jak2 v617f  (Quest Diagnostics)

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A ) Schematic illustration of the inducible and conditional Ulk1 knockout mouse MPN model used, and analyses performed. (BMT – bone marrow transplant) ( B ) Hemoglobin (Hb) levels, ( C ) hematocrit (HCT) percentage and ( D ) red blood cell (RBC) counts in peripheral blood of Ulk1 +/+ Jak2 V617F and Ulk1 FL/FL Jak2 V617F recipient mice are shown for pre- (day 21) and post- (day 42) pIpC injection. ( E ) Spleen weight and ( F ) percentage of GFP + splenocytes three weeks post pIpC injection of the recipient mice. ( G ) Percentage of GFP + ( G-i ) Ter119 med CD71 high proerythroblasts (R1), ( G-ii ) Ter119 high CD71 high basophilic erythroblasts (R2), and ( G-iii ) Ter119 high CD71 med late basophilic and polychromatophilic erythroblasts (R3) and Ter119 high CD71 low orthochromatophilic erythroblasts (R4) in the spleen of recipient mice three weeks post pIpC injection. ( H ) Percentage of GFP + bone marrow (BM) cells three weeks post pIpC injection of the recipient mice. Percentage of GFP + ( I-i ) R1, ( I-ii ) R2, and ( I-iii ) R3+R4 cells in the BM of recipient mice three weeks post pIpC injection. ( B-I ) Box plots show distribution of data from 6 mice per group. ( B-D ) Mixed effects models were used to analyze these data, with Hb, HCT or RBC counts as the outcome variable, and time (pre- and post-pIpC), genotype and their interaction as fixed effects, and a mouse random effect to account for within-mouse correlation between repeated measurements. Fisher’s LSD approach was used, and pairwise comparison p -values were not adjusted. ( E-I ) Statistical analyses were performed using Mann-Whitney test. ( B-I ) Statistically significant p -values are reported. ns - not significant. See also supplemental figures 1 – 3 .

Article Snippet: To assess the effects of drug-targeted inhibition of ULK1 on cellular viability of JAK2 V617F -positive leukemic cells, SET-2 cells (20,000 cells/well) or HEL cells (1,500 cells/well) were plated in quadruplicate in 96-well plates and treated with vehicle-control (DMSO), ULK101 (TargetMol), SBI-0206965 (Cayman Chemical) or SBP-7455 (TargetMol) at increasing concentrations, for 5 days.

Techniques: In Vivo, Knock-Out, Injection, Comparison, MANN-WHITNEY

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A ) Schematic illustration of the Jak2 V617F knock-in MPN transplant mouse model used, treatment regimen, and analyses performed. ( B ) Hemoglobin (Hb) levels, ( C ) hematocrit (HCT) percentage and ( D ) red blood cell (RBC) counts in peripheral blood of recipient mice are shown for each treatment group four weeks post treatment initiation. ( E ) Percentage of Ter119 med CD71 high proerythroblasts (R1), ( F ) Ter119 high CD71 high basophilic erythroblasts (R2) and ( G ) Ter119 high CD71 med late basophilic and polychromatophilic erythroblasts (R3) and Ter119 high CD71 low orthochromatophilic erythroblasts (R4) in the BM of recipient mice four weeks post treatment initiation. ( H ) Spleen weight and percentage of ( I ) R1, ( J ) R2 and ( K ) R3+R4 cells in the spleen of recipient mice four weeks post treatment initiation. ( B-K ) Box plots show distribution of data from 10 mice per group. Statistical analyses were performed using Mann-Whitney test. Statistically significant p -values are reported. See also supplemental figures 6 and 7 . ( L-M ) Cellular viability of JAK2 V617F -positive ( L ) HEL and ( M ) SET-2 cells treated with either DMSO-vehicle control (Ctrl) or with increasing doses of the ULK1 inhibitor SBP-7455 for five days, as indicated, was measured using WST-1 cell viability reagent. Data are expressed as percent cell viability relative to Ctrl-treated cells and represent means ± SEM of ( L ) four and ( M ) three independent experiments. IC50 values are shown. ( N ) Clonogenic capability of HEL cells treated with either vehicle-control (DMSO) or with increasing doses of the ULK1 inhibitor SBP-7455, as indicated. Data are expressed as percent colony formation relative to DMSO-treated cells (control) and represent means ± SEM of three independent experiments. Statistical analysis was performed using Welch’s ANOVA test that accounts for unequal variances followed by Dunnett’s T3 pairwise multiple comparisons test. Statistically significant p -values are reported. Test for linear trend based on the one-way ANOVA model showed a significant decrease in colony formation with increased dose of ULK1 inhibitor ( p <0.0001). See also supplemental figure 8 .

Article Snippet: To assess the effects of drug-targeted inhibition of ULK1 on cellular viability of JAK2 V617F -positive leukemic cells, SET-2 cells (20,000 cells/well) or HEL cells (1,500 cells/well) were plated in quadruplicate in 96-well plates and treated with vehicle-control (DMSO), ULK101 (TargetMol), SBI-0206965 (Cayman Chemical) or SBP-7455 (TargetMol) at increasing concentrations, for 5 days.

Techniques: Inhibition, Activity Assay, In Vivo, Knock-In, MANN-WHITNEY, Control

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-B ) Immunoblotting analysis of the indicated proteins in lysates from non-targeting (NT) control, Atg7 KO and Ulk1 KO Jak2 V617F -positive Ba/F3-EpoR + GFP-LC3-RFP-expressing cells. ( C ) Percentage of autophagy measured as percentage of GFP negative cells assessed by flow cytometry analyses of NT control, Atg7 KO and Ulk1 KO Jak2 V617F -positive Ba/F3-EpoR + GFP-LC3-RFP-expressing cells cultured under normal nutrient conditions (RPMI 1640 media supplemented with 10% FBS and 10 ng/mL of IL-3, no starvation) or under starvation conditions (RPMI 1640 media without serum and IL-3) for 48 hours. Scatter dot plot shows means ± SD. Statistical analysis was performed using Welch’s ANOVA test that accounts for unequal variances followed by Dunnett’s T3 pairwise multiple comparisons test. Statistically significant p -values are reported. ( D-F ) Immunoblotting analysis of the indicated proteins in lysates from ( D ) Cas9-control compared to ULK1 KO HEL cells and from ( E ) HEL cells treated with either DMSO-vehicle control (C), 5μM SBP-7455 (S), 100nM bafilomycin A1 (B) or with the combination of SBP-7455 and bafilomycin A1 (S+B) for 4 hours under either nutrient-starvation conditions (HBSS) or normal complete nutrient-conditions (RPMI). Immunoblots shown are representative of three independent experiments. ( F ) SET-2 cells treated with either DMSO-vehicle control (C), 5μM SBP-7455 (S), 100nM bafilomycin A1 (B) or with the combination of SBP-7455 and bafilomycin A1 (S+B) for 4 hours under either nutrient-starvation conditions (HBSS) or normal complete nutrient-conditions (RPMI). Immunoblots shown are representative of three independent experiments.

Article Snippet: To assess the effects of drug-targeted inhibition of ULK1 on cellular viability of JAK2 V617F -positive leukemic cells, SET-2 cells (20,000 cells/well) or HEL cells (1,500 cells/well) were plated in quadruplicate in 96-well plates and treated with vehicle-control (DMSO), ULK101 (TargetMol), SBI-0206965 (Cayman Chemical) or SBP-7455 (TargetMol) at increasing concentrations, for 5 days.

Techniques: Inhibition, Western Blot, Control, Expressing, Flow Cytometry, Cell Culture

Journal: Molecular and cellular biology

Article Title: Loss of function mouse models reveal a novel regulatory function for ULK1 in myeloproliferative neoplasms

doi: 10.1080/10985549.2025.2529837

Figure Lengend Snippet: ( A-E ) qRT-PCR analysis of the indicated genes mRNA expression in murine c-kit + bone marrow Jak2 V617F/+ VavCre + cells from 3 mice treated for 6 hours with either vehicle (DMSO, Control) or 5μM SBP-7455 (SBP), as indicated. Data are means ± SD across three different mice. Fold changes were calculated relative to gene expression from one randomly selected vehicle control-treated mouse sample. Statistical analyses were performed using two-tailed unpaired t test with Welch’s correction. Statistically significant p -values are reported. ( F-J ) qRT-PCR analysis of the indicated genes mRNA expression in Cas9-control and ULK1 KO HEL cells, as indicated. Data are means ± SD across three independent experiments. Fold changes were calculated relative to gene expression from one randomly selected Cas9 control sample. Statistical analyses were performed using two-tailed unpaired t test with Welch’s correction. Statistically significant p -values are reported. See also supplemental figures 13 – 15 .

Article Snippet: To assess the effects of drug-targeted inhibition of ULK1 on cellular viability of JAK2 V617F -positive leukemic cells, SET-2 cells (20,000 cells/well) or HEL cells (1,500 cells/well) were plated in quadruplicate in 96-well plates and treated with vehicle-control (DMSO), ULK101 (TargetMol), SBI-0206965 (Cayman Chemical) or SBP-7455 (TargetMol) at increasing concentrations, for 5 days.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, Gene Expression, Two Tailed Test

Journal: Pharmaceutical Science Advances

Article Title: Fufang Huangbo Formula mitigates myeloproliferative neoplasms by activating p53/p21 signaling axis and inhibiting STAT3 and NF-κB signaling pathways

doi: 10.1016/j.pscia.2026.100121

Figure Lengend Snippet: FHF provided therapeutic benefit in JAK2 V617F -induced MPN mice. (A) Schematic diagram of the JAK2 V617F -driven MPN mice and therapeutic intervention. Transplantation donors of bone marrow cells expressing JAK2 V617F were treated with vehicle or FHF for 6 weeks. (B) RBC, HGB, and HCT counts in PB from indicated mice after treatment. N = 6 mice per group. (C) Results of flow cytometry analysis of TER119 + cells in bone marrow. (D) Spleen size and weight of indicated mice after FHF treatment. (E-G) Representative H&E staining of spleen, BM and lung.

Article Snippet: JAK2 V617F -floxed mice and Vav-Cre mice were obtained from the Jackson Laboratory.

Techniques: Transplantation Assay, Expressing, Flow Cytometry, Staining

Journal: Pharmaceutical Science Advances

Article Title: Fufang Huangbo Formula mitigates myeloproliferative neoplasms by activating p53/p21 signaling axis and inhibiting STAT3 and NF-κB signaling pathways

doi: 10.1016/j.pscia.2026.100121

Figure Lengend Snippet: Schematic illustration of FHF treatment for MPNs. FHF treatment inhibits the JAK2/STAT3 and NF-κB signaling pathways. This suppresses the expression of pro-inflammatory cytokines and cell proliferation. On the other hand, FHF promotes cellular senescence by activating the p53/p21 signaling pathway.

Article Snippet: JAK2 V617F -floxed mice and Vav-Cre mice were obtained from the Jackson Laboratory.

Techniques: Protein-Protein interactions, Expressing

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A Percentage of CD45.1/2 Nras G12D cells among CD45.1/2 Nras G12D and CD45.1 Nras WT Lin − murine cells (50/50 ratio) treated with DMSO or ruxolitinib (0.25 µM). B Proliferation curves of Nras G12D or Nras WT Lin − cells after DMSO or ruxolitinib (0.25 µM). A, B Mean of n = 2 biological replicates. C In vivo Nras G12D competition model. Created in BioRender . D Percentage of CD45.1/2 Nras G12D cells in peripheral blood of mice transplanted with 10% CD45.1/2 Jak2 WT Nras G12D and 90% CD45.2 Jak2 V617F Nras WT Lin − cells ( n = 4 per group). Ruxolitinib (90 mg/kg twice daily) was started 4 weeks post-transplantation. E In vivo Nras Q61K competition model. Created in BioRender . F Percentage of GFP + Nras Q61K Jak2 V617F cells in the bone marrow of mice transplanted with Jak2 V617F cells expressing a GFP + Nras Q61K vector ( n = 5 per group). Ruxolitinib (90 mg/kg twice daily) was started 3 weeks post-transplantation. G , H Colony formation assay for cKit + bone marrow cells from Jak2 WT ( G ) or Jak2 V617F ( H ) mice expressing Empty or Nras Q61K vectors. Colony number after at least 6 days of DMSO or ruxolitinib (1 µM) ( n = 4 biological replicates). I Western blot for the indicated proteins in HEL cells expressing GFP, Empty or NRAS Q61K vectors, treated 24 h with ruxolitinib (3 µM). J In vitro competition model. Created in BioRender . K Percentage of GFP + and GFP − _Empty or GFP − _ NRAS Q61K HEL or UKE-1 cells. 24 days after ruxolitinib (3 µM and 15 µM for HEL and UKE-1, respectively), the indicated cells were treated 3 days with ruxolitinib or ruxolitinib (Ruxo.) and trametinib (Trame.) (10 µM and 0.1 µM for HEL and UKE-1, respectively) ( n = 3 biological replicates). L Western blot for the indicated proteins in Ba/F3 MPL-CALR WT and MPL-CALR del52 cells expressing Empty or NRAS Q61K vectors, treated 24 h with ruxolitinib (75 nM). M Percentage of Crimson + and Crimson − _Empty or Crimson − _ NRAS Q61K Ba/F3 MPL-CALR WT and MPL-CALR del52 cells after ruxolitinib ( n = 3 biological replicates). Statistical significance using two-tailed Mann–Whitney ( D, F ) or Welch’s t-test ( H, K, M ). Experiments( I, L ) were performed twice with similar results. Error bars represent mean ± SEM. P-values in the figure. Source data provided as Source Data file. Schemas created using BioRender.

Article Snippet: To obtain Jak2 V617F and Jak2 WT primary hematopoietic murine cells, total bone marrow cells were isolated from femurs, spine and tibias of 6 to 12 week-old Wild-Type C57BL/6JOlaHsd (Envigo) or Vav-cre X Floxed- Jak2 V617F C57BL/6 mice , by bone crushing then passed through a 70 μM cell strainer to obtain a single cell suspension, followed by red blood cell lysis (Sigma-Aldrich). c-Kit + cells were then magnetically sorted (CD117 MicroBeads, mouse, Cat #130-091-224, Miltenyi Biotec) and maintained in StemSpan SFEM (StemCell Technologies) supplemented with 1% penicillin-streptomycin (Gibco) and 10 ng/ml mIL-3, 10 ng/ml IL-6, 25 ng/ml mFLT3-ligand, and 25 ng/ml mSCF (Peprotech).

Techniques: In Vivo, Transplantation Assay, Expressing, Plasmid Preparation, Colony Assay, Western Blot, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A qRT-PCR for Nras and Jak2 expression level in murine 32D cells infected with an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 4 biological replicates ± SEM. B Growth inhibition of 32D murine cells expressing an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector 6 days after GFP sorting. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 6 biological replicates ± SEM. C Model of the in vitro Nras Q61K -sh Jak2 murine myeloid cells competition assay. Created in BioRender . D Percentage of GFP + _CRIMSON − 32D cells expressing an Empty, a Nras Q61K or a Nras Q61K _sh Jak2 encoding vector 3 days after sorting. Statistical significance was determined using two-tailed Welch’s t-test. Error bars represent mean of n = 3 biological replicates ± SEM. E –F Colony formation assay of Jak2 WT (E) or Jak2 V 617F (F) C57BL/6 primary bone marrow c-Kit + murine cells expressing an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector at least 6 days after GFP sorting. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 4 biological replicates ± SEM. G Genetic variants association with resistance to the JAK2 inhibitors ruxolitinib ( n = 497), momelotinib ( n = 476) and fedratinib ( n = 93) according to the Beat AML v2 human primary patient AML dataset. Data is presented as volcano plots for gene variants' effect size (Glass) on the x-axis versus –log10(P-value) on the y-axis. Significance was set at – log10(P-value)>1.3, based on the two-tailed Student t-test with Welch’s correction, with variants having a negative effect size associated with drug sensitivity (red), while those having a positive effect size associated with drug resistance (blue). P-values are reported in the figure. Source data provided as a Source Data file.

Article Snippet: To obtain Jak2 V617F and Jak2 WT primary hematopoietic murine cells, total bone marrow cells were isolated from femurs, spine and tibias of 6 to 12 week-old Wild-Type C57BL/6JOlaHsd (Envigo) or Vav-cre X Floxed- Jak2 V617F C57BL/6 mice , by bone crushing then passed through a 70 μM cell strainer to obtain a single cell suspension, followed by red blood cell lysis (Sigma-Aldrich). c-Kit + cells were then magnetically sorted (CD117 MicroBeads, mouse, Cat #130-091-224, Miltenyi Biotec) and maintained in StemSpan SFEM (StemCell Technologies) supplemented with 1% penicillin-streptomycin (Gibco) and 10 ng/ml mIL-3, 10 ng/ml IL-6, 25 ng/ml mFLT3-ligand, and 25 ng/ml mSCF (Peprotech).

Techniques: Quantitative RT-PCR, Expressing, Infection, Plasmid Preparation, Two Tailed Test, Inhibition, In Vitro, Competitive Binding Assay, Colony Assay

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A NRAS and JAK2 expression level qRT-PCR in Empty or NRAS Q61K HEL and UKE-1 cells. Error bars represent mean of n = 3 (HEL) and n = 4 (UKE-1) biological replicates ± SD. B Western blot for indicated proteins, in Empty or NRAS Q61K HEL and UKE-1. The experiment was performed twice with similar results. C , D Gene Set Enrichment Analysis (GSEA) for the AML dataset Beat_AML_v2, across the collection of MSigDB_v7.4 Hallmark (50) and additional RAS/MAPK (24) and cell cycle (11) gene sets. Data represented as volcano plots ( C ) of –log10(p-value + 0.0001) versus the Normalized Enrichment Score (NES) for each gene set. NES using Kolmogorov-Smirnov enrichment test. Gene sets related to RAS/MAPK and Cell Cycle highlighted in red and blue respectively. Gray dots indicate all other. Representative GSEA plots ( D ) for gene sets related to RAS/MAPK and Cell Cycle. E Heatmaps depicting relative gene expression changes for leading edge genes of the top RAS/MAPK and Cell Cycle enriched gene sets. Depleted and enriched genes are respectively in blue and red. Row-normalized data. F Propidium Iodide cell cycle analysis of Empty or NRAS Q61K HEL and UKE-1 cells after 11 or 9 days of ruxolitinib (3 and 15 µM, respectively). Cells in sub G1 not represented. Mean of n = 2 biological replicates. G EdU incorporation of Empty or NRAS Q61K HEL and UKE-1 cells after 12 days of ruxolitinib (3 and 15 µM, respectively). Error bars represent the mean of n = 3 biological replicates ± SEM. H , I Beta-galactosidase staining for Empty or NRAS Q61K HEL and UKE-1 cells after 6 days of ruxolitinib (3 and 15 µM, respectively). Quantification ( H ) and representative images ( I ). Error bars represent mean of n = 2 (HEL) and n = 3 (UKE-1) biological replicates ± SD. J Proliferation curves of HEL cells expressing Empty, Nras Q61K , JAK2 V617F or Nras Q61K and JAK2 V617F vectors. Error bars represent mean ± SD of n = 10 (day 0), n = 8 (days 3, 6), n = 4 (day 9) biological replicates. Statistical significance using two-tailed Welch’s t-test ( A, F –H, J ). P-values are reported in the figure. Source data provided as a Source Data file.

Article Snippet: To obtain Jak2 V617F and Jak2 WT primary hematopoietic murine cells, total bone marrow cells were isolated from femurs, spine and tibias of 6 to 12 week-old Wild-Type C57BL/6JOlaHsd (Envigo) or Vav-cre X Floxed- Jak2 V617F C57BL/6 mice , by bone crushing then passed through a 70 μM cell strainer to obtain a single cell suspension, followed by red blood cell lysis (Sigma-Aldrich). c-Kit + cells were then magnetically sorted (CD117 MicroBeads, mouse, Cat #130-091-224, Miltenyi Biotec) and maintained in StemSpan SFEM (StemCell Technologies) supplemented with 1% penicillin-streptomycin (Gibco) and 10 ng/ml mIL-3, 10 ng/ml IL-6, 25 ng/ml mFLT3-ligand, and 25 ng/ml mSCF (Peprotech).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Cell Cycle Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A Percentage of CD45.1/2 Nras G12D cells among CD45.1/2 Nras G12D and CD45.1 Nras WT Lin − murine cells (50/50 ratio) treated with DMSO or ruxolitinib (0.25 µM). B Proliferation curves of Nras G12D or Nras WT Lin − cells after DMSO or ruxolitinib (0.25 µM). A, B Mean of n = 2 biological replicates. C In vivo Nras G12D competition model. Created in BioRender . D Percentage of CD45.1/2 Nras G12D cells in peripheral blood of mice transplanted with 10% CD45.1/2 Jak2 WT Nras G12D and 90% CD45.2 Jak2 V617F Nras WT Lin − cells ( n = 4 per group). Ruxolitinib (90 mg/kg twice daily) was started 4 weeks post-transplantation. E In vivo Nras Q61K competition model. Created in BioRender . F Percentage of GFP + Nras Q61K Jak2 V617F cells in the bone marrow of mice transplanted with Jak2 V617F cells expressing a GFP + Nras Q61K vector ( n = 5 per group). Ruxolitinib (90 mg/kg twice daily) was started 3 weeks post-transplantation. G , H Colony formation assay for cKit + bone marrow cells from Jak2 WT ( G ) or Jak2 V617F ( H ) mice expressing Empty or Nras Q61K vectors. Colony number after at least 6 days of DMSO or ruxolitinib (1 µM) ( n = 4 biological replicates). I Western blot for the indicated proteins in HEL cells expressing GFP, Empty or NRAS Q61K vectors, treated 24 h with ruxolitinib (3 µM). J In vitro competition model. Created in BioRender . K Percentage of GFP + and GFP − _Empty or GFP − _ NRAS Q61K HEL or UKE-1 cells. 24 days after ruxolitinib (3 µM and 15 µM for HEL and UKE-1, respectively), the indicated cells were treated 3 days with ruxolitinib or ruxolitinib (Ruxo.) and trametinib (Trame.) (10 µM and 0.1 µM for HEL and UKE-1, respectively) ( n = 3 biological replicates). L Western blot for the indicated proteins in Ba/F3 MPL-CALR WT and MPL-CALR del52 cells expressing Empty or NRAS Q61K vectors, treated 24 h with ruxolitinib (75 nM). M Percentage of Crimson + and Crimson − _Empty or Crimson − _ NRAS Q61K Ba/F3 MPL-CALR WT and MPL-CALR del52 cells after ruxolitinib ( n = 3 biological replicates). Statistical significance using two-tailed Mann–Whitney ( D, F ) or Welch’s t-test ( H, K, M ). Experiments( I, L ) were performed twice with similar results. Error bars represent mean ± SEM. P-values in the figure. Source data provided as Source Data file. Schemas created using BioRender.

Article Snippet: MSCV-IRES-GFP (Empty Vector), MSCV-IRES-mCherry FP (mCherry Vector) and MSCV-IRES-GFP ASXL1 (ASXL1G646Wfs*12 Vector) were a gift from Tannishtha Reya, Dario Vignali & Anjana Rao (Addgene # 20672, # 52114 and # 81021). pcw107 (Empty Vector) and JAK2 (V617F)-pcw107-V5 ( JAK2 V 617F Vector) were a gift from John Doench, Kris Wood & David Sabatini (Addgene # 62511 and # 64610).

Techniques: In Vivo, Transplantation Assay, Expressing, Plasmid Preparation, Colony Assay, Western Blot, In Vitro, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A qRT-PCR for Nras and Jak2 expression level in murine 32D cells infected with an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 4 biological replicates ± SEM. B Growth inhibition of 32D murine cells expressing an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector 6 days after GFP sorting. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 6 biological replicates ± SEM. C Model of the in vitro Nras Q61K -sh Jak2 murine myeloid cells competition assay. Created in BioRender . D Percentage of GFP + _CRIMSON − 32D cells expressing an Empty, a Nras Q61K or a Nras Q61K _sh Jak2 encoding vector 3 days after sorting. Statistical significance was determined using two-tailed Welch’s t-test. Error bars represent mean of n = 3 biological replicates ± SEM. E –F Colony formation assay of Jak2 WT (E) or Jak2 V 617F (F) C57BL/6 primary bone marrow c-Kit + murine cells expressing an Empty, a Nras Q61K or a Nras Q61K -sh Jak2 encoding vector at least 6 days after GFP sorting. Statistical significance was determined using a two-tailed Welch’s t-test. Error bars represent the mean of n = 4 biological replicates ± SEM. G Genetic variants association with resistance to the JAK2 inhibitors ruxolitinib ( n = 497), momelotinib ( n = 476) and fedratinib ( n = 93) according to the Beat AML v2 human primary patient AML dataset. Data is presented as volcano plots for gene variants' effect size (Glass) on the x-axis versus –log10(P-value) on the y-axis. Significance was set at – log10(P-value)>1.3, based on the two-tailed Student t-test with Welch’s correction, with variants having a negative effect size associated with drug sensitivity (red), while those having a positive effect size associated with drug resistance (blue). P-values are reported in the figure. Source data provided as a Source Data file.

Article Snippet: MSCV-IRES-GFP (Empty Vector), MSCV-IRES-mCherry FP (mCherry Vector) and MSCV-IRES-GFP ASXL1 (ASXL1G646Wfs*12 Vector) were a gift from Tannishtha Reya, Dario Vignali & Anjana Rao (Addgene # 20672, # 52114 and # 81021). pcw107 (Empty Vector) and JAK2 (V617F)-pcw107-V5 ( JAK2 V 617F Vector) were a gift from John Doench, Kris Wood & David Sabatini (Addgene # 62511 and # 64610).

Techniques: Quantitative RT-PCR, Expressing, Infection, Plasmid Preparation, Two Tailed Test, Inhibition, In Vitro, Competitive Binding Assay, Colony Assay

Journal: Nature Communications

Article Title: JAK2 inhibition mediates clonal selection of RAS pathway mutations in myeloproliferative neoplasms

doi: 10.1038/s41467-025-60884-1

Figure Lengend Snippet: A NRAS and JAK2 expression level qRT-PCR in Empty or NRAS Q61K HEL and UKE-1 cells. Error bars represent mean of n = 3 (HEL) and n = 4 (UKE-1) biological replicates ± SD. B Western blot for indicated proteins, in Empty or NRAS Q61K HEL and UKE-1. The experiment was performed twice with similar results. C , D Gene Set Enrichment Analysis (GSEA) for the AML dataset Beat_AML_v2, across the collection of MSigDB_v7.4 Hallmark (50) and additional RAS/MAPK (24) and cell cycle (11) gene sets. Data represented as volcano plots ( C ) of –log10(p-value + 0.0001) versus the Normalized Enrichment Score (NES) for each gene set. NES using Kolmogorov-Smirnov enrichment test. Gene sets related to RAS/MAPK and Cell Cycle highlighted in red and blue respectively. Gray dots indicate all other. Representative GSEA plots ( D ) for gene sets related to RAS/MAPK and Cell Cycle. E Heatmaps depicting relative gene expression changes for leading edge genes of the top RAS/MAPK and Cell Cycle enriched gene sets. Depleted and enriched genes are respectively in blue and red. Row-normalized data. F Propidium Iodide cell cycle analysis of Empty or NRAS Q61K HEL and UKE-1 cells after 11 or 9 days of ruxolitinib (3 and 15 µM, respectively). Cells in sub G1 not represented. Mean of n = 2 biological replicates. G EdU incorporation of Empty or NRAS Q61K HEL and UKE-1 cells after 12 days of ruxolitinib (3 and 15 µM, respectively). Error bars represent the mean of n = 3 biological replicates ± SEM. H , I Beta-galactosidase staining for Empty or NRAS Q61K HEL and UKE-1 cells after 6 days of ruxolitinib (3 and 15 µM, respectively). Quantification ( H ) and representative images ( I ). Error bars represent mean of n = 2 (HEL) and n = 3 (UKE-1) biological replicates ± SD. J Proliferation curves of HEL cells expressing Empty, Nras Q61K , JAK2 V617F or Nras Q61K and JAK2 V617F vectors. Error bars represent mean ± SD of n = 10 (day 0), n = 8 (days 3, 6), n = 4 (day 9) biological replicates. Statistical significance using two-tailed Welch’s t-test ( A, F –H, J ). P-values are reported in the figure. Source data provided as a Source Data file.

Article Snippet: MSCV-IRES-GFP (Empty Vector), MSCV-IRES-mCherry FP (mCherry Vector) and MSCV-IRES-GFP ASXL1 (ASXL1G646Wfs*12 Vector) were a gift from Tannishtha Reya, Dario Vignali & Anjana Rao (Addgene # 20672, # 52114 and # 81021). pcw107 (Empty Vector) and JAK2 (V617F)-pcw107-V5 ( JAK2 V 617F Vector) were a gift from John Doench, Kris Wood & David Sabatini (Addgene # 62511 and # 64610).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Gene Expression, Cell Cycle Assay, Staining, Two Tailed Test