Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 mRNA expression in glomerular cells of human and mouse. A. The single-cell RNA-seq database search with KIT (http://humphreyslab.com/SingleCell/displaycharts.php) identified ANGPT2 mRNA expression in various kidney cell types, including glomerular cells (podocytes, mesangial cells and endothelial cells) and various tubular cell types. EC, endothelial cells; PT (S1), proximal tubular cells (segment 1); LH (DL), Loop of Henle (descending limb); LH (AL), Loop of Henle (ascending limb); DCT, distal convoluted tubule; PC, principal cells; IC, intercalated cells. B. GEO database search (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE123179) revealed ANGPT2 mRNA in podocytes (Pod), mesangial cells (Mes) and endothelial cells (Endo) from mice by bulk sequencing. N=3 for each group; *P=0.00046, mesangial cells vs. podocytes; P=0.00037, mesangial cells vs. endothelial cells.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, RNA Sequencing, Sequencing

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 protein expression in glomerular cells of human and mouse. A. Immunofluorescence staining of ANGPT2 in normal human glomeruli. Podocyte marker, SYNPO, was co-stained, thereby localizing ANGPT2 to podocytes and non-podocytes. B. Similar co-staining of ANGPT2 and SYNPO in mouse glomeruli showing ANGPT2 is present in both podocytes and non-podocytes in mice.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, Immunofluorescence, Staining, Marker

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 expression in glomeruli was upregulated in patients with diabetic nephropathy. From the gene expression dataset of glomeruli from diabetic patients (GSE96804), the glomerular ANGPT2 levels in DN patients at different stages of the diseases were obtained. Early: urinary albumin <0.5 g/24 h with normal serum creatinine; middle: urinary albumin >0.5 g/24 h with normal serum creatinine; late: serum creatinine >1.24 mg/dL. *P<0.05, **P<0.01, significant difference vs. normal subjects (Ctrl).

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing, Gene Expression

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: ANGPT2 expression in glomeruli was upregulated in patients with multiple glomerular diseases. A. Nephroseq searches identified ANGPT2 upregulation in glomeruli of patients with CKD (Normal n=3; CKD n=5; fold change 6.98, P=0.001). B. ANGPT2 upregulation in hypertension (Normal n=4; hypertension n=14; fold change 1.22, P=0.023). C. ANGPT2 upregulation in FSGS (Normal n=9; FSGS n=8; fold change 1.36, P=0.033). D. ANGPT2 in LN (Normal n=14; LN n=32; fold change 1.41, P=0.005). *P<0.05, significant vs. normal subjects.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: Gene expression regulation by ANGPT2 in podocytes and mesangial cells in culture. A. Volcano plot visualization of statistically significant gene expression changes in ANGPT2-treated podocytes (left) and mesangial cells (right) versus untreated cells, respectively. X-axis, log fold change; Y-axis, -log10 P value. B. Venn diagrams showing the huge difference in the list of genes regulated by ANGPT2 in podocytes vs. mesangial cells.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Gene Expression

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: GO analysis shows fundamental differences in enrichments between the regulated genes in podocytes (A) and those in mesangial cells (B) treated with ANGPT2.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques:

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: KEGG pathways analysis shows the difference in KEGG pathways between the group of genes regulated in podocytes (A) and that in mesangial cells (B) after treatment with ANGPT2.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques:

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: pERK was increased in ANGPT2-treated podocytes but decreased in mesangial cells in culture. ANGPT2 (500 ng/ml) was used to treat podocytes and mesangial cells for 24 h. The results represent the data from three independent experiments and are expressed as mean ± SD. *P<0.05; **P<0.01 vs. control.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Control

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: pAKT was increased in ANGPT2-treated podocytes but downregulated in mesangial cells in culture. Podocytes and mesangial cells were treated with 500 ng/ml ANGPT2 for 24 h and subject to immnunoblotting. The results represent the data from three independent experiments and are expressed as mean ± SD. **P<0.01 vs. control.

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Control

Journal: American Journal of Translational Research

Article Title: Distinct effects of ANGPT2 on gene expression of glomerular podocytes and mesangial cells

doi:

Figure Lengend Snippet: Expression of ANGPT2 interacting proteins in podocytes and mesangial cells

Article Snippet: The information of the reagents used in the present study is as follows: Antibodies against ANGPT2 (Affinity), SYNPO (Santa Cruz), GAPDH (Proteintech), total ERK1/2, phospho-ERK1/2, total AKT and phospho-AKT (Cell Signaling Technologies); Reverse transcription kit (DRR037A, Takara); Quantitative PCR kit (Thermo Fisher Scientific); RIPA cell lysis buffer, BCA protein quantification kit (Beyotime, Shanghai); RNA extraction kit (Takara); ANGPT2 (HY-P7510, MCE); Trizol (Invitrogen).

Techniques: Expressing

Journal: bioRxiv

Article Title: Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

doi: 10.1101/2021.02.15.431296

Figure Lengend Snippet: A, Cartoon model of 3-dimensional sprouting assay denoting imaging setup. B, Localization of transduced mCherry (Cherry)-Slp2a during lumen formation in sprouts stained for VE- Cadherin (VE-Cad), moesin, and podocalyxin (Podxl). C , Images of Slp2a siRNA (si) knockdown and scramble (scram) control sprouts stained for indicated proteins. D, Confirmation of Slp2a siRNA-mediated knockdown by western blot probed for Slp2a and alpha-tubulin (α-Tub). n=3. E, Quantification of sprout length for indicated groups. F, Quantification of non-lumenized sprouts between indicated groups. G, Mosaic rescue experiment in which cells were treated with indicated siRNAs and transduced with Cherry- Slp2a (red). Arrows indicate a lack of lumen in addition to a lack of Cherry-Slp2a expression. H, Quantification of percent non-lumenized sprouts between indicated groups. I, Mosaic knockdown experiment in which cells were treated with Slp2a siRNA (red) and then mixed with scramble-treated cells (non-fluorescent) and then challenged to sprout. Top row depicts non-opposing siRNA-treated cells. Bottom row depicts opposing siRNA-treated cells. Arrow denotes a lack of lumen. J, Quantification of mosaic KD sprouts with percent non-lumenized sprouts. All experiments used human umbilical vein endothelial cells. In all panels L denotes lumen; white box denotes magnification; white lines denotes exterior of sprout; values are means +/- SEM; n= individual sprouts across three experimental replicates; significance: *P<0.05, ***P<0.0005, NS=Not Significant. Statistical significance was assessed with an unpaired t-test or a 1-way ANOVA followed by a Dunnett multiple comparisons test.

Article Snippet: The following constructs were procured for the study: pGEX4T-1 (gift from Fernando Martin- Belmonte; Addgene plasmid #40059); pmCherry-C1 hSlp2-a (gift from Fernando Martin- Belmonte; Addgene plasmid #40056); pEGFP-C1 hSlp2-a C2AB (gift from Fernando Martin-Belmonte; Addgene plasmid #40051); pEGFP-C1 hSlp2-a del.SHD (gift from Fernando Martin-Belmonte; Addgene plasmid #40050); pEGFP-C1 hSlp4-a (gift from Fernando Martin-Belmonte; Addgene plasmid #40032); GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); pro- vWF-GFP/mCherry (gift from Tom Carter), and pCMV6-Angpt2 (Origene MR207970).

Techniques: Imaging, Staining, Western Blot, Transduction, Expressing

Journal: bioRxiv

Article Title: Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

doi: 10.1101/2021.02.15.431296

Figure Lengend Snippet: A, Cartoon model of Slp2a domains and mutants used for experimentation. Slp2a-ΔC2AB lacks two phospholipid binding C2 domains. Slp2a-C2AB mutant lacks the Rab-binding domain as well as residues linking it to the C2AB domains. B, GFP-Slp2a-C2AB expressing in both scramble (scram) and Slp2a siRNA(si) knockdown groups and stained for indicated proteins. Arrow indicates lack of lumen. C, MCherry(Cherry)-Slp2a-ΔC2AB expressing in both scramble and Slp2a siRNA knockdown groups and stained for indicated proteins. Arrow indicates lack of lumen. D, Quantification of lumen formation of individual sprouts. Cells were treated with scramble or Slp2a siRNA and then infected with indicated constructs. Green represents lumen formation and red represents non-lumenized sprouts. N-value represents individual sprouts across three experimental replicates. E, GFP-Slp2a-C2AB and Cherry-Slp2a-ΔC2AB expressing in sprouts stained for moesin and with Weibel-Palade body (WPB) marker, von Willebrand factor (vWF). F, Localization of Cherry-Slp2a-ΔC2AB prior to lumen opening (pre-lumen, top panels) and after lumen opening (lumenized, bottom panels). Arrows indicate heavy localization at the apical membrane. G, Quantification of Cherry-Slp2a-ΔC2AB localization pre-lumen and during lumenogenesis (lumenized). N-value represents individual sprouts across three experimental replicates. H, Live imaging of mCherry-Slp2a-WT and GFP-Slp4a-WT. Yellow arrow identifies future lumen expansion and white arrow indicates open lumen. All experiments use human umbilical vein endothelial cells. In all panels L denotes lumen; white box denotes magnification.

Article Snippet: The following constructs were procured for the study: pGEX4T-1 (gift from Fernando Martin- Belmonte; Addgene plasmid #40059); pmCherry-C1 hSlp2-a (gift from Fernando Martin- Belmonte; Addgene plasmid #40056); pEGFP-C1 hSlp2-a C2AB (gift from Fernando Martin-Belmonte; Addgene plasmid #40051); pEGFP-C1 hSlp2-a del.SHD (gift from Fernando Martin-Belmonte; Addgene plasmid #40050); pEGFP-C1 hSlp4-a (gift from Fernando Martin-Belmonte; Addgene plasmid #40032); GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); pro- vWF-GFP/mCherry (gift from Tom Carter), and pCMV6-Angpt2 (Origene MR207970).

Techniques: Binding Assay, Mutagenesis, Expressing, Staining, Infection, Construct, Marker, Imaging

Journal: bioRxiv

Article Title: Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

doi: 10.1101/2021.02.15.431296

Figure Lengend Snippet: A, 2-dimensional localization of mCherry(Cherry)-Slp2a and GFP-Rab27a in top panel. Bottom panel, localization of Cherry-Slp2a-ΔC2AB and GFP-Rab27a. B, Localization of Cherry-Slp2a and GFP-Rab27a in sprouts (top panels) and Cherry-Slp2a-ΔC2AB and GFP-Rab27a (bottom panels). C, Representative image of immunoprecipitation of GST-tagged Slp2a and GST (control) proteins used to probe for Rab27a binding. Image is one of three experimental replicates. D, Tom20-tagged GFP-Rab27a expressing cells also stained for mitochondria (Mito-tracker®). E, Tom20-GFP-Rab27a mis-localization experiments in 2D to test for binding interactions. Rab27a constitutively active (CA, Q78L) and dominant negative (DN, L130P) mutants were co-expressed with Cherry-Slp2a-ΔC2AB and Cherry-Slp2a. F, Quantification of localization of Cherry-Slp2a-ΔC2AB and mCherry-Slp2a in each of the 2D experiments presented in panel E. n=number of individual cells over three experimental repeats. All experiments use human umbilical vein endothelial cells. In all panels L denotes lumen; white box denotes magnification; white lines denote exterior of sprout.

Article Snippet: The following constructs were procured for the study: pGEX4T-1 (gift from Fernando Martin- Belmonte; Addgene plasmid #40059); pmCherry-C1 hSlp2-a (gift from Fernando Martin- Belmonte; Addgene plasmid #40056); pEGFP-C1 hSlp2-a C2AB (gift from Fernando Martin-Belmonte; Addgene plasmid #40051); pEGFP-C1 hSlp2-a del.SHD (gift from Fernando Martin-Belmonte; Addgene plasmid #40050); pEGFP-C1 hSlp4-a (gift from Fernando Martin-Belmonte; Addgene plasmid #40032); GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); pro- vWF-GFP/mCherry (gift from Tom Carter), and pCMV6-Angpt2 (Origene MR207970).

Techniques: Immunoprecipitation, Binding Assay, Expressing, Staining, Dominant Negative Mutation

Journal: bioRxiv

Article Title: Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

doi: 10.1101/2021.02.15.431296

Figure Lengend Snippet: A, Representative western blot confirmation of Rab27a siRNA (si) knockdown. n=3. B, Quantification of non-lumenized sprouts in indicated groups. n= individual sprouts over three experimental repeats. C, Quantification of lumen diameter at multiple locations within sprouts. Distances were measured proximally, at the mid- point, and distally from the bead. n=individual sprouts over three experimental repeats. D, Localization of Weibel-Palade body cargo von-Willebrand Factor (vWF), during lumen formation between siRNA-treated groups. Arrows indicate accumulation of vWF within the lumen. E , Quantification of vWF localization in indicated siRNA-treated groups. N= individual cells across three experimental repeats. F, Images of phorbol 12-myristate 13-acetate (PMA)- and vehicle (DMSO)-treated cells between indicated groups. G, Quantification of vWF fluorescent intensity between indicated conditions. n= individual cells across three experimental repeats. H, Mosaic rescue effect on vWF localization in sprouts between indicated groups. Cells were transduced with mCherry (Cherry)-Slp2a and treated with indicated siRNA. All experiments use human umbilical vein endothelial cells. AU= arbitrary unit. In all panels L denotes lumen; white box denotes magnification; white lines denote exterior of sprout; values are means +/- SEM; significance: *P<0.05, ***P<0.001, ****P<0.0001, NS=Not Significant. Statistical significance was assessed with a 1-way ANOVA followed by a Dunnett multiple comparisons test.

Article Snippet: The following constructs were procured for the study: pGEX4T-1 (gift from Fernando Martin- Belmonte; Addgene plasmid #40059); pmCherry-C1 hSlp2-a (gift from Fernando Martin- Belmonte; Addgene plasmid #40056); pEGFP-C1 hSlp2-a C2AB (gift from Fernando Martin-Belmonte; Addgene plasmid #40051); pEGFP-C1 hSlp2-a del.SHD (gift from Fernando Martin-Belmonte; Addgene plasmid #40050); pEGFP-C1 hSlp4-a (gift from Fernando Martin-Belmonte; Addgene plasmid #40032); GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); pro- vWF-GFP/mCherry (gift from Tom Carter), and pCMV6-Angpt2 (Origene MR207970).

Techniques: Western Blot, Transduction

Journal: bioRxiv

Article Title: Synaptotagmin-like protein 2a regulates lumen formation via Weibel-Palade body apical secretion of angiopoietin-2 during angiogenesis

doi: 10.1101/2021.02.15.431296

Figure Lengend Snippet: A, Angiopoietin-2 (Ang-2) colocalization experiments in 2-dimensional (2D) culture. Images show localization of Ang-2-RFP, GFP-Rab27a, von-Willebrand Factor (vWF), mCherry (Cherry)-Slp2a and Cherry- Slp2a-ΔC2AB. B, Ang-2 in cells with and without Weibel-Palade bodies (WPBs) denoted by vWF- positive staining. C, Quantification of Ang-2-GFP localization to WPBs between 2D culture and 3D sprouts. D, Ang-2-GFP and vWF localization in 3D sprouts. E, Ang-2-GFP localization at different time points during sprout development. The left panels are localization during the early stage of lumen formation and the right panels are after lumens are established. F, Quantification of Ang-2-GFP localization at different developmental time points. AU= arbitrary unit, AM= apical membrane and IC= intracellular. n=individual sprouts over three experimental repeats. All experiments use human umbilical vein endothelial cells. In all panels L denotes lumen; white box denotes magnification; white lines denote exterior of sprout; values are means +/- SEM; significance: *P<0.05, NS=Not Significant. Statistical significance was assessed with an unpaired t-test.

Article Snippet: The following constructs were procured for the study: pGEX4T-1 (gift from Fernando Martin- Belmonte; Addgene plasmid #40059); pmCherry-C1 hSlp2-a (gift from Fernando Martin- Belmonte; Addgene plasmid #40056); pEGFP-C1 hSlp2-a C2AB (gift from Fernando Martin-Belmonte; Addgene plasmid #40051); pEGFP-C1 hSlp2-a del.SHD (gift from Fernando Martin-Belmonte; Addgene plasmid #40050); pEGFP-C1 hSlp4-a (gift from Fernando Martin-Belmonte; Addgene plasmid #40032); GFP-Rab27A (gift from William Gahl; Addgene plasmid #89237); pro- vWF-GFP/mCherry (gift from Tom Carter), and pCMV6-Angpt2 (Origene MR207970).

Techniques: Staining