Agn193109

For inhibition of chromatin compaction, HeLa cells were incubated with 1 mM valproic acid (VPA) (P4543, Sigma–Aldrich). VNB treatment was done after 17 h of VPA incubation and VPA was kept on the cells during and after VNB treatment. Cells were fixed 6 h after VNB treatment, resulting in VPA being on the cells for a total time of 24 h before fixation. For inhibition of microtubule polymerisation, HeLa cells were incubated with 1 mM nocodazole (358240100, Acros). Cells were treated with VNB after 1 h of nocodazole incubation and nocodazole was kept on the cells during and after VNB treatment. Cells were fixed 6 h after VNB treatment, resulting in nocodazole being on the cells for a total time of ± 8 h before fixation. To increase LMNA transcription, HeLa cells were incubated with 3 mM AGN-193109 (SML-2034, Merck) for 48 h before VNB treatment. AGN was kept on the cells during and after VNB treatment. Cell viability and photoporation efficiency were determined 2 h after VNB treatment.

After a 36 h si-RNA transfection, cells were cultures in a serum-free medium for 12 h followed by culturing with mediums containing Rol (17772, 5 μg/L, Sigma-Aldrich, St. Louis, MO), atRA (R2625, 5 μmol/L, Sigma-Aldrich, St. Louis, MO), atRA and AGN193109 (SML2034, 1 μmol/L, Sigma-Aldrich, St. Louis, MO) mixture, or equivalent amounts of DMSO (Sigma -Aldrich, St. Louis, MO), respectively. Then, cells were fixed in 4% paraformaldehyde or collected for subsequent analysis 24 h later.

24 hours after splitting, organoids were treated with either ENR – Vitamin A containing 0.1% final volume DMSO, ENR + Vitamin A containing 0.1% final volume DMSO 5μM all-trans-Retinoic acid (ATRA, Cayman Chemical), 10μM 9- cis-Retinoic acid (9-cisRA, Cayman Chemical), 1μM AGN193109 (Sigma Aldrich), 1μM NRX194204 (Axon Medchem), 1μM HX531 (Sigma Aldrich) or 10 μM LG100268 (Sigma Aldrich). Drugs were stored under reduced light conditions and exposure to light was minimized. Media containing compounds was refreshed after 24h. Total treatment time was 48h for all drugs. All drugs were dissolved in DMSO and added to the culture medium at working concentration in 0.1% final volume DMSO. After treatment, organoids were harvested using Organoid Harvesting Solution (Cultrex, R&D Systems) and pelleted. Pellets were snap frozen and stored at −80 C for RNA-sequencing, or cryopreserved to maintain native chromatin context for ATAC-sequencing.

Sieved AGX1-2 beads (150 or 200 μm in diameter, Sigma) were soaked in a stable form of all-trans-retinoic acid, TTNPB (Sigma, 0.05 mg/ml dissolved in DMSO, Sigma) or AGN193109 (Sigma, 1 mg/ml dissolved in DMSO, Sigma) for 1 hour and then washed in DMEM before being grafted to the middle of wing buds using a sharp tungsten needle. TTNPB has been shown to diffuse from AGX1-2 beads over an approximate 12-20-hour period and can be used to model RA distribution in chick wing buds due to comparable patterning effects, kinetics and diffusion constants ((Eichele et al., 1984 (link)), (Eichele et al., 1985 (link)), (Eichele and Thaller, 1987 (link)).

To investigate the effects of IGFBP5 and RARα on senescence, the cells were treated with rIGFBP5 with or without 10 nM All-Trans Retinoic Acid (ATRA) from Sigma Aldrich (MO, USA). We used siRNA (sc-29465) obtained from Santa Cruz Biotechnology (TX, USA) to silence RARα. Additionally, we inhibited RARα receptors with 1 μM AGN 193109 from Sigma Aldrich (MO, USA).
After the treatments, the senescence-associated beta-galactosidase assay in combination with Ki67 immunocytochemistry was performed, as described above.

Acetylcholine, AGN193109, aldosterone, aristolochic acid I, cisplatin, CGRP, DEAB, dexamethasone, D-glucose, endothelin-1, gentamicin, human albumin, human angiotensin II, human IgG, human transferrin, LPS, mannitol, norepinephrine, tetracycline, atRA and vasopressin were purchased from Sigma-Aldrich Company Ltd, Gillingham, UK. RA-568 was synthesised by Sygnature Discovery, Nottingham, UK. All reagents dissolved in ethanol and/or dimethylsulphoxide (DMSO) were first diluted to 1000 × stock solution and then diluted 1000 × with culture medium to the working concentrations. Control groups were treated with 0.1% ethanol and/or DMSO.