Journal: International Journal of Molecular Sciences

Article Title: Pyk2/FAK Signaling Is Upregulated in Recurrent Glioblastoma Tumors in a C57BL/6/GL261 Glioma Implantation Model

doi: 10.3390/ijms241713467

Figure Lengend Snippet: PF-562271 treatment downregulates Cyclin D1 expression and the Ki67 proliferation index in tumors regrown after surgical resection in a GL261-C57BL/6 mouse glioma implantation model. ( A ) Representative Western blots and quantifications with individual values for the relative level of Cyclin D1 protein expression in the tumors of animals that received vehicle or PF-562271 for 14 days after tumor resection. The displayed values represent the relative differences in the PF-562271 treatment group compared to the vehicle group. Actin was used as a loading control. ( B ) Representative immunofluorescence confocal images, and ( C ) a quantification of cells expressing the Ki67 marker within regrown tumors following surgical resection. The tumors were treated with PF-562271 (50 mg/kg/day) or vehicle for a continuous 14-day duration post-resection, beginning 2 days prior to the tumor removal procedure. Arrows indicate cells expressing Ki67. Scale bar is 100 µm. Mean ± S.E. and significant differences from the control (*) ( p < 0.005). N = 5.

Article Snippet: Cell lysates (20 μg), separated by 10% SDS–PAGE, were transferred to PVDF membranes and probed with anti-phospho-Pyk2 (Tyr 579/580) (#44636G, Thermo Fisher Scientific, Invitrogen, MA, USA), anti-Pyk2, anti-phospho-FAK (Tyr 925), anti-FAK, and anti-Cyclin D1 (#3480, #3284, #3285, #55506 Cell Signaling, Danvers, MA, USA) antibodies, followed by the addition of the corresponding secondary antibodies (cat. #A9169, Sigma–Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Marker

Journal: Nature Communications

Article Title: Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence

doi: 10.1038/s41467-023-40596-0

Figure Lengend Snippet: a A comprehensive pipeline was settled to sequentially validate altered cell cycle and enhanced senescence in MA under either defective PKP2 (si- PKP2 ) or enduring sustained exposure to MCM, also characterized by impaired PKP2 gene expression. In the latter, we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Bar plots show the change for a set of genes previously identified as related to impaired PKP2 , including those (i) directly or (ii) inversely related to senescence, and (iii) biomarkers downstream E2F signaling, also bound to cell cycle, in b si-PKP2 and c MCM-treated adipocytes. SA-β-gal staining in MA under different conditions. Pictures show three representative images sorted out from four biological replicates in which measures of absorbance (546 nm) allowed the quantitative assessment plotted at the bottom right corner. e By using a BD Accuri C6 flow cytometer, the amounts of SA-β-gal positive adipocytes were assessed. Representative flow cytometric curves are provided for each treatment. The gray line shows cell distribution in unmarked control cells. The gating strategy is exemplified in supplementary Fig. , . The bean plot on the right indicates the amounts of SA-β-gal positive adipocytes for each condition ( n = 4 biological replicates/condition). f Western blot analysis for PKP2, Cyclin D1 and PAPPA in non-targeting controls (NTC) and si-PKP2-treated MA. The bar plots below indicate β-actin-normalized relative amounts of each protein. g The box plot (median values plus 25 th and 75 th percentiles, and whiskers bound to maximum and minimum values in each group) shows concentrations of hydrogen peroxide (H 2 O 2 ) measured in conditioned media supernatants collected from cell cultures of either NTC or si-PKP2 adipocytes (left boxes), or MCM-inflamed adipose cells (right boxes) containing a plasmid with an empty cassette (reference) or coding for PKP2 ( n = 8 biological replicates/condition). h Representative 20x immunofluorescent images (the scale bars denote 100 μm length) showing cell cycle driver Cyclin D1 signal in adipocyte cultures. The bean plot at the right-hand shows the Cyclin D1 signal after correcting for blue fluorescent stain DAPI in four biological replicates/conditions. Below, i the percentage of nuclei showing Protein Kinase C alpha (PKCα) immunofluorescence staining in adipocyte cultures following the treatments depicted above. Representative 20x immunofluorescent images are provided in Fig. (scale bars of 100 μm), and an example of image handling and data collection is shown in Fig. . j Gene expression patterns and k Western blot analysis of PKP2, PAPPA, and Cyclin D1 in cultures of adipocytes maintained undisturbed (Ctrl) and inflamed (MCM) adipocytes in which we used plasmids to restore PKP2 levels (MCM + OE_PKP2). Ctrl and MCM groups were treated with the negative empty control vector for pReceiver-M90 and transfection reagent, as per proper control conditions. Bar plots show results assessed in four biological replicates/conditions, and the mean ± SEM Statistical significance was assessed by two-tailed Fisher’s exact t -test. * p < 0.05; ** p < 0.01. Source data are provided as a Source data file.

Article Snippet: Fixed cells were incubated with primary antibodies against Cyclin D1 (Cell Signaling, #55506, clone E3P5S) or PKCα (#2056), followed by incubation with goat anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor™ 488 (Invitrogen, #A-11034).

Techniques: Expressing, Staining, Flow Cytometry, Western Blot, Plasmid Preparation, Immunofluorescence, Transfection, Two Tailed Test