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: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

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 Ulk1 wild type versus Ulk1 knockout mouse MPN model used, and analyses performed. ( B ) Percentage of engraftment of CD45.2+ cells, ( C ) hemoglobin (Hb) levels, ( D ) hematocrit (HCT) percentage and ( E ) red blood cell (RBC) counts in peripheral blood of recipient mice are shown for two- and four-weeks post bone marrow transplant (BMT). ( F ) Percentage of GFP + BM cells four weeks post BMT of the recipient mice. ( G ) Percentage of GFP + ( G-i ) Ter119 med CD71 high proerythroblasts (R1), ( G-ii ) Pre-CFU-E, and ( G-iii ) PreMegE in the BM of recipient mice four weeks post BMT. ( H ) Spleen weight and ( I ) percentage of GFP + splenocytes four weeks post BMT of the recipient mice. ( J ) Percentage of GFP + ( J-i ) R1, ( J-ii ) R2 and ( J-iii ) R3+R4 cells in the spleen of recipient mice four weeks post BMT. ( B-E ) Data shown are means ± SD (n = 10 per group). Mixed effects models were used to analyze these data, with engraftment, Hb, HCT or RBC counts as the outcome variable, and time post-bone marrow transplant (BMT) (2 and 4 weeks), 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. ( F-J ) Box plots show distribution of data from 10 mice per group. Statistical analyses were performed using Mann-Whitney test. ( B-J ) Statistically significant p -values are reported. See also supplemental figures 4 and 5 .

Article Snippet: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

Techniques: In Vivo, Knock-Out, 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: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

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: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

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-B ) HEL cells were treated with either vehicle (DMSO, Ctrl), 5μM SBI-0206965 (SBI) or 5μM ULK101 for 6 hours followed by RNA-seq and analysis of transcript expression. ( A ) Heatmap of differentially expressed genes between treatment groups (adj. p val. < 0.01). ( B ) MA plot of genes significantly changed by drug-targeted inhibition of ULK1 in HEL cells versus control (adj. p val. < 0.01). In purple are represented up-regulated genes and in green are represented down-regulated genes. ( C and D ) Enriched ontology clusters for genes that are ( C ) increased or ( D ) decreased after drug-targeted inhibition of ULK1 in HEL cells. ( A-D ) Data are from 4 biological replicates per condition. See also supplemental figures 9 – 11 .

Article Snippet: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

Techniques: Inhibition, Activity Assay, Gene Expression, RNA Sequencing, Expressing, 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-E ) Scatter dot plots of PIM1 , ID2 , HES1 , CTR9 and PDCD2 expression in healthy individuals (normal, n = 11) and patients with ET (n = 47), PV (n = 28) and MF (n = 18). Data extracted from GSE5464672. Shown are means ± SEM of Log2 mRNA expression. Statistical analyses were performed using one-way ANOVA followed by Dunnett’s multiple comparisons adjustment comparing each MPN group vs. Normal group. Statistically significant p -values are reported. ( F-J ) mRNA expression of the indicated genes was assessed by qRT-PCR analysis for HEL cells treated for 6 hours with either vehicle (DMSO, Control) or SBP-7455 (SBP), as indicated. Data are means ± SEM from three independent experiments. Fold changes relative to control (dashed line) were calculated for each experimental replicate (SBP/DSMO), and fold changes were compared to FC=1 using a one-sample t-test, p values are reported. See also supplemental figure 12 .

Article Snippet: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, 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-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: Breeding pairs were set using both male and female mice (6–12-week-old) between: 1) Ulk1 +/+ mice (C57BL/6J, The Jackson Laboratory, Strain #:000664, RRID:IMSR_JAX:000664) and MxCre + mice (B6.Cg-Tg(Mx1-cre)1Cgn/J, The Jackson Laboratory, Strain #:003556, RRID:IMSR_JAX:003556) to generate Ulk1 +/+ MxCre + donor mice, 2) Ulk1 FL/FL mice (B6.129- Ulk1 tm1Thsn /J, The Jackson Laboratory, Strain #:017976, RRID:IMSR_JAX:017976) 22 and MxCre + mice to generate Ulk1 FL/+ MxCre + that were used for breeding to then generate Ulk1 FL/FL MxCre + donor mice, and 3) Jak2 V617F/+ conditional knock-in (KI) mice 27 (kindly provided by B. L. Ebert and A. Mullally, Harvard Medical School) and VavCre + mice (B6.Cg- Commd10 Tg(Vav1-icre)A2Kio /J, The Jackson Laboratory, Strain #: 008610, RRID:IMSR_JAX:008610) to generate Jak2 V617F/+ VavCre + donor mice.

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

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) Cross-sections of quadriceps from control (Ulk1+/−Ulk2−/flox) and Ulk1/2 hypomorph (Ulk1−/− Ulk2−/flox) mice at 24 weeks were stained with H&E and modified Gomori trichrome staining. Arrow indicates a crescentic inclusion and arrowhead indicates a vacuole. Scale bars, 25 μm.

Article Snippet: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Staining, Modification

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: (A) HEK293T cells were transfected with ULK1-DDK or ULK2-DDK and subjected to heat shock at 43°C for 1 h. Immediately following heat shock, cells were lysed and immunoprecipitated for DDK, followed by immunoblot against the indicated proteins.

Article Snippet: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Molecular cell

Article Title: ULK1/2 Regulates Stress Granule Disassembly Through Phosphorylation and Activation of VCP/p97

doi: 10.1016/j.molcel.2019.03.027

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The pMXs-IP-EGFP-ULK1 (38193) and pMXs-IP-EGFP-ULK2 (38201) were obtained from Addgene.

Techniques: Recombinant, Western Blot, Software

Journal: Cell reports

Article Title: Selenium Deficiency Is Associated with Pro-longevity Mechanisms

doi: 10.1016/j.celrep.2019.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-p-ULK1 , Cell Signaling Technology, sampler kit (8359T) , Ser 555 (mAb 5869) Ser 757 (mAb 6888).

Techniques: Recombinant, Expressing, Purification, Sequencing, Software