Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 1. Differential expression and roles for the IFI16 and AIM2 proteins during cellular senescence of HDFs. A, total cell extracts prepared from young (Y), old (O), or senescent (S) WI-38 HDFs were analyzed by immunoblotting using antibodies specific to the indicated proteins. B, total RNA isolated from young (Y), old (O), or senescent (S) WI-38 HDFs were analyzed by semiquantitative PCR (left) or quantitative real-time PCR (right) for the indicated genes. The ratio of the test gene to actin mRNA was calculated in units (1 unit being the ratio of the test gene to actin mRNA). The relative steady-state levels of IFNB mRNA in young HDFs are indicated as 1. Results are mean values of triplicate experiments, and error bars represent SD (*,P < 0.05; **, P < 0.01). C, subconfluent cultures of young (Y) or old (O) WI-38 HDFs were infected with Cignal Lenti ISRE Reporter (Luc) virus as suggested by the supplier. The infected cells were harvested after 40 to 44 hours to assays for the firefly and Renilla luciferase activities as described in Materials and Methods. Normalized relative firefly luciferase activity is shown.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Quantitative Proteomics, Western Blot, Isolation, Real-time Polymerase Chain Reaction, Infection, Virus, Luciferase, Activity Assay

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 2. The knockdown of the IFNa/bRa subunit expression in HDFs decreases IFI16 expression and delays the onset of a senescence phenotype. A, subconfluent cultures of young WI-38 HDFs infected with control lentivirus (control) or virus encoding for shIFNa/bRa RNA (shIFNa/bRa) were either left untreated or treated with IFN-a for 18 hours. After the treatment, total cell extracts were prepared and analyzed by immunoblotting using antibodies specific to the indicated proteins. B, control or shIFNa/bRa HDFs described in A were either left untreated or treated with IFN-b for 18 hours. Total cell extracts were analyzed by immunoblotting. C, total cell extracts from control or shIFNa/ bRa HDFs were analyzed by immunoblotting for the indicated proteins. D, phase-contrast photographs indicating morphologic changes (top) and differences in the number of SA-b-gal–positive cells (bottom) between control HDFs and HDFs after the knockdown of the IFNa/ bRa subunit expression.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Knockdown, Expressing, Infection, Control, Virus, Western Blot

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 3. DNA damage response also contributes to the constitutive levels of the IFI16 protein. A, subconfluent cultures of young WI-38 or IMR-90 HDFs were either left untreated (lanes 1 and 4) or treated with increasing units of bleomycin (lanes 2, 3, 5, and 6) for 24 hours. After the treatment, total cell extracts were analyzed by immunoblotting for the indicated proteins. B, subconfluent cultures of young WI-38 were either left untreated (lane 1) or treated with increasing units of bleomycin (lanes 2 and 3) for 24 hours. After the treatment, total cell extracts were analyzed by immunoblotting for the indicated proteins. C, subconfluent cultures of young WI-38 were either left untreated or treated with bleomycin for 24 hours. After the treatment, total RNA was analyzed by quantitative real-time PCR for IFI16 and AIM2 mRNA levels. The ratio between the test gene (the IFI16 or AIM2) mRNA levels to actin mRNA was calculated in units (1 unit being the ratio of the test gene to actin mRNA). The relative steady-state levels of IFI16 mRNA in control HDFs are indicated as 1.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 4. Constitutively increased levels of the IFI16 protein in ATM HDFs are associated with the activation of IFN signaling. A, subconfluent cultures of young WI-38 or ATM HDFs were either left untreated or treated with IFN-b for 18 hours. After the treatment, total RNA was extracted and analyzed by quantitative real-time PCR for steady-state levels of IFI16 mRNA. The ratio between the test gene IFI16 mRNA levels to actin mRNA was calculated in units (1 unit being the ratio of the test gene to actin mRNA). The relative steady-state levels of IFI16 mRNA in WI-38 control HDFs are indicated as 1. B, subconfluent culture of young WI-38 or ATM HDFs was transfected with either ISRE-luc or IFI16-luc reporter plasmid (1.8 mg) along with a second pRL-TK reporter (0.2 mg). The transfected cells were harvested after 40 to 44 hours to conduct assays for the firefly and Renilla luciferase activities as described in Materials and Methods. Normalized relative firefly luciferase activity in WI-38 cells is shown as 1. C, subconfluent cultures of young WI-38 or ATM HDFs were either left untreated (lanes 1 and 3) or treated with IFN-b (1,000 units/mL) for 18 hours. After the treatment, total cell extracts were analyzed by immunoblotting for the indicated proteins. D, subconfluent cultures of young WI-38 or ATM HDFs were either left untreated (lanes 1 and 3) or treated with IFN-b (1,000 units/mL) for 18 hours. After the treatment, total cell extracts were analyzed by immunoblotting for the indicated proteins. E, subconfluent cultures of young WI-38 HDFs that were infected with control virus (Vector) or shATM virus (shATM) were left untreated (lanes 1 and 4), treated with IFN-b (lanes 2 and 5), or treated with bleomycin (lanes 3 and 6) for 18 hours. After the treatment, total cell extracts were analyzed by immunoblotting for the indicated proteins.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Control, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Infection, Virus

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 5. Prolonged IFN-b treatment of young HDFs increases levels of IFI16, AIM2, and proinflammatory cytokine IL-1b. A and B, for treatment of young WI-38 HDFs with IFN-b, cultures at approximately 20% confluence were treated with IFN-b for the indicated days (medium was changed after every 2 days and fresh IFN-b was added). After the treatment, HDFs were harvested and total cell lysates were analyzed by immunoblotting for the levels of the indicated proteins. C, cultures of young WI-38 were either left untreated or treated with the indicated amount of the human recombinant IL-b for 18 hours. After the treatment, total RNA was extracted and analyzed by quantitative real-time PCR for steady-state levels of IFI16 (left) or AIM2 (right) mRNA. The ratio between the test gene (the IFI16 or AIM2) mRNA levels to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). The relative steady-state levels of IFI16 or AIM2 mRNA in WI-38 control HDFs are indicated as 1.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Western Blot, Recombinant, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 6. IFI16 protein is primarily detected in the cytoplasm of old or IFN-b–treated HDFs, and nucleofection of young HDFs with dsDNA induces the IFN-b expression. A, cultures of young (PDL 30) proliferating or old (PDL 50) WI-38 HDFs were harvested, and cells were subjected to the nuclear and cytoplasmic fractionation. The fractions containing equal amounts of proteins were analyzed by immunoblotting using antibodies specific to the indicated proteins. B, cultures of young proliferating WI-38 HDFs were either left untreated or treated with IFN-b for 24 hours. After the treatment, cells were subjected to the nuclear and cytoplasmic fractionation. The fractions containing approximately equal amounts of proteins were analyzed by immunoblotting. C, young proliferating (PDL 30) WI-38 HDFs were nucleofected without DNA (control) or with the indicated amounts of plasmid DNA (GFP plasmid) as described in Materials and Methods. After 18 hours of nucleofection, total RNA was extracted and analyzed by quantitative real-time PCR for steady-state levels of IFNB mRNA. The ratio between the IFNB mRNA levels to actin mRNA was calculated in units (1 unit being the ratio of the IFNB mRNA to actin mRNA). The relative steady-state levels of IFNB mRNA in control HDFs are indicated as 1. D, young proliferating WI-38 HDFs were either left without nucleofection or nucleofected without DNA or nucleofected with 2 mg of plasmid DNA. After 18 hours of nucleofection, total cell lysates were analyzed by immunoblotting for the indicated proteins.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Expressing, Fractionation, Western Blot, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 8. Knockdown of IFI16 expression in HDFs reduces the constitutive and dsDNA-induced activation of IFN signaling. A, total cell extracts prepared from the young WI-38 HDFs that were infected with control virus (Vector) or shIFI16 virus (shIFI16) were analyzed by immunoblotting for the indicated proteins. B, young WI-38 HDFs that were infected with control virus (Vector) or shIFI16 virus (shIFI16) were either nucleofected without (lanes 1 and 3) or with (lanes 2 and 4) dsDNA. Eighteen hours after nucleofections, cells were harvested and total cell extracts were analyzed by immunoblotting for the indicated proteins.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques: Knockdown, Expressing, Activation Assay, Infection, Control, Virus, Plasmid Preparation, Western Blot

Journal: Molecular Cancer Research

Article Title: Differential Roles for the Interferon-Inducible IFI16 and AIM2 Innate Immune Sensors for Cytosolic DNA in Cellular Senescence of Human Fibroblasts

doi: 10.1158/1541-7786.mcr-10-0565

Figure Lengend Snippet: Figure 9. Proposed roles of the IFI16 and AIM2 proteins in cellular senescence-associated cell growth arrest and secretory phenotype in HDFs. CASP-1, caspase-1.

Article Snippet: To knockdown the expression of the indicated genes in young WI-38 HDFs, subconfluent cultures of cells (in a 6-well plate) were infected with lentivirus (purchased from Santa Cruz Biotechnology) encoding short hairpin RNA (shRNA) to the IFI16 (sc-35633-V), AIM2 (sc-88166-V), IFNa/bRa (sc-35637-V), or ATM (sc-29761-V) gene.

Techniques:

Journal: Molecular Metabolism

Article Title: Mitochondrial protein deacetylation by SIRT3 in osteoclasts promotes bone resorption with aging in female mice

doi: 10.1016/j.molmet.2024.102012

Figure Lengend Snippet: SIRT3-induced ATPIF1 deacetylation activates osteoclast mitophagy in aged mice. (A–E, not B) BMMs from 24-month-old female C57BL/6 mice transduced with lentiviral vectors expressing control sh-RNA or sh-RNAs targeting the hyperacetylated proteins and cultured with RANKL for 5 days (A and C) or 3 days (D–G). (A) Representative pictures (left) and number (right) of TRAP + multinucleated osteoclasts and Von Kossa–stained bone biomaterial surface. (triplicate cultures). Scale bar: 500 μm. (B) BMMs were isolated from 24-month-old female C57BL/6 mice and were cultured with M-CSF (30 ng/mL) and RANKL (30 ng/mL) for 3 days. Western blot analysis of expression of ATPIF1. (triplicate cultures). (C) Levels of mRNA of an osteoclast marker in cultured osteoclasts measured by qPCR. (triplicate cultures). (D–F) Different fractions of mitochondrial respiration per cell measured with Seahorse (n = 10–14 wells/group). (G) Western blot analysis of expression of mitophagy marker. (triplicate cultures). Line and error bars represent mean ± SD. P values were determined using Student's t-test. All in vitro assays were performed in cultured BMMs pooled from more than 3 mice and repeated at least twice.

Article Snippet: Twenty-four hours later, non-adherent cells were submitted to a Ficoll-Hypaque gradient (Sigma–Aldrich), and cells at the interface were cultured in the presence of 30 ng/mL M-CSF, 8 μg/mL polybrene (Santa Cruz Biotechnology), and the lentiviral particles for 16 h. We further cultured the infected bone marrow macrophages with M-CSF for 24 h and then added 2 μg/mL puromycin (Santa Cruz Biotechnology) for 48 h to remove uninfected cells.

Techniques: Transduction, Expressing, Control, Cell Culture, Staining, Isolation, Western Blot, Marker, In Vitro