Journal: Journal of Cardiothoracic Surgery

Article Title: POLR1D silencing suppresses lung cancer cells proliferation and migration via inhibition of PI3K-Akt pathway

doi: 10.1186/s13019-024-02791-y

Figure Lengend Snippet: Expression of POLR1D in lung cancer cells. a The mRNA expression of POLR1D in human embryonic lung fibroblasts (HFL1) and five-lung cancer cell lines (H2170, SK-MES-1, H226, PC-9, H1975) by RT‑qPCR. b The representative bands of POLR1D protein in six-lung cancer cell lines. c Quantification of POLR1D protein expression determined by Western blot. Data represent the average of three independent experiments (mean ± SD). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. HFL1 group

Article Snippet: Human embryonic lung fibroblasts (HFL1) and lung cancer cell lines (H2170, H226, SK-MES-1, PC-9, and H1975) were obtained from the American Type Tissue Culture Collection (ATCC; Rockville, MD, USA).

Techniques: Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: To senesce or not to senesce: how primary human fibroblasts decide their cell fate after DNA damage

doi:

Figure Lengend Snippet: Time series for G1-S arrest and senescence for different γ-irradiation regimes in MRC5 fibroblasts. ( A ) Relative abundance of EdU positive cells (mean ± SEM (n≥3)). ( B ) Population doublings (mean ± SEM for at least three independent cell counts with >100 cells each) ( C ) SA-βG activity (mean ± SEM (n=3)). ( D ) Percentage of live cells (negative for both Annexin V and propidium iodide (PI), early apoptotic cells (positive for Annexin V and negative for PI), late apoptotic/necrotic cells (positive for both Annexin V and PI) and dead cells (negative for Annexin V and positive for PI (mean ± SE (n=3)). Representative FACS scatter plots for EdU and SA-βG measurements are shown in .

Article Snippet: MRC5 primary human embryonic lung fibroblasts (ATCC, Cat. No. CCL-171TM) at population doubling ~22 were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% foetal bovine serum (FBS)(Gibco), 100 units/ml MEM non-essential amino acids solution (Gibco) and 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco).

Techniques: Irradiation, Activity Assay

Journal: Aging (Albany NY)

Article Title: To senesce or not to senesce: how primary human fibroblasts decide their cell fate after DNA damage

doi:

Figure Lengend Snippet: Dynamics of p16, Rb, Cyclin A2 and Cdc25A expression after γ-irradiation in MRC5 human fibroblasts. ( A ) Relative p16 abundance. ( B ) Relative Rb1(Ser780) abundance ( C ) Total and hypo-phosphorylated Rb1 protein for 2.5 Gy IR. ( D ) Total and hypo-phosphorylated Rb1 protein for 10 Gy IR. ( E ) Relative Cyclin A2 abundance ( F ) Relative Cdc25A abundance. Error bars indicate SEM (n≥3). Representative Western Blots are shown in .

Article Snippet: MRC5 primary human embryonic lung fibroblasts (ATCC, Cat. No. CCL-171TM) at population doubling ~22 were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% foetal bovine serum (FBS)(Gibco), 100 units/ml MEM non-essential amino acids solution (Gibco) and 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco).

Techniques: Expressing, Irradiation, Western Blot

Journal: Aging (Albany NY)

Article Title: To senesce or not to senesce: how primary human fibroblasts decide their cell fate after DNA damage

doi:

Figure Lengend Snippet: G1-S dynamics and model fits in MRC5 cells after IR. ( A ) Relative abundance of EdU positive cells and simulated active Cyclin E/Cdk2 complex ( CycECdk2-a in panel F). ( B ) Measured and simulated relative total p21 abundance ( p21 in F).( C ) Measured and simulated relative total Cyclin E1 abundance ( CycE + CycE/Cdk2 + CycE/Cdk2-a in panel F). ( D ) Measured and simulated relative total Cdk2 abundance ( Cdk2 + CycE/Cdk2 + CycE/Cdk2-a in panel F). ( E ) Measured and simulated relative phosphorylated (Thr160) Cdk2 abundance ( CycECdk2 + CycECdk2-a in panel F). ( F ) Wiring scheme of the best approximating p21-dependent G1-S transition model. ( G ) Steady state analysis of active Cdk2 ( CycE/Cdk2-a in F of the parameterized combined DNA damage-G1-S arrest model as a function of DNA damage response (DDR), i.e. γH2AX foci, including free parameter perturbations by sampling 50 times from a uniform distribution within an interval of plus/minus 20% around the original parameter value. Solid line: Stable steady state of CycE/Cdk2-a of the parameterized model as a function of DNA damage (DDR). Light gray region: 5-95% of stable steady states of CycE/Cdk2-a of the parameterized model with perturbed free parameters. Dark gray region: First to third quartile of steady states of CycE/Cdk2-a of the parameterized model with perturbed free parameters. Inset : Steady state γH2AX foci, i.e. BASE+TAF from , as a function of IR [Gy]. A - D : Lines indicate simulations of the fitted model. Symbols indicate mean measured values ± SEM (n≥3) scaled to day 0. Representative Western Blots are shown in . The corresponding data are provided in -13.

Article Snippet: MRC5 primary human embryonic lung fibroblasts (ATCC, Cat. No. CCL-171TM) at population doubling ~22 were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% foetal bovine serum (FBS)(Gibco), 100 units/ml MEM non-essential amino acids solution (Gibco) and 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco).

Techniques: Sampling, Western Blot

Journal: Aging (Albany NY)

Article Title: To senesce or not to senesce: how primary human fibroblasts decide their cell fate after DNA damage

doi:

Figure Lengend Snippet: p21 silencing 11 days after IR with sample collection 2 days later (13d). ( A ) relative p21 levels. ( B ) relative EdU incorporation. ( C ) relative Cdk2 levels. All values are scaled to day 0. Light and dark gray bars indicate 10 and 20 Gy radiation, respectively. Error bars indicate standard error of the mean (SEM) (n≥3). ( D ) representative western blot demonstrating validation of the p21 silencing in irradiated MRC5 cells (20 Gy).

Article Snippet: MRC5 primary human embryonic lung fibroblasts (ATCC, Cat. No. CCL-171TM) at population doubling ~22 were cultured in Dulbecco's modified Eagle's medium (D-MEM) supplemented with 10% foetal bovine serum (FBS)(Gibco), 100 units/ml MEM non-essential amino acids solution (Gibco) and 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco).

Techniques: Western Blot, Biomarker Discovery, Irradiation

Journal: Science advances

Article Title: Restructuring of the plasma membrane upon damage by LC3-associated macropinocytosis.

doi: 10.1126/sciadv.abg1969

Figure Lengend Snippet: Fig. 1. Laser injury leads to ATG7-dependent formation of LC3-positive vesicles around the plasma membrane repair area. (A) MCF7 cells were injured by ablation laser in medium with or without Ca2+. Mean ± SEM of normalized FM1-43 cytoplasmic dye levels, 11 cells per condition from three experiments, P < 0.0001 (AUC analysis, unpaired t test with Welsh’s correction). Black arrow, injury time point. (B) Representative images for FM1-43 intensity of MCF7 cells injured in Ca2+-containing medium (left) and Ca2+-deficient medium (right) before and after injury (61 s). White arrows, injury site. Blue arrows point to FM1-43 dye accumulation. (C) Representative sequen- tial images of ANXA4-tRFP–transfected MCF7 eGFP-LC3 cells exposed to laser injury. Yellow arrow, LC3 puncta formation. White circles, eGFP-LC3 quantification. Also see movie S1. (D) eGFP-LC3 intensity after laser injury in injured areas compared to control. Fifteen individual cells from three experiments (mean ± SEM, paired t test of AUC values). Black arrow, injury time point. (E) Immunoblot for ATG7, ATG5, and the ATG5/ATG12 complex (Hsp90, loading control) of lysates from MCF7 eGFP-LC3 CRISPR KO cells: nontargeted control, ATG5 CRISPR KO, and ATG7 CRISPR KO. (F) Cell membrane repair kinetic upon laser injury in ATG7 CRISPR KO and Ctrl cells as measured by FM4-64 cytoplasmic dye levels from 11 cells per cell line (from three experiments). Mean ± SEM. Black arrow, injury time point. (G) Analysis of eGFP-LC3 intensity at the injury site in MCF7 ATG7 CRISPR KO and Ctrl cells (mean ± SEM, 16 cells from three experiments per cell line, unpaired t test of AUC values, Welsh’s correction). (H) Repre- sentative sequential images of eGFP-LC3 puncta formation in Ctrl (left) and in ATG7 CRISPR KO cells (right) after laser injury. White arrows indicate injury sites, yellow ar- rows indicate area of LC3 puncta formation, and white circles indicate the area used for eGFP-LC3 quantification. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: ANXA4-tRFP had been generated by cloning ANXA4 (Origene, #RG203229) into the pCMV6-AC-tRFP vector (Origene, #PS100034) (2).

Techniques: Clinical Proteomics, Membrane, Transfection, Control, Western Blot, CRISPR