phospho cdk2  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Dual targeting of PI3Kδ and PPARα enhances antitumor activity via FoxO1 activation in follicular lymphoma

doi: 10.1038/s41419-026-08593-5

Figure Lengend Snippet: A - C EdU flow plots for Karpas-422, RL, and Sc-1 cells after 24-h treatment (Linperlisib 32 μM; Chiglitazar 16 μM). D – F Quantification of EdU-positive cells showing reduced DNA synthesis with the combination ( n = 3). G – L ( G , H ) for Karpas-422; ( I , J ) for RL; ( K , L ) for Sc-1. Cell-cycle distribution by PI staining, demonstrating G0/G1 accumulation with Linperlisib and further enhancement by the combination (Linperlisib 16 μM; Chiglitazar 8 μM; n = 3). M GSEA indicating negative enrichment of the G1/S transition pathway (NES = − 2.85865, p < 0.00001). N – P Western blots of G1/S checkpoint proteins (p27, Cyclin E1, CDK2, p-CDK2); combination treatment increased p27 and decreased Cyclin E1, CDK2, and p-CDK2 (representative of three independent experiments).

Article Snippet: The primary antibodies used were as follows: PI3K p110δ (A19742; ABclonal), pan-Akt (#4691; CST), phospho-Akt (Ser473, #4060; CST), PPARα (ab227074; Abcam), FoxO1 (#2880; CST), phospho-FoxO1 (Thr24, #9464; CST) and (Ser256, #9461; CST), FoxO3a (#12829; CST), FoxO4 (#9472; CST), Mcl-1 (#16225-1-AP; Proteintech), Bcl-2 (#12789-1-AP; Proteintech), Bim (A19702; ABclonal), Bax (#50599-2-Ig; Proteintech), Cleaved PARP(#5625; CST), P21(#2947; CST), p27 (#3686; CST), Cyclin E1 (#11935-1-AP; Proteintech), CDK2 (#10122-1-AP; Proteintech), phospho-CDK2 (#4539; CST), GLUT1(#73015; CST), PGK1(A12686; ABclonal), β-Tubulin (#10094-1-AP; Proteintech), Lamin B1 (#13435; CST), and β-Actin (#66009-1-Ig; Proteintech).

Techniques: DNA Synthesis, Staining, Western Blot

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

Journal: iScience

Article Title: CDK7 is a novel therapeutic target in fibrolamellar carcinoma

doi: 10.1016/j.isci.2025.113925

Figure Lengend Snippet: CDK7 regulation of RNA Polymerase II phosphorylation and super-enhancer gene expression (A) Protein analysis for phosphorylated RNA polymerase II (serine-2, serine-5, and serine-7), CDK2 and CDK7 was performed and phosphorylation levels were quantified using ImageJ. Shown are two separate biological replicates for each line. Statistically significant differences between HepG2 and H33 are shown ( t test, ∗ p < 0.05). (B) RNA sequencing of HepG2 cells and H33 cells treated at varying doses of SY-5609 for 24 h ( n = 6 biological replicates per group) were evaluated for prominent super-enhancer-associated genes, including SLC16A14 and LINC00473 . Findings from the H33 clone were confirmed in a separate DNAJB1-PRKACA -expressing clone (H12) ( t test, ∗∗ p < 0.01). (C) DNAJB1-PRKACA -expressing H33 cells were treated with a selective CDK7 inhibitor, SY5609 (100 nM, 1 μM, and 10 μM), or DMSO control (0) for 24 h ( n = 3 biological replicates per group, two representative images shown per group for western blot). Known substrate targets of CDK7 were assessed including RNA Pol II CTD (Ser 2, 5, and 7), Thr160 phosphorylated CDK2 (pCDK2) and Thr170 phosphorylated CDK7 (pCDK7) ( t test, ∗ p < 0.05). (D and E) To assess for CDK7-dependent expression of FLC-specific genes, H33 cells were treated with SY-5609 (1–300 nM) for 24 h and levels of mRNA expression (RT-qPCR) versus DMSO control were evaluated, including SLC16A14 and LINC00473 . This was repeated with a separate covalent-binding selective and specific CDK7 inhibitor (YKL-5-124). Shown are three biological replicates per drug dose per mRNA with statistical significance denoted as compared to DMSO control ( t test, ∗ p < 0.05, ∗∗ p < 0.01).

Article Snippet: phospho-CDK2 (Thr160) , Cell Signaling Technology , Cell Signaling Technology #2561; RRID: AB_2078685.

Techniques: Phospho-proteomics, Gene Expression, RNA Sequencing, Expressing, Control, Western Blot, Quantitative RT-PCR, Binding Assay