dapi thermofisher cat. no. d1306  (Thermo Fisher)

Journal: Molecular Oncology

Article Title: Inhibition of acyl‐ CoA synthetase long‐chain isozymes decreases multiple myeloma cell proliferation and causes mitochondrial dysfunction

doi: 10.1002/1878-0261.13794

Figure Lengend Snippet: Targeting the ACSLs is a Novel Target to Inhibit Myeloma Cell Proliferation and Survival. (A) Overall survival of patients from CoMMPass trial with high or low ACSL1 expression (relative to median, ENSG00000151726) for all patients ( n = 754). P ‐values are calculated from log‐rank Kaplan–Meier test. (B) Hallmark Fatty Acid Metabolism genes from Gene Set Enrichment Analysis (M5935) displayed as the average gene fitness (Chronos Score) data from the Cancer Dependency Map of 21 human myeloma cell lines. Blue bars represent targets of interest with average Chronos scores < 0 (fitness defect upon CRISPR knockout), while red bars are Chronos scores > 0. ACSL family members are highlighted in yellow. (C) ACSL activity in MM.1S cells after treatment with a range of Triacsin C (TriC) doses; IC 50 values were calculated using non‐linear regression (four parameter, variable slope) based on the relative ACSL Activity to the vehicle; n = 3. (D) MM.1S, MM.1R, RPMI‐8226, OPM2 and U266B1 cells were incubated with various doses of TriC for 48 h and stained with Trypan Blue to quantify viable cells·mL −1 . EC 50 values were calculated using non‐linear regression (four parameter, variable slope) in graphpad prism v9.4.1. n = 3. (E) Proliferation of human myeloma cell lines MM.1S, OPM‐2 and RPMI‐8226 treated with various doses of TriC for 48 h and stained with AF647 anti‐human Ki67 (% positive); MM.1S ( n = 5), OPM‐2 ( n = 6), RPMI‐8226 ( n = 3). (F) Cell cycle distribution of MM.1S cells treated with various doses of TriC for 48 h and stained with DAPI; n = 3. (G) Apoptosis assay using Annexin V/DAPI staining of MM.1S and OPM2 cells treated with various doses of TriC for 48 h; n = 3. (H) Intracellular BAX (AF488 mouse anti‐human BAX antibody) levels in MM.1S cells treated with various doses of TriC for 48h or 72h; data displayed as FITC MFI; n = 3. Statistics: Two‐way ANOVA with Tukey's multiple comparison test (E–G) or Šídák's multiple comparisons test (H). All data are mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001.

Article Snippet: To characterize apoptosis, MM cells were collected, washed 3 times with Cell Staining Buffer (BioLegend, Cat. No. 420201, San Diego, CA, USA) and stained with APC‐Annexin V (1 : 20, BioLegend Cat. no. 640920), DAPI (0.004 μg·mL −1 , ThermoFisher, Cat. No. D1306) in Annexin V Binding Buffer (BioLegend, Cat. no. 422201) for 15 min at room temperature.

Techniques: Expressing, CRISPR, Knock-Out, Activity Assay, Incubation, Staining, Apoptosis Assay, Comparison