4 6 diamidino 2 phenylindole dapi thermofisher scientific d1306 to protm 3 thermofisher scientific s33025 7 aminoactinomycin d  (Thermo Fisher)

Journal: bioRxiv

Article Title: Divergent Cell-Type Specific Hypoxia Responses in Human Stem Cell–Derived and Primary Islets

doi: 10.1101/2025.07.16.665006

Figure Lengend Snippet: Divergent functional, metabolic, and cellular responses of primary and SC-islets to acute hypoxia. Functional, metabolic, and immunohistochemical analyses of primary islets (top row, A-E) and SC-islets (bottom row, F-J) after exposure to 1% O 2 for 0, 6, 24, or 48 hours. (A, F) Representative immunohistochemical images of (A) primary and (F) SC-islet sections at each timepoint. Panels show co-staining for DAPI (blue), C-peptide (Cpep, green), and PDX1 (red); DAPI, glucagon (GCG, green), and somatostatin (SST, cyan); and DAPI and BNIP3L (red). Scale bars = 200 µm. (B, G) Absolute insulin secretion in response to low (2 mM, grey bars) and high (20 mM, dark bars) glucose for (B) primary and (G) SC-islets. (C, H) Oxygen consumption rate (OCR) of (C) primary and (H) SC-islets, measured via Seahorse metabolic flux analysis. Different colored lines represent islets recovered after the indicated duration of hypoxia. The traces show metabolic responses to sequential injections (indicated by dashed lines) of glucose (20 mM), oligomycin (1.5 µM), FCCP (0.5 µM), and finally rotenone/antimycin A (0.5 µM). (D, I) Glucose-stimulated insulin secretion (GSIS) stimulation index (SI) for (D) primary and (I) SC-islets, calculated as the ratio of high-to-low glucose secretion. (E, J) Extracellular acidification rate (ECAR) of (E) primary and (J) SC-islets, measured concurrently with OCR. *Data in (B, D, G, I) are presented as mean ± SEM of n=3 (primary) or n=4 (SC-islets) biological replicates; individual data points are shown as open circles. Data in (B, E, G, J) are representative traces from a single experiment, with lines showing the mean ± SEM of technical replicates. For insulin secretion (B, G) and stimulation index (D, I), statistical significance was assessed using repeated measures ANOVA followed by pairwise t-tests with Bonferroni correction for post-hoc analysis. Significance is denoted as *p < 0.05, **p < 0.01.

Article Snippet: DAPI (ThermoFisher; D1306) was used for nuclear staining.

Techniques: Functional Assay, Immunohistochemical staining, Staining

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