The proteins were resolved by SDS-PAGE and transferred to membranes, that were incubated with the respective antibodies. The band’s intensity was quantified using ImageLab software, normalized to PonceauS staining intensity and represented as fold change to control (untreated cells). The cell viability was determined using PrestoBlue cell viability reagent assay, considering untreated cells as 100% viability. The results are expressed as mean ± SD from at least three replicates. * p < 0.05; **** p < 0.0001.

Article Snippet: The primary antibodies were diluted in either 5%non-fat milk or 5% BSA and incubated for 1 h or ON at 4 °C: anti-p(Ser473)-AKT (rabbit, 1:2000, 4060S, Cell Signaling), anti-AKT (mouse, 1:500, sc-5298, Santa Cruz Technologies, Santa Cruz, CA, USA), anti-p(Ser9)-GSK3β (mouse, 1:1000, sc-373800, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-GSK3β (rabbit, 1:1000, 9315S, Cell Signaling), anti-p(Thr320)-PP1α (rabbit, 1:1000, ab62334, Abcam, Cambridge, UK), anti-PP1α (rabbit, 1:2500, homemade), anti-PP1β (mouse, 1:1000, sc-365678, Santa Cruz Biotechnologies), anti-PP1γ (rabbit, 1:1000, homemade), anti-p(Thr37/46)-4E-BP1 (rabbit, 1:1000, 236B4, Cell Signaling), anti-4E-BP1 (rabbit, 1:1000), anti-HK2 (mouse, 1:500, sc-374091, Santa Cruz Biotechnologies), anti-FASN (mouse, 1:500, sc-55580, Santa Cruz Biotechnologies); anti-CD36 (rabbit, 1:2000, NB400-144, Novus Biologicals, Centennial, CO, USA) and anti-CPT1A (mouse, 1:500, sc-393070, Santa Cruz Biotechnologies).

Techniques: Expressing, Incubation, Lysis, SDS Page, Software, Staining, Control

Journal: Pharmaceutics

Article Title: CAVPENET Peptide Inhibits Prostate Cancer Cells Proliferation and Migration through PP1γ-Dependent Inhibition of AKT Signaling

doi: 10.3390/pharmaceutics16091199

Figure Lengend Snippet: Proposed molecular mechanisms that drive the effect of CAVPENET bioportide. Briefly, CAVPENET is taken up by the prostate cancer cells and increases the activity of PP1, probably by disrupting the interaction with CAV1. The increased activity of this phosphatase decreases the phosphorylation and activity of AKT, ultimately leading to blockage of glycolysis, by decreasing HK2 expression, and decreases fatty acid synthesis and uptake, by decreasing FASN and CD36 expression. To compensate for these alterations, PCa cells increase lipid oxidation by increasing CPT1 expression. The alterations observed in this work are highlighted in bold.

Article Snippet: The primary antibodies were diluted in either 5%non-fat milk or 5% BSA and incubated for 1 h or ON at 4 °C: anti-p(Ser473)-AKT (rabbit, 1:2000, 4060S, Cell Signaling), anti-AKT (mouse, 1:500, sc-5298, Santa Cruz Technologies, Santa Cruz, CA, USA), anti-p(Ser9)-GSK3β (mouse, 1:1000, sc-373800, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), anti-GSK3β (rabbit, 1:1000, 9315S, Cell Signaling), anti-p(Thr320)-PP1α (rabbit, 1:1000, ab62334, Abcam, Cambridge, UK), anti-PP1α (rabbit, 1:2500, homemade), anti-PP1β (mouse, 1:1000, sc-365678, Santa Cruz Biotechnologies), anti-PP1γ (rabbit, 1:1000, homemade), anti-p(Thr37/46)-4E-BP1 (rabbit, 1:1000, 236B4, Cell Signaling), anti-4E-BP1 (rabbit, 1:1000), anti-HK2 (mouse, 1:500, sc-374091, Santa Cruz Biotechnologies), anti-FASN (mouse, 1:500, sc-55580, Santa Cruz Biotechnologies); anti-CD36 (rabbit, 1:2000, NB400-144, Novus Biologicals, Centennial, CO, USA) and anti-CPT1A (mouse, 1:500, sc-393070, Santa Cruz Biotechnologies).

Techniques: Activity Assay, Expressing

Journal: Molecular Therapy

Article Title: Targeting Rap1b signaling cascades with CDNF: Mitigating platelet activation, plasma oxylipins and reperfusion injury in stroke

doi: 10.1016/j.ymthe.2024.09.005

Figure Lengend Snippet: Systemic administration of CDNF decreased infarction volume, accelerated neurobehavioral recovery, attenuated neuroinflammation, and preserved BBB integrity and junction protein expressions in the lesioned cortex after dMCAo (A) Representative images of brain sections stained with TTC showing infarction area. (B) Quantification of infarction volume using TTC staining after 48 h of reperfusion. n = 8–10 per group; ∗ p < 0.05 vs. dMCAo + vehicle; Student’s t test was used for the analysis of statistical significance. (C) Modified neurological severity scores (mNSS) were examined at 2 days post-dMCAo n = 8–10 per group. ∗∗ p < 0.01 vs. dMCAo + vehicle; Student’s t test was used for the analysis of statistical significance. (D) Forepaw use bias of the rats was assessed in the cylinder test at 2, 7, and 14 days after dMCAo. n = 5–6 per group. ∗∗ p < 0.01 by Fisher’s LSD test, following two-way ANOVA (effect of treatment: F(1,28) = 11.77, p < 0.0019). (E) Horizontal distance traveled for 30 min on days 2, 7, and 14 post-dMCAo. ∗ p < 0.05 by original FDR method of Benjamini and Hochberg test, following two-way ANOVA (effect of treatment: F(2,31) = 11.97, p < 0.0001). (F and G) Body asymmetry test and Bederson’s score were analyzed on days 2, 7, and 14 after dMCAo, ∗∗ p < 0.01, ∗∗∗ p < 0.001 indicate comparison with vehicle with Bonferroni’s post hoc test following two-way ANOVA. (H) Western blot bands of iNOS, COX-2, IL-1β, TNF-α, IL-10, CD163, CD36, and GAPDH at 2 days after dMCAo. (I) Representative brain coronal sections (2 mm thickness) show Evans blue extravasation on day 2 post-dMCAo. (J and K) Comparison of dye concentrations in the ipsilateral (J) and contralateral (K) cortex between dMCAo + vehicle and dMCAo + CDNF groups. Dye concentration is presented as μg/g of tissue weight and calculated from a standard curve obtained from known amounts of dye. ∗ p < 0.05, paired t test. Mean ± SEM is shown. (L) Western blotting showed the levels of tight junction proteins ZO-1, claudin-5, LRP-1, and 92 kDa type IV collagenase, MMP-9, within the peri-infarct cortical area. (M) At 2 days post-stroke, the decreased levels of iNOS, COX-2, IL-1β, TNF-α, and MMP-9, but increased levels of IL-10, CD163, CD36, ZO-1, Claudin-5, and LRP-1 were detected in the ischemic cortex of CDNF treatment group. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 by multiple unpaired t test. Mean ± SEM is shown.

Article Snippet: Primary antibodies and concentrations were used as follows: iNOS (1:500, GeneTex, GTX130246), CD163 (1:250, Abcam, ab87099, ab182422), CD91 (1:1,000, Abcam, ab92544), CD36 (1:500, Novus, NB400-144), GAPDH (1:1,000, Abcam, ab181602), IL-10 (1:500, Santa Cruz, SC-32815), CD11b (1:1,000, GeneTex, GTX134493), TNF-α (1:500, Santa Cruz, SC-12744), IL-1β (1:500, Santa Cruz, SC-12742), ZO-1 (1:250, Santa Cruz, SC-33725), Claudin-5 (1:500, Invitrogen, 35–2500), and MMP-9 (1:250, Abcam, ab19016).

Techniques: Staining, Modification, Comparison, Western Blot, Concentration Assay