Journal: Frontiers in Aging

Article Title: Membrane transporter progressive ankylosis protein homologue ( ANKH / Ank ) partially mediates senescence-derived extracellular citrate and is regulated by DNA damage, inflammation, and ageing

doi: 10.3389/fragi.2025.1583288

Figure Lengend Snippet: ANKH transcript is upregulated in PEsen human fibroblasts and downregulated by telomerase. (A) Expression of ANKH mRNA in growing and PEsen NHOF-1 cells using primer set 1. (B) This is similar to (A) but shows the results of primer set 2 in the same experiments. (C) This is similar to (A) but shows the expression of the senescence marker p16INK4A in the same experiments. (D) This is similar to (A) but shows the expression of the senescence marker p21WAF1 in the same experiments. (E) This is similar to (A) but shows SA-βGal expression. (A–E) Yellow plain bars, growing young cells 20.2-21 MPDs; blue horizontal striped bars, PEsen cells 68.2 MPDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; *P > 0.05 < 0.1; ns, not significant. The results are presented as the averages +/- standard deviation. N = 3. (F) Expression of ANKH mRNA in NHOF-1 PURO, TERT-HA, DNTERT, and TERT cell using primer set 1. N = 3. (G) This is similar to (F) but shows the results of primer set 2 in the same experiments. (H) This is similar to (F) but shows the expression of the senescence marker p16INK4A in the same experiments. (I) This is similar to (F) but shows the expression of the senescence marker p21WAF1 in the same experiments. (J) This is similar to (F) but shows SA-βGal expression. (K,L) Representative images of SA-βGal staining in (E). (K) Young growing NHOF-1 fibroblasts (20.2 MPDs). (L) . NHOF-1 cells (68.2 MPDs). Bar, 100 µm. (F–J) (A–E) Plain yellow bars, LATE PURO; blue horizontal striped bars, TERT-HA; brown stippled bars, DNTERT; and purple left-right diagonally striped bars, TERT all after completing 60 MPDs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; * >0.05 < 0.1; ns, not significant. The results are presented as the averages +/- standard deviation. N = 4, except (J) .

Article Snippet: pBABE retroviral vectors on the puromycin-resistant backbone expressing TERT and TERT-HA , the dominant-negative catalytically dead TERT DNTERT , or the empty vector were obtained from Addgene Europe (Teddington, Middlesex, UK; Cat# 1771, 1772 and 1775, respectively).

Techniques: Expressing, Marker, Standard Deviation, Staining

Journal: Viruses

Article Title: HTLV-1 p13 Protein Hijacks Macrophage Polarization and Promotes T-Cell Recruitment

doi: 10.3390/v17040471

Figure Lengend Snippet: P13 targets mitochondria in monocytic cells. ( A ) Schematic representation of p13-HA-GFP viral protein. The p13 protein fused to HA and GFP carboxy terminal tags was cloned in a retroviral vector pBABE. The N-terminal region of p13 has a mitochondrial targeting sequence (MTS) from amino acids 21 to 35. ( B ) Immunoblot for HA expression from total cellular extracts of THP-1 cells transduced with retrovirus expressing p13-HA-GFP. Cells transduced with empty retrovirus were used as control (Ctrl). β-Actin expression was used as a loading control. ( C ) Stable cell lines THP-Ctrl or THP-p13 were adhered to glass slides by cytospin and stained with an antibody to complex IV (COXIV). Scale bar: 10 µm. ( D ) Proliferation assay of THP-Ctrl or THP-p13. Cells were stained with CellTrace™ Far Red, and MFI was measured by flow cytometry every 24 h for three days. The results of three independent experiments were graphed; Ctrl is labeled in blue, and p13-expressing cells in red. Statistical significance was verified with a Student’s t -test. No statistical significance was noted between p13-expressing cells and Ctrl. ( E ) THP-Ctrl and THP-p13 cells following the treatment with increased concentration of Staurosporine (0.01, 0.1, 1 μM). Cells were stained with Live/Dead Fixable Blue dye (ThermoFisher Scientific) to measure the percentage of live cells every 24 h for three days by flow cytometry. The results of three independent experiments were graphed; Ctrl and p13 cells are labeled in blue and red, respectively. Statistical significance was verified with a Student’s t -test. No statistical significance was noted between p13-expressing cells and Ctrl. ( F ) Seahorse extracellular flux analysis measured the oxygen consumption rate in THP-p13 or THP-Ctrl cells. A representative rate of spare respiratory capacity is shown. Different mitochondrial parameters are as follows: basal respiration (yellow), ATP-linked respiration (green), proton leak (magenta), maximal respiratory capacity (blue), and non-mitochondrial respiration (gray). ( G ) The maximal respiratory capacity rate was graphed for THP-Ctrl (blue) and THP-p13 (red). Statistical significance was verified with a Student’s t -test and reported in the figure. The p -values are summarized with asterisks, *** ( p ≤ 0.001). ( H ) Seahorse extracellular flux analysis measured the oxygen consumption rate in p13-expressing and control THP cells.

Article Snippet: The p13-HA-GFP construct encodes the mammalian codon-optimized p13 (with a modification in amino acid number 13 from glutamic acid [E] to glycine [G]) in-frame with an HA and GFP tag in a retroviral pBABE-puro vector. pBABE-puro was kindly provided by Dr. Hartmut Land, Dr. Jay Morgenstern, and Dr. Robert Weinberg (Addgene plasmid # 1764; https://www.addgene.org/1764/ [accessed on 24 March 2025]; RRID: Addgene_1764) [ ].

Techniques: Clone Assay, Retroviral, Plasmid Preparation, Sequencing, Western Blot, Expressing, Transduction, Control, Stable Transfection, Staining, Proliferation Assay, Flow Cytometry, Labeling, Concentration Assay