ice cold flow cytometry buffer  (Thermo Fisher)

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1 -mutated acute myeloid leukemia

doi: 10.1016/j.apsb.2022.10.018

Figure Lengend Snippet: HEL inhibits cell proliferation, induces apoptosis and differentiation in AML cells. (A) Structure of HEL highlighting (in red) three potentially thiol reactive sites. (B, C) Calculation of IC 50 for HEL using MTT dose response curves expressed as the log of concentration vs. cell viability of the AML or HMSC cells ( n = 5). (D) the effect of HEL (5 μmol/L) on cell apoptosis of multiple AML cells was analyzed by FCM with AraC (5 μmol/L) as the positive group ( n = 3). (E) AML cells were treated with DMSO, HEL (5 μmol/L) or ATRA (5 μmol/L) for 5 days as indicated. CD11b or CD14 expression was measured by FCM, the positive cell population percentage of CD11b or CD14 cells analysis results were presented ( n = 3). Representative flow cytometry charts of experiments D and E are shown in , CTR means control group. All data are presented as Median ± IQR, ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.001.

Article Snippet: For the FCM differentiation assay, cells were washed with ice-cold flow cytometry staining buffer (FCS, #00-4222-26, eBioscience) twice, resuspended in 100 μL FCS buffer, and then stained with FITC-anti-human CD11b or APC-anti-human CD14 for 45 min on ice.

Techniques: Concentration Assay, Expressing, Flow Cytometry, Control

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1 -mutated acute myeloid leukemia

doi: 10.1016/j.apsb.2022.10.018

Figure Lengend Snippet: The in vivo effect of HEL on NPM1 mutant AML cells. (A) Experiment schema. After confirmation of bone marrow AML engraftment to >10% in 3 randomly selected mice, remaining mice were randomized to model, 50 mg/kg HEL, or 50 mg/kg AraC, by i. p. every 48 h for 14 days (B) Bone marrow AML burden. Representative flow cytometry for Human CD45 + (AML) and mouse CD45 + (normal) cells. (C) Granulocyte (CD11b) and monocyte (CD14) lineage differentiation marker expression in marrow AML cells using FCM analysis. (D, E) Photos show spleens from model M-NSG mice and different treatment groups, with H&E-stained spleen sections showing splenic lesions (yellow arrow). Scale bar, 12 μm. (F) Serial blood counts. The increasing of WBC and decreasing of Hb or PLT was due to myeloblasts. All the above experiments were analyzed by ten mice per group (vehicle group, n = 5), presented as Median ± IQR, ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: For the FCM differentiation assay, cells were washed with ice-cold flow cytometry staining buffer (FCS, #00-4222-26, eBioscience) twice, resuspended in 100 μL FCS buffer, and then stained with FITC-anti-human CD11b or APC-anti-human CD14 for 45 min on ice.

Techniques: In Vivo, Mutagenesis, Flow Cytometry, Marker, Expressing, Staining

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1 -mutated acute myeloid leukemia

doi: 10.1016/j.apsb.2022.10.018

Figure Lengend Snippet: HEL impairs NPM1 mutant AML cells and induces cell differentiation through targeting RPS2. (A) Anti-FLAG, anti-RPS2 and loading control anti- β -actin protein expression in OCI-AML3 cells stably expressing FLAG-GFP, FLAG-RPS2 WT or FLAG-RPS2 C222S mutant assessed using western blotting. (B)–(E) Cell viability (B, n = 6); cycles (C, n = 5), apoptotic rates (D, n = 3) and differentiation status (E, n = 3) of OCI-AML3 cells transiently expressing pcDNA3.1 vector, FLAG-RPS2 WT or a FLAG-RPS2 C222S mutant treated with DMSO or HEL (5 μmol/L) for 48 h, cell viability was tested by MTT method and cell cycle, apoptosis, and differentiation were all tested by flow cytometry method. Representative flow cytometry charts of experiments C&D are shown in . All the above experiments were analyzed by at least three biological replicates, presented as Median ± IQR; ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: For the FCM differentiation assay, cells were washed with ice-cold flow cytometry staining buffer (FCS, #00-4222-26, eBioscience) twice, resuspended in 100 μL FCS buffer, and then stained with FITC-anti-human CD11b or APC-anti-human CD14 for 45 min on ice.

Techniques: Mutagenesis, Cell Differentiation, Control, Expressing, Stable Transfection, Western Blot, Plasmid Preparation, Flow Cytometry

Journal: Acta Pharmaceutica Sinica. B

Article Title: Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1 -mutated acute myeloid leukemia

doi: 10.1016/j.apsb.2022.10.018

Figure Lengend Snippet: RPS2 is an essential target for NPM1 mutant AML treatment, and this target is p53 dependent. OCI-AML3 sh NC (blue) or sh p53 (red) cells transfected with RPS2 siRNA or si NC (control) and treated with DMSO or HEL (5 μmol/L), respectively for 48 h, the cells were then tested for the (A) proliferation, (B) apoptosis, (C) cycle arrest, and (D) differentiation status [a, DMSO; b, si NC ; c, HEL (5 μmol/L)+si NC ; d, si RPS2 ; e, HEL (5 μmol/L)+si RPS2 ] and (E) expressions of p53, RPS2, caspase 3, C-caspase 3, p21 and CEBP/ α proteins. Experiments (A ) was analyzed by five biological replicates; experiments (B–D ) were analyzed by three biological replicates. Representative flow cytometry charts of experiments B&C are shown in . All data are presented as Median ± IQR; ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, unpaired t test; gels shown in experiment (E) are representative blots for three biological independent samples per group; Protein level quantification results are shown in Supporting Information Fig. S15. (F) Expression of RPS2 protein and loading control actin levels in OCI-AML3 cells transfected with RPS2 siRNA and siNC (control), assessed using Western blot. (G, H) Northern blot analysis of pre-rRNA processing phenotypes after transfected with RPS2 siRNA or si NC (control) treated with DMSO or HEL (10 μmol/L) for 48 h, respectively. Mature rRNAs were shown on the EtBr-stained gel. Bands on the Northern blots or EtBr gels ( n = 3) were quantitated by ImageJ, presented as Median ± IQR; ns, not significant, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗∗ P < 0.0001. (I) FBL (red) and NPM1 (green) localization in the RPS2 knock-down OCI-AML3 cells by IF. Hoechst 33342 (blue) was used to stain for nuclei. Images by Leica SP8 confocal microscope. Scale bar, 12.3 μm. Quantification of percentage of cells with nucleolar stress is shown in Supporting Information Fig. S16 .

Article Snippet: For the FCM differentiation assay, cells were washed with ice-cold flow cytometry staining buffer (FCS, #00-4222-26, eBioscience) twice, resuspended in 100 μL FCS buffer, and then stained with FITC-anti-human CD11b or APC-anti-human CD14 for 45 min on ice.

Techniques: Mutagenesis, Transfection, Control, Flow Cytometry, Expressing, Western Blot, Northern Blot, Staining, Knockdown, Microscopy