mouse anti cdkn1a p21  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

doi: 10.64898/2026.03.18.712515

Figure Lengend Snippet: (A-D) The analysis of male 19 months WT or Gzmk -/- mice (male). (A) Staining of SA-β-gal in liver and hippocampus. (B) H&E staining in liver, lung and kidney. (C) SA-β-gal staining for WAT from indicated mice. (D) Western blot analysis of the expression of p16, p21 and p53 in liver. (E) The bioluminescence imaging of indicated mice (15 months, female) by injecting luciferase substrates. (F-G) CD8 T cells (4 x10 6 ) from old or old Gzmk -/- mice were adoptively transferred into young p16 Ink4a -luciferase reporter mice, one month later, the luciferase activity was measured (F), and the expression of the p16 -driven luciferase reporter in indicated tissues were measured by RT-qPCR.

Article Snippet: Primary antibodies used in this study: Mouse anti-CDKN2A/p16 (Santa Cruz Biotechnology, cat# sc-1661, 1:1000) Mouse anti-CDKN1A p21 (Santa Cruz Biotechnology, cat# sc-6246, 1:1000) Mouse anti-p53 (Santa Cruz Biotechnology, cat# sc-98, 1:1000) Mouse anti-GAPDH (Absin, abs830030,1:10000) Rabbit anti-γH2A.X (Cell Signaling Technology, cat# 9718S, 1:1000)

Techniques: Staining, Western Blot, Expressing, Imaging, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

doi: 10.64898/2026.03.18.712515

Figure Lengend Snippet: (A, B) Primary human bladder fibroblasts were isolated from surgical excision from bladder cancer patients and subjected to GZMK treatment. (A) HBFs were treated with 100nM GZMK for 5 days, and SA-β-gal staining were performed. (B) HBFs were treated with 100nM GZMK in medium without FBS for one day, IL-6 and TNFα in supernatant were measured by ELISA. (C, D) MEFs were isolated and subjected to GZMK treatment as same with . (E-H) BMDMs were subjected to Gzmk or indicated inhibitors treatment. (E) BMDMs were stimulated with Gzmk for 30 minutes and phosphorylated ERK and p38 were determined by flow cytometry. (F) BMDMs were cultured with Gzmk or combined with indicated inhibitors for three days, SA-β-gal staining were performed. BMDMs were cultured with Gzmk or combined with indicated inhibitors for one day, expression of P16 (G) and P21 (H) were analyzed by flow cytometry, IL-6 and TNFα (I) in supernatant were measured by ELISA.

Article Snippet: Primary antibodies used in this study: Mouse anti-CDKN2A/p16 (Santa Cruz Biotechnology, cat# sc-1661, 1:1000) Mouse anti-CDKN1A p21 (Santa Cruz Biotechnology, cat# sc-6246, 1:1000) Mouse anti-p53 (Santa Cruz Biotechnology, cat# sc-98, 1:1000) Mouse anti-GAPDH (Absin, abs830030,1:10000) Rabbit anti-γH2A.X (Cell Signaling Technology, cat# 9718S, 1:1000)

Techniques: Isolation, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Culture, Expressing

Journal: bioRxiv

Article Title: A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

doi: 10.64898/2026.03.18.712515

Figure Lengend Snippet: Old mice (19 months) were administrated with SCH or PPACK for one-month. The level of IL-6 and TNFα (A), ALT and AST (B) in plasma from indicated mice were determined by ELSIA. (C) Staining of SA-β-gal in liver. (D) H&E staining of lungs from indicated mice. (E) Staining of SA-β-gal in hippocampus from indicated mice. (F-G) Western blot analysis of P16, P21 and P53 at liver and brain from indicated mice.

Article Snippet: Primary antibodies used in this study: Mouse anti-CDKN2A/p16 (Santa Cruz Biotechnology, cat# sc-1661, 1:1000) Mouse anti-CDKN1A p21 (Santa Cruz Biotechnology, cat# sc-6246, 1:1000) Mouse anti-p53 (Santa Cruz Biotechnology, cat# sc-98, 1:1000) Mouse anti-GAPDH (Absin, abs830030,1:10000) Rabbit anti-γH2A.X (Cell Signaling Technology, cat# 9718S, 1:1000)

Techniques: Clinical Proteomics, Staining, Western Blot

Journal: Archives of Pharmacal Research

Article Title: cis -Clerodane-type diterpenoids from Tinospora crispa and their anticancer potential

doi: 10.1007/s12272-026-01596-y

Figure Lengend Snippet: Effects of compounds 1 – 5 on cancer signaling pathways in liver and lung cancer cell lines. A Hepa1c1c7, B Hepa1-6, C LLC1, and D A549 cells were treated with 100 μM of compounds 1 – 5 (A = 5 , B = 2 , C = 1 , D = 3 , E = 4 ) for 24 h. In liver cancer models, compounds 1 and 2 increased SAV1 and p-LATS1, while compound 3 reduced YAP, TAZ, and pan-TEAD expression. Compounds 1 , 2 , 4 , and 5 elevated p-YAP. In lung cancer models, all compounds suppressed p-AKT; compounds 3 and 4 most reduced p-AKT, STAT3, Cyclin D1 and increased p21

Article Snippet: Membranes were blocked with 2% skim milk in TBST at 4 °C and then incubated overnight at 4 °C with the following primary antibodies: MST1 (1:1000, #14,946, Cell Signaling Technology), SAV1 (1:1000, #13301S, Cell Signaling Technology), MOB1 (1:1000, #13730S, Cell Signaling Technology), LATS1 (1:1000, #3477S, Cell Signaling Technology), phospho-YAP (p-YAP; 1:1000, #4911S, Cell Signaling Technology), YAP (1:1000, #14,074, Cell Signaling Technology), TAZ (1:1000, #83669S, Cell Signaling Technology), pan-TEAD (1:1000, #13295S, Cell Signaling Technology), phospho-AKT (p-AKT; 1:1000, #9275S, Cell Signaling Technology), AKT (1:1000, #9272S, Cell Signaling Technology), phospho-ERK1/2 (p-ERK1/2; 1:1000, # PAB16949 , Abnova), total ERK1/2 (1:1000, #9102S, Cell Signaling Technology), STAT3 (1:1000, #9139S, Cell Signaling Technology), Cyclin D1 (1:1000, sc-8396, Santa Cruz Biotechnology), Waf1/Cip1/CDKN1A p21 (1:1000, sc-53870, Santa Cruz Biotechnology), Bax (1:1000, sc-23959, Santa Cruz Biotechnology), Bcl-2 (1:1000, sc-23960, Santa Cruz Biotechnology), Caspase-3 (1:1000, sc-56053, Santa Cruz Biotechnology), and E-cadherin (1:1000, #3195, Cell Signaling Technology).

Techniques: Protein-Protein interactions, Expressing

Journal: bioRxiv

Article Title: Targeting CyclinD1-CDK6 to Mitigate Senescence-Driven Inflammation and Age-Associated Functional Decline

doi: 10.1101/2025.08.01.668243

Figure Lengend Snippet: a, RNA-seq of IMR90 fibroblasts 10 days after ionizing radiation (IR) shows CCND1 among a small subset of proliferation-associated genes upregulated in senescence. b, Western blot time course (0–11 days post-IR) shows progressive accumulation of cyclin D1 protein in senescent cells. Ponceau staining was used as a loading control c, Genome browser tracks show CCND1 upregulation in replication-induced and oncogene-induced senescence. d, Western blot of senescent IMR90s shows increased CCND1 and CCND2, decreased CDK6, and no change in CDK4. Phosphorylated pRB (ppRB) and total pRB were both reduced. Senescence markers CCNA2, CCNB1 and LMNB1 also decreased, while CDKN1A and phospho-p65 (pp65) were increased. Total p65 was unchanged. Ponceau staining was used as a loading control. e , Representative immunofluorescence images of proliferating (Pro) and senescent (Sen) IMR90s. In senescent cells, CCND1 localizes to nuclei that are EdU-negative, ppRB–negative, and express high CDKN1A and IL-8. CCND1+ cells also exhibit cytoplasmic chromatin fragments (CCFs; γH2AX/DAPI-positive puncta), enlarged nuclei, and reduced Lamin B1 at the nuclear periphery. f, Quantification of: percentage of total nuclei that are CCND1+; percentage of CCND1+ nuclei that are EdU+, ppRB+ or CDKN1A+; number of CCFs per nucleus; and nuclear area. Each dot represents an independent biological replicate (separate irradiation). For immunofluorescence quantifications, each dot is the average of ≥3 technical replicates from the same irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using t-test. P < 0.05 was considered significant.

Article Snippet: The following primary antibodies were used: Cyclin D1 (Thermo Fisher Scientific, Cat# MA5-16356), Cyclin D2 (Cell Signaling Technology, Cat# 3741S), Cyclin A2 (Abcam, Cat# ab38), Cyclin B1 (Millipore, Cat# 4220), CDKN1A/p21 (Santa Cruz Biotechnology, Cat# sc-817), Lamin B1 (ProteinTech, Cat# 12987-1-AP), CDK4 (Cell Signaling Technology, Clone D93GE), CDK6 (Cell Signaling Technology, Cat# 13331), pp65 (Ser536) (Cell Signaling Technology, Cat# 3033S), p65 (Santa Cruz Biotechnology, Cat# sc-8008), STAT1 (Cell Signaling Technology, Cat# 9172S), phospho-STAT1 (Ser727) (Cell Signaling Technology, Cat# 9177S), pRb (Cell Signaling Technology, Cat# 9313S), ppRb (Ser807/811) (Cell Signaling Technology, Cat# 8516S), phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, Cat# 9718S), and 53BP1 (Millipore, Cat# MAB3802).

Techniques: RNA Sequencing, Western Blot, Staining, Control, Immunofluorescence, Irradiation

Journal: bioRxiv

Article Title: Targeting CyclinD1-CDK6 to Mitigate Senescence-Driven Inflammation and Age-Associated Functional Decline

doi: 10.1101/2025.08.01.668243

Figure Lengend Snippet: a, Representative comet assay images and quantification of comet tail length and olive moment in IR-induced senescent IMR90 fibroblasts, showing reduced DNA damage with Palbociclib treatment. b, Western blots show decreased 53BP1 and γH2AX protein levels following Palbociclib; Ponceau staining was used as a loading control. c, Representative immunofluorescence images and quantification of nuclear γH2AX intensity and CCF frequency, both reduced in Palbociclib-treated senescent cells. d, ELISA for 2′3′-cGAMP shows reduced cGAS–STING activation after Palbociclib. e–f, Immunoprecipitation–mass spectrometry (IP–MS) using two CCND1 antibodies identifies top CCND1 interactors in senescent IMR90s, shown as ranked bar plots of log10(LFQ+1) intensity comparing D1 IP versus IgG control. g, Co-immunoprecipitation confirms interaction between CCND1 and CDKN1A in senescent cells. h, Knockdown of CDKN1A increases nuclear γH2AX and CCF formation. i, Palbociclib rescues the elevated DNA damage and CCFs caused by CDKN1A knockdown. j, Representative immunofluorescence images and quantification showing that KIF4A knockdown reduces CCF frequency. k, qPCR analysis showing that KIF4A knockdown suppresses SASP and ISG gene expression. Expression values are shown as fold change relative to non-targeting control siRNA in senescent cells, normalized to the geometric mean of GAPDH and RPL13. Each biological replicate represents an independent irradiation. For immunofluorescence quantifications, each dot reflects the average of ≥3 technical replicates per irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using one-way ANOVA. P < 0.05 was considered significant.

Article Snippet: The following primary antibodies were used: Cyclin D1 (Thermo Fisher Scientific, Cat# MA5-16356), Cyclin D2 (Cell Signaling Technology, Cat# 3741S), Cyclin A2 (Abcam, Cat# ab38), Cyclin B1 (Millipore, Cat# 4220), CDKN1A/p21 (Santa Cruz Biotechnology, Cat# sc-817), Lamin B1 (ProteinTech, Cat# 12987-1-AP), CDK4 (Cell Signaling Technology, Clone D93GE), CDK6 (Cell Signaling Technology, Cat# 13331), pp65 (Ser536) (Cell Signaling Technology, Cat# 3033S), p65 (Santa Cruz Biotechnology, Cat# sc-8008), STAT1 (Cell Signaling Technology, Cat# 9172S), phospho-STAT1 (Ser727) (Cell Signaling Technology, Cat# 9177S), pRb (Cell Signaling Technology, Cat# 9313S), ppRb (Ser807/811) (Cell Signaling Technology, Cat# 8516S), phospho-Histone H2A.X (Ser139) (Cell Signaling Technology, Cat# 9718S), and 53BP1 (Millipore, Cat# MAB3802).

Techniques: Single Cell Gel Electrophoresis, Western Blot, Staining, Control, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Activation Assay, Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Knockdown, Gene Expression, Expressing, Irradiation