Fmk001

[4-(2-methyl-1-(4-methylpiperazin-1-yl)-1-oxopropan-2-yl)-N-(6-(pyridin-4-yl)imidazo [1,2-a]pyridin-2-yl)benzamide] (T3) was synthesised and purified as described previously [15 (link)]. [N-(8-fluoro-6-(pyridin-4-yl)imidazo[1,2-a]pyridin-2-yl)-4-(2-methyl-1-(4- methylpiperazin-1-yl)-1-oxopropan-2-yl)benzamide trihydrochloride] (T3-1) was synthesised and purified as shown in Supporting Information (S1 File). The Bcl-2 family protein inhibitor ABT-263 was purchased from MedChemExpress (HY-10087; Monmouth Junction, NJ, USA). The pan-caspase inhibitor Z-VAD-FMK was purchased from R&D Systems (FMK001; Minneapolis, MN, USA) and used at 25 μM in all the experiments described in the present study.

VSMCs were pre-treated for 1 h with 50µM Z-VAD.FMK (R&D Systems FMK001) prior to addition of Stau and α-Fas/CHX and incubated for the indicated time points. VSMCs were also pre-treated with 10µM SB203580 (CELL, SM32) and 25µM SP600125 (Sigma, S5567). Recombinant proteins were purchased from Peprotech. Cells were treated with 100 ng/ml IL-6 (216 − 16) and GM-CSF (315-03) or 50 ng/ml of each for combined treatment. Neutralization of IL-6 and GM-CSF in conditioned medium was achieved with 0.75 µg/ml of anti-GM-CSF (R&D MAB415-SP) and anti-IL-6 (R&D MAB406-SP). Inhibition of transcription was performed with 2 µg/ml of Actinomycin-D (NovusBio, NB1229).

Human dermal fibroblasts (fibroblasts established from a skin biopsy) from five control subjects and five patients who carried the HGPS p.Gly608Gly mutation were obtained from the Coriell Cell Repository. Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies) supplemented with 15% fetal bovine serum (Life Technologies), 2 mM l‐glutamine (Life Technologies), and 1× penicillin–streptomycin (Life Technologies) at 37°C in a humidified atmosphere containing 5% CO₂. Testing for mycoplasma contamination was performed regularly. Fibroblasts were cultured in the presence or absence of MG132 (474790, Merck Chemical LTD), MG115 (SCP0005, Sigma), MG262 (I‐120‐200, R&D Systems), bortezomib (S1013, Euromedex), carfilzomib (S2853, Euromedex), chloroquine diphosphate crystalline (C6628, Sigma), bafilomycin A1 (B1793, Sigma), caspase‐6 inhibitor Z‐VEID‐FMK (FMK006, R&D Systems), pan‐caspase inhibitor Z‐VAD‐FMK (FMK001, R&D Systems), leptomycin B (L2913, Sigma), 2‐D08 (SML1052, Sigma), and ginkgolic acid C15:1 (74741, Sigma). The experiments were performed on fibroblasts of HGPS patients and healthy subjects matched for age and passage.

Recombinant human caspase-1 (1 unit/rx, #1081, BioVision Inc., CA. USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the presence of lentinan or Z-VAD-FMK (FMK001, R&D Systems). Caspase-1 activity was measured using a Caspase-1/ICE Fluorometric Assay Kit (#K110, BioVision Inc.) according to the manufacturer’s protocol.

Caspase-1 activity was measured using a Caspase-1/ICE Fluorometric Assay Kit (K110, BioVison Inc.) according to the manufacturer’s protocol. Briefly, human recombinant caspase-1 (1 unit/rx, 1081, BioVison Inc., CA. USA) was incubated with YVAD-pNA, a substrate of caspase-1, in the presence of MB or Z-VAD-FMK (a pan caspase inhibitor, FMK001, R&D Systems).

Stocks of PKC inhibitor Gö6976 (10 mm, Tocris 2253), Trk receptor inhibitor K252a (200 μm, Calbiochem #420298), pan-caspase inhibitors Boc-D-fmk (10 mm, Abcam ab142036), zVAD-fmk (20 mm, R&D Systems FMK001), and necroptosis inhibitor necrostatin-1 (NEC-1; 100 mm, Sigma-Aldrich N9037) were prepared in dimethylsulfoxide (DMSO) and used at 1:1000 dilution (final concentration of DMSO below 0.1%). The translation inhibitor cycloheximide (CHX; R&D Systems 0970/100) was dissolved at 1.0 g/l in water and used at 1:1000. Autophagy inhibitor 3-methyladenine (3-MA; Sigma-Aldrich M9281) was dissolved at 10 mm in phenol-red neurobasal media. Drugs were applied at the same time that the trophic factor withdrawal was initiated.

To determine the cytotoxicity induced by AraC, Aza, Gal-9, and CQ (from the LC3B Antibody Kit for Autophagy, L10382, Invitrogen^TM, Carlsbad, CA, USA), 5 × 10⁴ cells (either cell lines or primary patient-derived material) were plated in a 48 well plate with 200 µL RPMI supplemented with 10% FCS (for cell lines) or Gartner’s medium (for patient-derived AML cells or CB cells) and treated with the indicated concentrations of Gal-9, AraC, Aza or CQ. In the case of combinational treatments, Gal-9 and AraC were added simultaneously, whereas cells were pre-incubated with Aza for 16 h before adding Gal-9. For experiments with Z-VAD-fmk (R&D Systems, Inc., FMK001, Wiesbaden, Germany), cells were pre-treated with 20 µM Z-VAD-fmk for 16 h and again treated with 20 µM Z-VAD-fmk freshly added 1 h before adding Gal-9 or Staurosporine (Sigma Aldrich, St. Louis, MO, USA). For long-term assays using patient-derived AML cells (5–7 days), wells were first coated with MS5 cells (pre-plated 1 day before the assay to reach confluency at the day of use) before AML cells were layered on top of the stromal layer. For the CQ assays, cells were incubated on top of MS5 cells. Cytotoxicity was assessed using either flow cytometry-based cell counts, Annexin-V staining, DioC6 staining, or MTS assay.