Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: In order to inducibly delete RUNX1 in adult endothelium, we crossbred Cdh5(PAC)-CreERT2 mice (Taconic Cat# 13073, Rensselaer, NY) with Runx1(flox/flox) mice (Jackson Laboratory Cat# 008772, Bar Harbor, ME) and generated Cdh5-CreERT2;Runx1(flox/flox) mice in our lab . Mice with correct genotype were each treated with 2 mg of tamoxifen daily via intraperitoneal injection for 5 days followed by 1 week of incubation.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Experimental protocol for intervention of SuHx-PH in SOX17enhKO mice shows administration of the RUNX1 inhibitor Ro5-3335 or Ro24-7429 every other day for 6 times 1 week after the beginning of mild SuHx treatment. (B and C) RVSP (B) and RV/LV+S ratio (C) were measured at the end of week 3. Data in (B) and (C) are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques:

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Representative Western blot analyses of endothelial and hematopoietic markers in control versus RUNX1 overexpression HPAECs, with quantification of relative band intensities normalized to loading controls. (B and C) Human Endothelial Cell Biology RT²-PCR Array analyses in HPAECs overexpressing RUNX1 compared to control vector transduced cells. Gene expression values were normalized to housekeeping genes and represented as fold change relative to controls. Bar graphs and Volcano plots highlight differentially expressed genes meeting the significance criteria of fold change > 1.5 and FDR < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Gene Expression

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: HPAECs transduced with RUNX1 overexpressing (RUNX1 OE) or control lentivirus were subjected to quantitative assays to evaluate RUNX1-driven phenotypic and functional changes. (A) Morphometric assessment (length/width ratio) demonstrated pronounced alterations in cell morphology in RUNX1 OE cells. (B) Tube formation assays demonstrated impaired angiogenic network assembly (C) Migration assays showed increased gap closure over time in RUNX1 OE cells. (D) MTS proliferation assays indicated enhanced growth in RUNX1 OE cells. (E) Reduced Caspase-3/7 activity following TNF-α treatment demonstrated decreased apoptosis in RUNX1 OE cells. All image-based analyses (A-C) were quantified using ImageJ software. Statistical significance for (D and E) was determined by two-way ANOVA with multiple comparisons, while all other comparisons were performed using unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transduction, Control, Functional Assay, Migration, Activity Assay, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Western blot analyses show (A) increased RUNX1 protein levels in SOX17 KO HPAECs, with quantification of relative band intensity normalized to β-ACTIN. (B and C) Treatment of SOX17 KO HPAECs with RUNX1 inhibitor Ro5-3335 at 0, 10 or 25 µM (B) or RUNX1-targeting siRNA (C) leads to partial restoration of endothelial marker expression in the absence of SOX17. *p < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A and B) RT-PCR analyses (A) and Western blot analyses (B) demonstrate increased RUNX1 expression and reduced endothelial marker levels in iPSC-ECs derived from SOX17 mutant patients compared with healthy controls. (C) Western blots shows in a dose-dependent manner RUNX1 inhibitor Ro5-3335 partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. (D) Western blots shows RUNX1 targeting siRNA downregulation partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 denote statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: RUNX1 expression in whole bone marrow (A) and whole lung (B) in SOX17enhKO and WT mice after 3 weeks of normoxia (Norm) or Sugen/hypoxia (SuHx). N = 5-15 mice per group, * P < 0.05, ** P < 0.01, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Out of the 359 PAH patients, SOX17 expression was undetectable in 206 and detectable in 153 patients, whereas the expression of RUNX1 could be seen in all subjects. RUNX1 expression was significantly higher in patients with undetectable expression of SOX17 compared to those with detectable SOX17 expression. * P < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Experimental protocol for intervention of SuHx-PH in SOX17enhKO mice shows administration of the RUNX1 inhibitor Ro5-3335 or Ro24-7429 every other day for 6 times 1 week after the beginning of mild SuHx treatment. (B and C) RVSP (B) and RV/LV+S ratio (C) were measured at the end of week 3. Data in (B) and (C) are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques:

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Representative Western blot analyses of endothelial and hematopoietic markers in control versus RUNX1 overexpression HPAECs, with quantification of relative band intensities normalized to loading controls. (B and C) Human Endothelial Cell Biology RT²-PCR Array analyses in HPAECs overexpressing RUNX1 compared to control vector transduced cells. Gene expression values were normalized to housekeeping genes and represented as fold change relative to controls. Bar graphs and Volcano plots highlight differentially expressed genes meeting the significance criteria of fold change > 1.5 and FDR < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Western Blot, Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Gene Expression

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: HPAECs transduced with RUNX1 overexpressing (RUNX1 OE) or control lentivirus were subjected to quantitative assays to evaluate RUNX1-driven phenotypic and functional changes. (A) Morphometric assessment (length/width ratio) demonstrated pronounced alterations in cell morphology in RUNX1 OE cells. (B) Tube formation assays demonstrated impaired angiogenic network assembly (C) Migration assays showed increased gap closure over time in RUNX1 OE cells. (D) MTS proliferation assays indicated enhanced growth in RUNX1 OE cells. (E) Reduced Caspase-3/7 activity following TNF-α treatment demonstrated decreased apoptosis in RUNX1 OE cells. All image-based analyses (A-C) were quantified using ImageJ software. Statistical significance for (D and E) was determined by two-way ANOVA with multiple comparisons, while all other comparisons were performed using unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Transduction, Control, Functional Assay, Migration, Activity Assay, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Western blot analyses show (A) increased RUNX1 protein levels in SOX17 KO HPAECs, with quantification of relative band intensity normalized to β-ACTIN. (B and C) Treatment of SOX17 KO HPAECs with RUNX1 inhibitor Ro5-3335 at 0, 10 or 25 µM (B) or RUNX1-targeting siRNA (C) leads to partial restoration of endothelial marker expression in the absence of SOX17. *p < 0.05.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A and B) RT-PCR analyses (A) and Western blot analyses (B) demonstrate increased RUNX1 expression and reduced endothelial marker levels in iPSC-ECs derived from SOX17 mutant patients compared with healthy controls. (C) Western blots shows in a dose-dependent manner RUNX1 inhibitor Ro5-3335 partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. (D) Western blots shows RUNX1 targeting siRNA downregulation partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 denote statistical significance.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: RUNX1 expression in whole bone marrow (A) and whole lung (B) in SOX17enhKO and WT mice after 3 weeks of normoxia (Norm) or Sugen/hypoxia (SuHx). N = 5-15 mice per group, * P < 0.05, ** P < 0.01, ns: not significant.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Out of the 359 PAH patients, SOX17 expression was undetectable in 206 and detectable in 153 patients, whereas the expression of RUNX1 could be seen in all subjects. RUNX1 expression was significantly higher in patients with undetectable expression of SOX17 compared to those with detectable SOX17 expression. * P < 0.05.

Article Snippet: Treatment with mild Sugen/hypoxia decreased RUNX1 expression in the bone marrow of wild-type mice, but increased RUNX1 in bone marrow of SOX17enhKO mice.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Experimental protocol for intervention of SuHx-PH in SOX17enhKO mice shows administration of the RUNX1 inhibitor Ro5-3335 or Ro24-7429 every other day for 6 times 1 week after the beginning of mild SuHx treatment. (B and C) RVSP (B) and RV/LV+S ratio (C) were measured at the end of week 3. Data in (B) and (C) are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques:

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Representative Western blot analyses of endothelial and hematopoietic markers in control versus RUNX1 overexpression HPAECs, with quantification of relative band intensities normalized to loading controls. (B and C) Human Endothelial Cell Biology RT²-PCR Array analyses in HPAECs overexpressing RUNX1 compared to control vector transduced cells. Gene expression values were normalized to housekeeping genes and represented as fold change relative to controls. Bar graphs and Volcano plots highlight differentially expressed genes meeting the significance criteria of fold change > 1.5 and FDR < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Western Blot, Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Gene Expression

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: HPAECs transduced with RUNX1 overexpressing (RUNX1 OE) or control lentivirus were subjected to quantitative assays to evaluate RUNX1-driven phenotypic and functional changes. (A) Morphometric assessment (length/width ratio) demonstrated pronounced alterations in cell morphology in RUNX1 OE cells. (B) Tube formation assays demonstrated impaired angiogenic network assembly (C) Migration assays showed increased gap closure over time in RUNX1 OE cells. (D) MTS proliferation assays indicated enhanced growth in RUNX1 OE cells. (E) Reduced Caspase-3/7 activity following TNF-α treatment demonstrated decreased apoptosis in RUNX1 OE cells. All image-based analyses (A-C) were quantified using ImageJ software. Statistical significance for (D and E) was determined by two-way ANOVA with multiple comparisons, while all other comparisons were performed using unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Transduction, Control, Functional Assay, Migration, Activity Assay, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Western blot analyses show (A) increased RUNX1 protein levels in SOX17 KO HPAECs, with quantification of relative band intensity normalized to β-ACTIN. (B and C) Treatment of SOX17 KO HPAECs with RUNX1 inhibitor Ro5-3335 at 0, 10 or 25 µM (B) or RUNX1-targeting siRNA (C) leads to partial restoration of endothelial marker expression in the absence of SOX17. *p < 0.05.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A and B) RT-PCR analyses (A) and Western blot analyses (B) demonstrate increased RUNX1 expression and reduced endothelial marker levels in iPSC-ECs derived from SOX17 mutant patients compared with healthy controls. (C) Western blots shows in a dose-dependent manner RUNX1 inhibitor Ro5-3335 partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. (D) Western blots shows RUNX1 targeting siRNA downregulation partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 denote statistical significance.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: RUNX1 expression in whole bone marrow (A) and whole lung (B) in SOX17enhKO and WT mice after 3 weeks of normoxia (Norm) or Sugen/hypoxia (SuHx). N = 5-15 mice per group, * P < 0.05, ** P < 0.01, ns: not significant.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Out of the 359 PAH patients, SOX17 expression was undetectable in 206 and detectable in 153 patients, whereas the expression of RUNX1 could be seen in all subjects. RUNX1 expression was significantly higher in patients with undetectable expression of SOX17 compared to those with detectable SOX17 expression. * P < 0.05.

Article Snippet: Similarly, RUNX1 expression in the lung did not differ between SOX17enhKO and wild-type mice under normoxic conditions and treatment with low dose Sugen/hypoxia increased RUNX1 expression in the lungs of SOX17enhKO mice but not in wild-type mice ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Experimental protocol for intervention of SuHx-PH in SOX17enhKO mice shows administration of the RUNX1 inhibitor Ro5-3335 or Ro24-7429 every other day for 6 times 1 week after the beginning of mild SuHx treatment. (B and C) RVSP (B) and RV/LV+S ratio (C) were measured at the end of week 3. Data in (B) and (C) are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques:

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A) Representative Western blot analyses of endothelial and hematopoietic markers in control versus RUNX1 overexpression HPAECs, with quantification of relative band intensities normalized to loading controls. (B and C) Human Endothelial Cell Biology RT²-PCR Array analyses in HPAECs overexpressing RUNX1 compared to control vector transduced cells. Gene expression values were normalized to housekeeping genes and represented as fold change relative to controls. Bar graphs and Volcano plots highlight differentially expressed genes meeting the significance criteria of fold change > 1.5 and FDR < 0.05. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Gene Expression

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: HPAECs transduced with RUNX1 overexpressing (RUNX1 OE) or control lentivirus were subjected to quantitative assays to evaluate RUNX1-driven phenotypic and functional changes. (A) Morphometric assessment (length/width ratio) demonstrated pronounced alterations in cell morphology in RUNX1 OE cells. (B) Tube formation assays demonstrated impaired angiogenic network assembly (C) Migration assays showed increased gap closure over time in RUNX1 OE cells. (D) MTS proliferation assays indicated enhanced growth in RUNX1 OE cells. (E) Reduced Caspase-3/7 activity following TNF-α treatment demonstrated decreased apoptosis in RUNX1 OE cells. All image-based analyses (A-C) were quantified using ImageJ software. Statistical significance for (D and E) was determined by two-way ANOVA with multiple comparisons, while all other comparisons were performed using unpaired two-tailed t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transduction, Control, Functional Assay, Migration, Activity Assay, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Western blot analyses show (A) increased RUNX1 protein levels in SOX17 KO HPAECs, with quantification of relative band intensity normalized to β-ACTIN. (B and C) Treatment of SOX17 KO HPAECs with RUNX1 inhibitor Ro5-3335 at 0, 10 or 25 µM (B) or RUNX1-targeting siRNA (C) leads to partial restoration of endothelial marker expression in the absence of SOX17. *p < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Western Blot, Marker, Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: (A and B) RT-PCR analyses (A) and Western blot analyses (B) demonstrate increased RUNX1 expression and reduced endothelial marker levels in iPSC-ECs derived from SOX17 mutant patients compared with healthy controls. (C) Western blots shows in a dose-dependent manner RUNX1 inhibitor Ro5-3335 partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. (D) Western blots shows RUNX1 targeting siRNA downregulation partially restored endothelial marker expression in iPSC-ECs derived from SOX17 mutant PAH patients. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 denote statistical significance.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Marker, Derivative Assay, Mutagenesis

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: RUNX1 expression in whole bone marrow (A) and whole lung (B) in SOX17enhKO and WT mice after 3 weeks of normoxia (Norm) or Sugen/hypoxia (SuHx). N = 5-15 mice per group, * P < 0.05, ** P < 0.01, ns: not significant.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Cdh5-CreERT2;Runx1(flox/flox);SOX17enhKO triple transgenic SRV mice were subjected to mild SuHx treatment. When the SRV mice were treated with corn oil without tamoxifen, they developed significantly elevated RVSP. When the SRV mice were treated with tamoxifen and placed under mild SuHx conditions, they exhibited normal RVSP. **** P < 0.0001.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Impaired SOX17 Expression Causes Endothelial Dysfunction and Pulmonary Arterial Hypertension by Insufficient Suppression of RUNX1

doi: 10.64898/2026.05.14.725187

Figure Lengend Snippet: Out of the 359 PAH patients, SOX17 expression was undetectable in 206 and detectable in 153 patients, whereas the expression of RUNX1 could be seen in all subjects. RUNX1 expression was significantly higher in patients with undetectable expression of SOX17 compared to those with detectable SOX17 expression. * P < 0.05.

Article Snippet: To generate stable RUNX1 overexpression HPAECs, full-length human RUNX1 cDNA (OriGene, Cat# SC123977) was cloned into the pHAGE-EF1α-IRES-blast vector from our lab. High titer lentiviruses were produced at Brown University Legorreta Cancer Center Cell & Genome Engineering Shared Resource.

Techniques: Expressing

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection (UMAP) clustering of 434,810 cells from eight public scRNA-seq datasets. ( B ) Dot plot showing relative Cdh5 and Runx1 co-expression across clusters; clusters 8 and 50 co-express both genes. ( C ) UMAP highlighting clusters 8 and 50; all other clusters shown in grey. ( D ) Violin plots of doublet scores across Leiden clusters. Clusters 50 and 8 show no evidence of doublet enrichment. ( E ) Datasets proportional contribution to clusters 50 and 8; each dataset is color-coded. ( F ) Dot plot showing expression of selected marker genes in clusters 50 and 8 (from the public sc RNA-seq datasets listed in ) and from clusters 0, 1, 13, 22, and 14 (from Polylox scRNA-seq; ). Results reflect mean expression and fraction of cells in group. ( G ) Cdh5, Runx1, and Col1a2 co-expression in the indicated clusters as a fraction of cells in the cluster. ( H, I ) t-SNE plot of ECs from 11 murine tissues ( G ) and Venn diagram ( H ) showing rare co-expression of Cdh5, Runx1, and Col1a2 in these tissues.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Expressing, Marker, RNA Sequencing

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Schematic representation of the Col1a2 tracking lines. ( B ) Representative confocal image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mouse, showing widespread distribution of ZsGreen + cells. Flow cytometric identification of RUNX1 + VE-Cadherin + CD45 − endothelial cells (ECs) in the BM of peanut oil-treated ( n = 6) and tamoxifen-treated ( n = 6) Col1a2-Cre ERT2 /ZsGreen adult mice; Cre − mice ( n = 5) ( C ); WT C57Bl/6 mice ( n = 6) and Fluorescence Minus One (FMO) control ( n = 5) ( D ); and Cdh5-Cre ERT2 (PAC)/ZsGreen mice treated with peanut oil ( n = 6) or tamoxifen ( n = 6); Cre − mice ( n = 5) ( E ). Left panels: quantification of cells identified by the indicated gates as a percentage of total BM ECs; each dot represents one mouse (1 femur + 1 tibia). Middle and right panels: representative gating strategies. ( F ) Representative confocal microscopy image of a BM section from a tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen adult mouse showing a ZsGreen + Endomucin + cell lining a vascular structure (white arrows). ( G ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /ZsGreen mouse treated with tamoxifen showing the presence of a nucleate CD45 + cell tracked by ZsGreen/Col1a2 fluorescence (pointed by the arrow). ( H ) Representative confocal image of a blood smear from a Col1a2-Cre ERT2 /mTmG mouse treated with tamoxifen showing the presence of a nucleated CD45 + cell tracked by EGFP/Col1a2 fluorescence (pointed by the arrow). Dots represent individual mice. Data are shown as mean ± SD. ***p < 0.001 by Student’s t -test.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Fluorescence, Control, Confocal Microscopy

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Percent EGFP + CD45 + cells in bone marrow (BM) and blood of tamoxifen-treated ( n = 6) or oil-treated ( n = 5) Col1a2-Cre ERT2 /mTmG mice ( A ) and tamoxifen-treated ( n = 4) or oil-treated ( n = 3) Col1a2-Cre ERT2 /ZsGreen mice ( B ). Cre-control mice ( n = 5 in A, and n = 2 in B). ( C ) Transplant experiment: sorted VE-Cadherin + CD45 − ZsGreen + /Col1a2 + cells from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice are transplanted into 5-FU-conditioned WT recipients. Detection ( D ) and characterization ( E ) of ZsGreen + CD45 + cells in BM and blood of WT 5-FU-conditioned mice ( n = 5), 4 weeks post-transplant of VE-Cadherin + CD45⁻ZsGreen + /Col1a2 + cells. Control FU-conditioned WT mice ( n = 4) received no cell transplant ( D ). ( F ) Time course of ZsGreen + peripheral blood mononuclear cell (PBMC) detection in control (Cdh5-Cre + /ZsGreen + ) and Runx1 EC-KI (Cdh5-Cre + /ZsGreen + /Runx1-KI) mice ( n = 10 per group). Representative images ( G ) and quantification ( H ) of ZsGreen + cells from OP9 cell-supported cultures of BM cells from tamoxifen-treated Cdh5-Cre + /ZsGreen + ( n = 11) and Runx1 EC-KI mice ( n = 5). Representative flow cytometry plots ( I ) and quantification ( J ) of CD45 + ZsGreen + cells from OP9 cell-supported BM cell cultures ( n = 5/group). Dots represent individual mice. Data are shown as mean ± SD. **p < 0.01, ***p < 0.001 by Student’s t -test.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Control, Flow Cytometry

Journal: eLife

Article Title: Single-cell lineage tracing identifies hemogenic endothelial cells in the adult mouse bone marrow

doi: 10.7554/eLife.109553

Figure Lengend Snippet: Related to . ( A ) Representative FACS gating strategy used to isolate BM hematopoietic cells, ECs, stromal cells, and Col1a2-tracked ECs from tamoxifen-treated Col1a2-Cre ERT2 /ZsGreen mice. ( B ) Gene expression profiling of unsorted BM and sorted BM populations defined in ( A ). Results from qRT-PCR are normalized by Gapdh and unsorted BM. Dots reflect experimental triplicates. ( C ) Representative confocal image of a BM section from a transplant recipient showing a CD45 + CD11b + ZsGreen + cell (arrow), indicating hematopoietic derivation from transplanted ZsGreen + VE-Cadherin + CD45 − (Col1a2 + ) cells. ( D ) Representative image of a blood smear from a WT recipient mouse transplanted with ZsGreen (Col1a2) + BM ECs from Col1a2-Cre ERT2 /ZsGreen mice. ZsGreen tracked cells are pointed by the arrows. ( E ) Schematic diagram of the Cdh5-Cre ERT2 /ZsGreen/Runx1-Knock-in (Runx1 EC-KI ) mouse line used to trace ECs with Runx1 expression induced upon tamoxifen treatment.

Article Snippet: TaqMan probes used were purchased from Applied Biosystems: Spp1 (Mm00436767_m1); Cxcl12 (Mm00445553_m1); Col1a2 (Mm00483888_m1); Cdh5 (Mm00486938_m1); Runx1 (Mm01213404_m1); Ptprc (Mm01293577_m1); Gapdh (Mm99999915_g1); Actb (Mm02619580_g1).

Techniques: Gene Expression, Quantitative RT-PCR, Knock-In, Expressing