imr90 human lung fibroblast  (ATCC)

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: IMR90 primary human lung fibroblasts (ATCC CCL-186) were cultured as described previously in Dulbecco’s Modified Eagle Medium (DMEM; Gibco 10313-121) supplemented with 10% fetal bovine serum (FBS; Corning 35-010-CV), 1% penicillin-streptomycin (Gibco 15140-122), and 2 mM glutamine (Gibco 25030-081).

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, Experimental design for siRNA knockdown experiment: IMR90 fibroblasts were induced senescent by ionizing radiation (IR) and transfected with siRNAs targeting CCND1, CDK4, CDK6, or both CDK4 and CDK6. Non-targeting controls (siNT1, siNT2) and proliferating (non-irradiated) cells were included. b, Experimental design for Palbociclib experiment: IMR90 fibroblasts were induced senescent by IR and treated with DMSO or Palbociclib. Proliferating (non-irradiated) controls were included. c–d, Western blots validating knockdown of CCND1, CDK4 and CDK6. e, Heatmap of all differentially expressed genes across siRNA conditions. A red box highlights a senescence-associated gene cluster selectively suppressed by CCND1 or CDK6 knockdown. f, Top enriched pathways in the red-box cluster include interferon-α response, TNFα signaling via NFκB, and interferon-γ response. g–h, Expression of SASP and ISG genes that are differentially expressed between proliferating cells and senescent siNT controls. i, Heatmap of all differentially expressed genes across Palbociclib conditions, with a red box indicating a cluster suppressed by Palbociclib treatment. j, Enrichment analysis of the Palbociclib-suppressed cluster shows reduced interferon-α response, TNFα signaling via NFκB, and interferon-γ response — the same top pathways as in f. k–l, Expression of SASP and ISG genes differentially expressed between proliferating and senescent DMSO-treated controls, showing suppression with Palbociclib. m–n , qPCR validation of SASP and ISG repression following Palbociclib treatment. Gene expression values are shown as fold change relative to senescent DMSO-treated controls, normalized to the geometric mean of GAPDH and RPL13. Each biological replicate represents an independent irradiation. Error bars denote mean ± s.d. Statistical analysis was performed using two-way ANOVA with Tukey’s post hoc test. P < 0.05 was considered significant.

Article Snippet: IMR90 primary human lung fibroblasts (ATCC CCL-186) were cultured as described previously in Dulbecco’s Modified Eagle Medium (DMEM; Gibco 10313-121) supplemented with 10% fetal bovine serum (FBS; Corning 35-010-CV), 1% penicillin-streptomycin (Gibco 15140-122), and 2 mM glutamine (Gibco 25030-081).

Techniques: Knockdown, Transfection, Irradiation, Western Blot, Expressing, Biomarker Discovery, Gene Expression

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: IMR90 primary human lung fibroblasts (ATCC CCL-186) were cultured as described previously in Dulbecco’s Modified Eagle Medium (DMEM; Gibco 10313-121) supplemented with 10% fetal bovine serum (FBS; Corning 35-010-CV), 1% penicillin-streptomycin (Gibco 15140-122), and 2 mM glutamine (Gibco 25030-081).

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