human methylcellulose based medium  (R&D Systems)

Journal: Signal Transduction and Targeted Therapy

Article Title: The deacetylases HDAC1/HDAC2 control JAK2 V617F -STAT signaling through the ubiquitin ligase SIAH2

doi: 10.1038/s41392-025-02369-7

Figure Lengend Snippet: Inhibition of class I HDACs induces apoptosis in JAK2 V617F -positive cells. a HEL cells were treated with 5 nM FK228 for 24 h (-, untreated control cells; +, treated cells). Immunoblotting shows full-length (fl.) and cleaved (cf.) PARP1 and cleaved forms of caspase-3; β-actin was used as a loading control; kDa, kilo Daltons. b HEL cells were treated with 5 nM FK228 for 24–48 h, stained with annexin-V-FITC/PI, and analyzed for early (annexin-V-positive) and late (annexin-V/PI-positive) apoptosis by flow cytometry. Left shows representative flow cytometry scans; right shows bar graphs with statistical evaluations. c HEL cells were incubated with 5 nM FK228 and/or 50 μM z-VAD-FMK for 24 h. PARP1 was detected by immunoblotting; α-tubulin served as a loading control. d HEL cells were treated as described in ( c ) and subsequently stained with annexin-V-FITC/PI and analyzed for apoptosis. e HEL cells were treated as described in ( c ), fixed, subsequently stained with PI, and analyzed for subG1 fraction accumulation via flow cytometry. f HEL cells were treated with 5 nM FK228 for 24–48 h. The indicated proteins were revealed by immunoblotting; β-actin was used as a loading control. g HEL cells were treated with 5 nM FK228 for 24 h and/or proteasome inhibitors (50 nM bortezomib; 10 μM lactacystin) for 6 h before being harvested. Immunoblotting shows the indicated proteins. h HEL cells were treated as described in ( g ) and analyzed for apoptosis via flow cytometry. i HEL cells were incubated with 5 nM FK228 or 5 µM MS-275 for 24 h, washed, plated into methylcellulose medium, and tested for colony formation. Left, representative images are shown. Scale bar, 200 μm. Right, graph depicts the numbers of colonies. The data are presented as mean ± SD of at least three independent experiments. Statistical analyses (unpaired t -test; one-way ANOVA; two-way ANOVA; Bonferroni correction; ns not significant; p values are as follows: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: Subsequently, cells were plated evenly as triplicates (1 × 10 3 cells/ml for HEL cells or 5 × 10 3 cells/ml for primary cells) in methylcellulose medium (Human Methylcellulose Base Media, Catalog #HSC002, R&D Systems, MN, USA).

Techniques: Inhibition, Control, Western Blot, Staining, Flow Cytometry, Incubation

Journal: Signal Transduction and Targeted Therapy

Article Title: The deacetylases HDAC1/HDAC2 control JAK2 V617F -STAT signaling through the ubiquitin ligase SIAH2

doi: 10.1038/s41392-025-02369-7

Figure Lengend Snippet: HDAC1 and HDAC2 protect JAK2 V617F -positive cells from apoptosis. a HEL cells were incubated with 5 μM MERCK60 for 24 h. PARP1 and caspase-3 were detected by immunoblotting; β-actin, loading control. b HEL cells were treated as described in ( a ), stained with annexin-V-FITC/PI, and analyzed for apoptosis. c HEL cells were transfected with siRNAs against HDAC1 or HDAC2 for 48 h. The cells were stained with annexin-V-FITC/PI and analyzed for apoptosis. d GSEA analyses revealed the upregulation of proapoptotic gene signatures in HEL cells that were treated with 5 μM MERCK60 for 24 h, with a normalized enrichment score (NES) of 1.38 and an FDR q value < 0.05. e HEL cells were treated with 5 μM MERCK60 for 48 h, washed, plated in methylcellulose medium, and analyzed for colony formation after 14 days. Left, representative images are shown. Scale bar, 200 μm. Right, graph depicts the numbers of colonies. f Scheme depicting the experiment for which ( g ) shows the outcome; created with BioRender ( https://BioRender.com ). g MPN patient-derived JAK2-mutated PBMCs and HSPCs from healthy donors (HD) were treated with 0.5 or 1 μM MERCK60 or with DMSO as a control and plated in methylcellulose. The number of colonies and total number of cells were counted on day 10. Representative images are shown (upper panel). Scale bar, 200 μm. h Scheme depicting the experiment for which ( i , j ) show the outcome; created with BioRender ( https://BioRender.com ). i , j Hematopoietic stem and progenitor cells were isolated from the bone marrow of three mice with PV. Leukemic stem cells were enriched and tested for c-Kit and Sca-1 expression. The cells were treated with 5 µM MERCK60 for 24 h, washed, plated in methylcellulose, and analyzed for colony formation after 10 days; scale bar, 100 μm ( i ) or fixed, subsequently stained with PI, and analyzed for subG1 fraction accumulation via flow cytometry ( j ). k TCGA dataset analysis revealed that the overall survival of AML patients inversely correlates with the expression of HDAC1 mRNA. The data are presented as mean ± SD of three independent experiments. Statistical analyses (unpaired t -test; one-way ANOVA; two-way ANOVA; Bonferroni correction; ns not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001)

Article Snippet: Subsequently, cells were plated evenly as triplicates (1 × 10 3 cells/ml for HEL cells or 5 × 10 3 cells/ml for primary cells) in methylcellulose medium (Human Methylcellulose Base Media, Catalog #HSC002, R&D Systems, MN, USA).

Techniques: Incubation, Western Blot, Control, Staining, Transfection, Derivative Assay, Isolation, Expressing, Flow Cytometry