mrc5 primary human lung fibroblasts  (ATCC)

Journal: Molecular Medicine Reports

Article Title: Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence

doi: 10.3892/mmr.2022.12861

Figure Lengend Snippet: Senescence markers in MRC5 cells. MRC5 cells were cultured for 2, 4, 6 and 8 weeks to induce replicative senescence in vitro . (A) SA-β-galactosidase staining of non-senescent (2 and 4 weeks) and senescent MRC5 cells (6 and 8 weeks). Arrows indicate positive blue staining for SA-β-galactosidase. After staining, cells were imaged using phase-contrast microscopy (40× magnification). (B) Quantitative analysis of SA-β-stained cells. Each measurement was performed in triplicate. Total mRNA expression levels of (C) lamin B and (D) CDKN2A/p16 were assessed by reverse transcription-quantitative PCR. Values were normalized to GAPDH mRNA expression. ***P<0.0002 and ****P<0.0001 vs. 2 weeks. SA-β-gal, senescence-associated β-galactosidase; CDKN2A, cyclin dependent kinase inhibitor 2A.

Article Snippet: Primary human lung fibroblast MRC5 cells (derived from 14-week-old male fetus normal lung tissue) were purchased from American Type Culture Collection.

Techniques: Cell Culture, In Vitro, Staining, Microscopy, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence

doi: 10.3892/mmr.2022.12861

Figure Lengend Snippet: Histone acetylation of CDKN2A/p16 promoter region changes in senescent MRC5 cells. MRC5 cultured cells at 4 (n-sc) and 8 weeks (sc) were collected and chromatin immunoprecipitation assay was performed using antibodies against (A) H3K9Ac, (B) H3K18Ac and (C) H3K56Ac. Data are presented as % input ± SEM using normal IgG as a specific control. **P<0.01 vs. n-sc. CDKN2A, cyclin dependent kinase inhibitor 2A; sc, senescent cell; n-sc, non-sc.

Article Snippet: Primary human lung fibroblast MRC5 cells (derived from 14-week-old male fetus normal lung tissue) were purchased from American Type Culture Collection.

Techniques: Cell Culture, Chromatin Immunoprecipitation, Control

Journal: Molecular Medicine Reports

Article Title: Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence

doi: 10.3892/mmr.2022.12861

Figure Lengend Snippet: SIRT7 binding to the CDKN2A/p16 promoter is lost in senescent MRC5 cells. MRC5 cells were cultured at 4 (n-sc) and 8 weeks (sc) and chromatin samples were collected. Chromatin immunoprecipitation assay was performed using antibodies against (A) SIRT1, (B) SIRT2, (C) SIRT6 and (D) SIRT7 chromatin-modifying proteins. Data are presented as % input ± SEM using normal IgG as a specific control. **P<0.01, ***P<0.001, ****P<0.0001 vs. n-sc. sc, senescent cell; n-sc, non-sc; SIRT; sirtuin.

Article Snippet: Primary human lung fibroblast MRC5 cells (derived from 14-week-old male fetus normal lung tissue) were purchased from American Type Culture Collection.

Techniques: Binding Assay, Cell Culture, Chromatin Immunoprecipitation, Control

Journal: Molecular Medicine Reports

Article Title: Transcriptional regulation of CDKN2A/p16 by sirtuin 7 in senescence

doi: 10.3892/mmr.2022.12861

Figure Lengend Snippet: SIRT7 knockdown deacetylates the CDKN2A/p16 promoter and upregulates p16 mRNA expression levels in senescent MRC5 cells. Non-senescent MRC5 cultured cells (2 weeks) were transfected with si-SIRT7. Effective downregulation was demonstrated using RT-qPCR and western blotting 48 h post-transfection. (A) SIRT7 and (B) CDKN2A/p16 mRNA expression levels were assessed using qPCR 48 h after transfection and normalized against GAPDH mRNA expression levels. ****P<0.0001 (C) SIRT7 Protein expression was assessed by Western blot. TFIIB protein was used as a loading control. Chromatin immunoprecipitation assay was performed using antibodies against (D) SIRT7 or (E) H3K18Ac. Data are presented as % input ± SEM using normal IgG as a specific control. ***P<0.001 vs. siCtrl. SIRT; sirtuin; CDKN2A, cyclin dependent kinase inhibitor 2A; si, small interfering; Ctrl, control; RT-qPCR, reverse transcription-quantitative PCR; TFIIB, transcription factor IIB).

Article Snippet: Primary human lung fibroblast MRC5 cells (derived from 14-week-old male fetus normal lung tissue) were purchased from American Type Culture Collection.

Techniques: Knockdown, Expressing, Cell Culture, Transfection, Quantitative RT-PCR, Western Blot, Control, Chromatin Immunoprecipitation, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Constitutive TRIM22 Expression in the Respiratory Tract Confers a Pre-Existing Defence Against Influenza A Virus Infection

doi: 10.3389/fcimb.2021.689707

Figure Lengend Snippet: TRIM22 is differentially expressed in a cell-type dependent manner. (A–C) Primary and hTERT immortalized human lung fibroblast (MRC5 and MRC5t, respectively), lung adenocarcinoma epithelial (A549), and SV40-transformed human kidney epithelial (HEK 293T; 293T) cells were treated with (+) or without (-) IFN-β (100 IU/ml) for 24 h. (A) Western blots of WCLs probed for TRIM22 expression. Actin is shown as a loading control. (B, C) qRT-PCR quantitation of TRIM22 and Mx1 mRNA transcript expression levels, respectively. Values normalized to MRC5t cells without IFN treatment (dotted line); n=3, means and SD shown. (D) Confocal microscopy images of MRC5t and A549 cells with or without IFN treatment (as in A ). TRIM22 was labelled by indirect immunofluorescence. Nuclei were stained with DAPI. (E) MRC5t and A549 cells were mock-treated, IFN- β (100 IU/ml) stimulated or infected with IAV (A/WSN/1933(H1N1), WSN) at a MOI of 1 PFU/cell (based on MDCK titres) for the indicated times (hours; h). WCLs were analyzed by western blots for TRIM22, Mx1, and viral protein (NP and NS1) expression. Actin is shown as a loading control. (F) A549 cells were infected with IAV (WSN; MOI of 0.01 PFU/cell based on MDCK titres) and harvested at the indicated times prior to western blotting (as in E ).

Article Snippet: Primary human foetal lung fibroblast (MRC5) cells were purchased from the European Collection of Authenticated Cell Cultures (ECACC; 05072101).

Techniques: Transformation Assay, Western Blot, Expressing, Control, Quantitative RT-PCR, Quantitation Assay, Confocal Microscopy, Immunofluorescence, Staining, Infection