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

Journal: PLoS ONE

Article Title: IFI16 Protein Mediates the Anti-inflammatory Actions of the Type-I Interferons through Suppression of Activation of Caspase-1 by Inflammasomes

doi: 10.1371/journal.pone.0027040

Figure Lengend Snippet: ( A ) Puromycin-resistant THP-1 cells either infected with control lentivirus (lanes 1 an2) or the virus expressing the shIFI16 RNA (lanes 3–8; cell populations from three different wells) were either left untreated (lanes 1, 3, 5, 7) or treated with IFN-α for 14 h. After treatment, total cell extracts containing increased amounts of proteins (∼100 µg/lane) were analyzed for the constitutive and induced levels of IFI16 and actin proteins. A long exposure was taken to detect the signal in all lanes. ( B ) Control THP-1 cells (lanes 1 and 2) or cell population from well # 9 (lanes 3 and 4) as shown in the panel (a) were either left untreated (lanes 1 and 3) or treated with IFN-α for 14 h (lanes 2 and 4). After the treatment, total RNA was analyzed for the steady-state levels of mRNA for the indicated genes. ( C ) Control THP-1 cells (lanes 1–4) or cells infected with virus expressing the shIFI16 mRNA (lanes 5–8; population # 9) were either left untreated (lanes 1 and 5) or treated with IFN-β (1,000 u/ml; lanes 2 and 6), LPS (100 ng/ml; lanes 3 and 7), or dsRNA (10 µg/ml; lanes 4 and 8) for 14 h. After the treatment, total cell lysates containing equal amounts of protein (∼100 µg/lane) were analyzed by immunoblotting using specific antibodies to the indicated proteins. FC, indicates the fold change in the levels of the activated caspase-1 (the p20 band) with respect to the control (lane 1). ( D ) Control THP-1 cells or cells infected with virus expressing the shIFI16 mRNA (population # 9) were either left untreated (white columns) or treated with IFN-β (1,000 u/ml; columns 2–5 and 7–10) for 14 h. After the treatment, total RNA levels were analyzed for the indicated genes by the quantitative TaqMan real-time PCR. The ratio of the test gene to actin mRNA was calculated in units (one unit being the ratio of the test gene to actin mRNA). Results are mean values of triplicate experiments and error bars represent standard deviation.

Article Snippet: THP-1 cells were either infected with control lentiviral particles (sc-108080; from Santa Cruz Biotech, Santa Cruz,, CA) or the lentiviral particles expressing shIFI16 RNA (sc-35633-V, Santa Cruz Biotech) in a six well plate as suggested by the supplier.

Techniques: Infection, Control, Virus, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation