Journal: Aging and Disease

Article Title: AI-Driven Robotics Laboratory Identifies Pharmacological TNIK Inhibition as a Potent Senomorphic Agent

doi: 10.14336/AD.2024.1492

Figure Lengend Snippet: Identification and testing of INS018_055 senomorphic activity in the chemotherapy-induced IMR-90 senescence model . ( A ) Schematic of the chemotherapy-induced IMR-90 senescence model. IMR-90 cells were treated with doxorubicin for 2 hours to induce cellular senescence, and cells were then treated with INS018_055 and respective controls for an additional 72 hours. Cellular senescence was evaluated by the detection of SA-β-gal and SASP factor expression. ( B ) Representative staining images of doxorubicin-induced senescent IMR-90 cells treated with DMSO, 100nM rapamycin (RAPA), 1.25 and 10µM INS018_055. Top Row: Hoechst middle row: grey-scale SA-β-gal signal, bottom row: bright field signal. White arrows are SA-β-gal positive cells. Scale bar = 200 μm. ( C ) SA-β-gal positive cell number quantitation in samples treated with DMSO, RAPA, and INS018_055 at indicated concentrations. DMSO group: n=6; Other groups: n=3; * p < 0.05; Mann-Whitney test. ( D ) Percentage of SA-β-gal positive cells in different groups. DMSO group: n=6; Other groups: n=3; * p < 0.05; Mann-Whitney test. ( E ) Total cell number quantitation from groups analyzed in (C-D). DMSO group: n=6; Other groups: n=3; Mann-Whitney test. ( F ) Expression analysis of cell cycle regulators and SASP detected by RT-qPCR. Relative expression of IL6 , IL8 , TGFB1 , IL1A , and IL1B were normalized against β-actin. n=9 per group; * p < 0.05; ** p < 0.01; unpaired two-tailed Student's t-test.

Article Snippet: Quantitative RT-PCRs were performed in triplicate using ChamQ SYBR qPCR Master Mix (Vazyme, cat# Q321-03).

Techniques: Activity Assay, Expressing, Staining, Quantitation Assay, MANN-WHITNEY, Quantitative RT-PCR, Two Tailed Test

Journal: Aging and Disease

Article Title: AI-Driven Robotics Laboratory Identifies Pharmacological TNIK Inhibition as a Potent Senomorphic Agent

doi: 10.14336/AD.2024.1492

Figure Lengend Snippet: Long-term treatment of INS018_055 prevented replicative senescence by suppressing SASP factors . ( A ) Schematic outline of long-term replicative senescence in IMR-90 cells. IMR-90 cells were cultured for 8 weeks and passaged to induce senescence. The cells were treated with three concentrations of INS018_055 at 0.3μM, 1μM, and 3μM, as well as corresponding DMSO controls. The cellular senescence was evaluated by the SA-β-gal staining and SASP factor expression detection at different passages as indicated. Moreover, cells treated with DMSO and INS018_055 (1μM) were collected for transcriptomic analysis. ( B ) Representative staining images of senescent IMR-90 cells at P15, P17, and P18 treated with DMSO and 1µM INS018_055. Top Row: Hoechst, middle row: grey-scale SA-β-gal signal, bottom row: bright field signal. White arrows are SA-β-gal positive cells. Scale bar = 200 μm. ( C ) SA-β-gal positive cell number quantitation in samples treated with 0.3, 1, and 3μM INS018_055 at different passages. P15: n=3 per group; P16, 17, 18: n=4 per group; * p < 0.05; Mann-Whitney test. ( D ) RT-qPCR analysis of cell cycle regulators and SASP. Relative expression of P16, P21, IL1B, IL6 , IL8 and TGFB1 were normalized against β-actin. P12, 15, 16, 17, P18: n=6 per group; * p < 0.05; ** p < 0.01; unpaired two-tailed Student's t-test.

Article Snippet: Quantitative RT-PCRs were performed in triplicate using ChamQ SYBR qPCR Master Mix (Vazyme, cat# Q321-03).

Techniques: Cell Culture, Staining, Expressing, Quantitation Assay, MANN-WHITNEY, Quantitative RT-PCR, Two Tailed Test