p cdk2  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: An inducer of snail hibernation causes quiescence and hibernation-like cardioprotection, through metabolic rewiring and autophagy, in mice hearts

doi: 10.64898/2026.01.08.698452

Figure Lengend Snippet: (A) The protocol for stressed mouse fibroblasts studies: after seeding, the cells were cultured in stress conditions for 24 or 72 hours with DMSO or SNAP, then returned to normal conditions without DMSO or SNAP for another 2 days. (B) Representative live images of mitochondria and lysosomes stained with MitoTracker and LysoTracker under stress conditions for 24 hours (Scale bar: 50 μm) shows early activation of lysosomes suggesting autophagy. (C) The protein levels of the autophagy markers LC-3B II and LAMP-1 between DMSO-treated and SNAP-treated cells under stress conditions for 24 hours were detected by immunoblots and quantified with Image J. (D) Cleaved caspase-3 protein levels were detected after a 72-hours of stress using immunoblots and quantified with Image J. (E) Cell numbers were detected and calculated using a Holomonitor microscope, which allows imaging of live cells based on optical density, avoiding the additional stress of staining: survival rate was calculated after stress for 3 days and population doubling time was calculated after returning to normal conditions for 2 days. (F) Ki-67, p-CDK2, t-CDK2, p-Rb and t-Rb protein levels were detected at day 0, 3 and 5 by immunoblots, and their expression levels were quantified using Image J. (G) Schematic shows the SNAP protects mouse fibroblasts during stress by putting them into reversible quiescence and allow them to re-enter the cell cycle after stress (unlike the irreversible state of senescence. (H) After seeding fibroblasts, cells were cultured in stress for 72 hours with DMSO or SNAP and then immediately studied as shown in subsequent panels. (I) OCR (oxygen consumption rate) of cells after normal vs stress conditions for 72 hours was assessed using a Seahorse XF24 Extracellular Flux Analyzer and SRC (the difference between maximal OCR triggered by FCCP and minimum OCR triggered by Oligomycin) were calculated. (J) Representative live images of TMRM and MitoSOX staining with cells after culturing in stress conditions for 72 hours (Scale bar: 20 μm) with TMRM and MitoSOX fluorescence intensity quantification. (K) p-PDHE1α Ser232 , p-PDHE1α Ser300 , p-PDHE1α Ser293 , (all potential sites of inhibiting phosphorylation) and t-PDH protein levels between DMSO and SNAP-treated cells were detected by immunoblots and quantified using Image J. All data are shown as mean ± S.D. and represent three ( B , C , E , F , I and L ) and eight ( J ) independent experiments. P values were calculated by one-way ANOVA with Tukey’s multiple comparisons post hoc tests ( F, J and I ) or two-sided unpaired Student’s t-tests ( B, C, E, and K ). * p <0.05, ** p < 0.01, *** p < 0.001; ns, no statistical significance.

Article Snippet: Antibody dilution was 1:1000 for the following immunoblots: p-Rb ser807/811 (Cell Signaling Technology, 8516), Rb (Invitrogen, SY63-03), p-CDK2 Thr160 (Cell Signaling Technology, 2561), t-CDK2 (Abcam, ab32147), β-Actin (Cell Signaling Technology, 3700), t-AMPK (Cell Signaling Technology, 5831), p-AMPK Thr172 (Cell Signaling Technology, 2535), AKT (Cell Signaling Technology, 9272), p-AKT Ser473 (Cell Signaling Technology, 9271), t-S6K1 (Cell Signaling Technology, 2708), and p-S6K1 Thr389 (Cell Signaling Technology, 9205), p-PDHE1α ser232 (Millipore Sigma, AP1063), p-PDHE1α ser300 (Millipore Sigma, AP1064), t-PERK (Cell Signaling Technology, 3192), p-PERK Thr982 (Invitrogen, PA5-40294), ATF6 (Abcam, ab37149), LC3B (Cell Signaling Technology, 2775), cleaved caspase-3 (Cell signaling Technology, 9664), t-mTOR (Cell Signaling Technology, 2983), p-mTOR ser2448 (Cell Signaling Technology, 5536), Ki67 (Abcam, ab16667), p-eIF2α Ser51 (Cell Signaling Technology, 9721), t-eIF2α (Cell Signaling Technology, 5324), Custom p-PDK1 Thr344 (generated by Neobiolab), t-PDK1 (Abcam, ab110025), LDHA (Cell Signaling Technology, 2012), p-JNK Thr183/Tyr185 (Cell Signaling Technology, 9251), t-JNK (Cell Signaling Technology, 3708), α-Tubulin (Cell Signaling Technology, 2144), NHERF-1 (Santa Cruz Biotechnology, sc-271552).

Techniques: Cell Culture, Staining, Activation Assay, Western Blot, Microscopy, Imaging, Expressing, Fluorescence, Phospho-proteomics