jak2 v617f  (TargetMol)

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: Viruses

Article Title: TNF-Mediated Inhibition of Classical Swine Fever Virus Replication Is IRF1-, NF-κB- and JAK/STAT Signaling-Dependent

doi: 10.3390/v13102017

Figure Lengend Snippet: The anti-CSFV activity of TNF involves JAK/STAT signaling. ( a , b ) The PEDSV.15 cells were treated with pTNF ( a ) or IFN-β ( b ) in the presence of ruxolitinib (2 µM) or DMSO for six hours prior to CSFV-luc infection (MOI, 0.1 TCID 50 /cell) and measurement of firefly luciferase activity 22 h later. The values are shown as the percentage of the DMSO control in the absence of pTNF or IFN-β, respectively. ( c , d ) JAK/STAT inhibitor Compound Library screens were performed with CSFV-luc-infected cells pre-stimulated with the medium, LPS or pTNF in the presence of the individual JAK/STAT inhibitors at the concentration of 0.5 µM ( c ) or 5 µM ( d ). The data due to cytotoxic and antiviral activity of the compounds were eliminated from the analysis. ( e ) The PEDSV.15 cells were treated with pTNF or mock in the presence or absence of ruxolitinib for four hours, and the IFN-β mRNA to 18S ribosomal RNA ratio was quantified by means of RT-qPCR and plotted as log 10 fold induction. ( f ) The PEDSV.15 cells were transfected with an NF-κB-dependent firefly luciferase gene reporter plasmid and treated with IFN-β, pTNF, LPS in the presence of the NF-κB signaling inhibitor TPCA-1 or the JAK/STAT inhibitor ruxolitinib. The NF-κB activity is represented as fold luciferase induction related to the medium treatment. The data represent the means and the standard deviations of three independent experimental replicas. The differences were considered to be statistically significant at p < 0.05 using the Student’s t -test (ns, nonsignificant).

Article Snippet: The PEDSV.15 cells seeded in 96-well plates (3 × 10 4 cells/100 µL/well) were treated with small-molecule compounds of the JAK/STAT Compound Library (Targetmol, Wellesley Hills, MA, USA, cat. No. L3700) at two concentrations (0.5 µM and 5 µM) for approximately one hour prior to stimulation with either LPS (100 ng/mL), pTNF (5 ng/mL) or the medium.

Techniques: Activity Assay, Infection, Luciferase, Concentration Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot, Comparison

Journal: Blood

Article Title: MDH1-mediated malate-aspartate NADH shuttle maintains the activity levels of fetal liver hematopoietic stem cells

doi: 10.1182/blood.2019003940

Figure Lengend Snippet: STAT3 transactivates Mdh1 expression to maintain FL-HSC activities. (A) Mdh1 luciferase reporter and different doses of Stat3 were cotransfected into 293T cells, followed by the determination of luciferase activities (n = 3). (B) ChIP assays were analyzed with 293T cells cotransfected with pGL4.27-mdh1 promoter vector and Stat3 plasmid or control plasmid. Input control and amplification of the Stat3-binding sequence of Mdh1 were determined by semiquantitative PCR. (C) Bisulfite sequencing analysis of the methylation patterns of the CpG islands in the Mdh1 promoter in CD45+ SoNar-high and -low FL hematopoietic cell fractions is shown. Each row represents a unique DNA clone; filled and open circles represent methylated and unmethylated CpGs, respectively. (D) Quantification data of methylated and unmethylated CpGs (n = 3). (E) Protein levels of pSTAT3, STAT3, and MDH1 were measured in CD45+ SoNar-high and -low FL cells. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against SoNar-high#1 cells. (F) Representative flow cytometric analysis of intracellular level of pSTAT3 of total CD45+ FL hematopoietic cells and FL-HSCs. Mean fluorescence intensity (MFI) of each group was shown (isotype control, yellow and green lines). (G) Quantification of MFI of intracellular level of pSTAT3 in panel F (n = 3). (H) Immunoblotting assay showed protein levels of pSTAT3, STAT3, and MDH1 in total CD45+ FL hematopoietic cells and FL-HSCs. Ratios of pSTAT3/STAT3 and MDH1/actin were quantified and normalized against total cells. (I) Representative flow cytometric analysis of intracellular level of pSTAT3 of CD45+ SoNar-high and -low FL-HSCs. MFI of each group is shown (isotype control, yellow and green lines). (J) Quantification of MFI of intracellular level of pSTAT3 in panel I (n = 3). (K) Protein level of MDH1 was measured in Stat3-overexpressed SoNar 32D cells and control cells by immunoblotting. Ratios of STAT3/actin and MDH1/actin were quantified and normalized against control cells. (L) Stat3-overexpressed SoNar 32D cells and control cells were evaluated for the ratios of SoNar fluorescence, and representative images are shown. Scale bar, 10 μm. (M) Quantification of the ratios of SoNar fluorescence in panel L. A total of 30 SoNar 32D cells were analyzed (n = 3). (N) Working model for the connections between FL-HSC activities and Mdh1-mediated malate-aspartate shuttle as indicated by SoNar. Data are represented as mean ± standard error of the mean. Student 2-tailed unpaired t test (G,J,M) and 2-way analysis of variance with Sidak’s multiple comparison test (D) were used for the comparison. *P < .05, ***P < .001.

Article Snippet: To test the effect of STAT3 inhibitor on MDH1 expression, purified Lin − Sca-1 + c-Kit + FL-HSCs and adult HSCs were treated with 10 or 20 μM of STAT3 inhibitor C188-9 (TargetMol) for 24 hours according to the previous study 37 and subjected to determination of pSTAT3, STAT3, or MDH1 level by western blot.

Techniques: Expressing, Luciferase, Plasmid Preparation, Control, Amplification, Binding Assay, Sequencing, Methylation Sequencing, Methylation, Fluorescence, Western Blot, Comparison