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

Journal: eLife

Article Title: Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex

doi: 10.7554/eLife.61432

Figure Lengend Snippet:

Article Snippet: The following plasmids were used in this study: pCDNA (control vector) (Clontech), pCDNA3-EGFP (Addgene Plasmid #13031), pCMV-mCherry-Mem (Addgene Plasmid #55779), pcDNA-Wnt3A (Addgene Plasmid #35908), pIRES-Jag1-HA (Addgene Plasmid #17336), pCMV-R-GECO1 (Addgene Plasmid #32444), pCMV-mCherry-CD9 (Addgene Plasmid #55013), pCMV-mCherry-CD81 (Addgene Plasmid #55012), pEGFP-CD63-C2 (Addgene Plasmid #62964), pCMV-EGFP-Rab18 (Addgene Plasmid #49550), pEGFP-C1-hMyoX (Addgene Plasmid #47608), pCMV6-hGAS1-Myc-DDK (Origene Cat: RC224804), pCMV3-mFGF2-N-GFPSpark (SinoBiological Cat: MG50037-ANG), pCMV3-mBMP2-C-GFPSpark (SinoBiological Cat: MG51115-ACG), pCMV-mCherry2, pCDNA3-mSHH-FL, pCDNA3-mSHH-N, pCDNA3-mSHH-FL-EGFP, pCDNA3-mSHH-FL-mCherry2, pCDNA3-V5-Disp-HA, pCS2-hBOC-EGFP and pCS2-hCDON-EGFP (a gift from A. Salic), pEGFP-C2-bMyo10-HMM ( ) and pEGFP-C2-bMyo10-Δ3PH, a modified version of pEGFP-bMyo10 where the 3 PH domains were removed via deletion of aa 1168–1491.

Techniques: Derivative Assay, Transfection, Construct, Recombinant, In Situ, Software, Imaging, Luciferase, Reporter Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks

doi: 10.1038/cddis.2013.443

Figure Lengend Snippet: Tanshinone-1 reduced Tyr705 phosphorylation of cellular Stat3, which contributed to apoptotic induction. ( a – d ) Tanshinone-1 (Tan-1) led to the dephosphorylation of Stat3 at Tyr705 in a time- and concentration-dependent manner in MDR and corresponding parental cells. The cells were treated with 40 μ M tanshinone-1 for the indicated time ( a , c and d ) or with gradient concentrations of tanshinone-1 for 4 h ( b ), and then detected by western blotting. ( e ) Tanshinone-1 reduced the protein expression of the Stat3-targeted genes. KB and KB/VCR cells were treated with or without 20 μ M tanshinone-1 for the indicated time and then western blotting for cellular levels of Mcl-1, cIAP-2, cyclin D1, c-Myc, and Survivin proteins was done. Basal levels (left) and reduced levels (right) of the examined proteins. ( f ) The siRNA for Stat3 (siStat3) decreased Stat3 expression and reversed the cleavage of procaspase 3 and PARP induced by tanshinone-1. KB/VCR cells were transfected with siStat3 for 48 h and then treated with 20 μ M tanshinone-1 for another 24 h. The cell lysates were subjected to western blotting. ( g and h ) siStat3 rescued apoptotic induction by tanshinone-1. KB/VCR cells were transfected with siStat3 for 48 h and then treated with 20 μ M tanshinone-1 for another 18 h. The resulting cells were stained with Annexin V/PI and analyzed by flow cytometry. The apoptosis rates from three independent experiments were expressed as mean±S.D.; * P <0.05 ( g ). Representative histograms are shown in h

Article Snippet: It is reported that residue substitutions of the mouse Stat3 at A661 and A663 with cysteine result in constitutive activation of Stat3., We amplified the human wild-type stat 3 expression plasmid from the HG10034-M-H clone (Sino Biological Inc., Beijing, China), and constructed an expression plasmid containing constitutively activated human Stat3 (pCMV-H3C) by site-directed mutagenesis as reported previously; , the mouse constitutively activated Stat3 plasmids were gifts from Drs Hua Yu and Jie-Hui Deng (Beckman Research Institute, USA) and Dr. James Darnell (The Rockefeller University, USA).

Techniques: De-Phosphorylation Assay, Concentration Assay, Western Blot, Expressing, Transfection, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks

doi: 10.1038/cddis.2013.443

Figure Lengend Snippet: Tanshinone-1 (Tan-1) activated cellular phosphatases. ( a ) Tan-1 did not inhibit upstream kinases of Stat3. Cells were treated with 40 μ M Tan-1 and analyzed by western blotting. ( b ) Na 3 VO 4 prevented the decrease of phospho-705-Stat3. Cells were pretreated with 20 μ M Na 3 VO 4 for 30 min, followed by treatment with 40 μ M Tan-1 for 4 h, and were then analyzed by western blotting. ( c ) Na 3 VO 4 completely prevented the Tan-1-mediated proliferation inhibition. Cells were pretreated with 20 μ M Na 3 VO 4 for 30 min, followed by the treatment with Tan-1 for 72 h, and were then analyzed by SRB assays and expressed as mean±S.D. ( d ) Na 3 VO 4 decreased cleavage of procaspase 3 and PARP and the reduction of cyclin D1, Mcl-1, and cIAP-2. Cells were pretreated with 20 μ M Na 3 VO 4 for 30 min, exposed to 20 μ M Tan-1 for 24 h, and then analyzed by western blotting. ( e and f ) Knockdown of Shp2 or PTP1B reversed the reduction of phospho-705-Stat3. Cells were transfected with siRNA against Shp2 and PTP1B for 48 h ( e ). The cells were then treated with 20 μ M Tan-1 for 4 h ( f ) and analyzed by western blotting. ( g and h ) Knockdown of Shp2 and PTP1B reduced the number of Annexin V/PI-positive cells. KB/VCR cells were transfected with siRNA against Shp2 and PTP1B for 48 h, followed by treatment with 20 μ M Tan-1 for 18 h, stained with Annexin V/PI, and then analyzed by flow cytometry. The apoptosis rates from three independent experiments were expressed as mean±S.D.; * P <0.05 ( g ). Representative histograms are shown in h

Article Snippet: It is reported that residue substitutions of the mouse Stat3 at A661 and A663 with cysteine result in constitutive activation of Stat3., We amplified the human wild-type stat 3 expression plasmid from the HG10034-M-H clone (Sino Biological Inc., Beijing, China), and constructed an expression plasmid containing constitutively activated human Stat3 (pCMV-H3C) by site-directed mutagenesis as reported previously; , the mouse constitutively activated Stat3 plasmids were gifts from Drs Hua Yu and Jie-Hui Deng (Beckman Research Institute, USA) and Dr. James Darnell (The Rockefeller University, USA).

Techniques: Western Blot, Inhibition, Transfection, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Tanshinone-1 induces tumor cell killing, enhanced by inhibition of secondary activation of signaling networks

doi: 10.1038/cddis.2013.443

Figure Lengend Snippet: Tanshinone-1 activated p38, AKT, and ERK signaling. ( a ) Signaling pathways consisting of Stat3, p38, AKT, and ERK, may crosstalk and influence their respective biological effects. ( b and c ) Phosphorylation was analyzed by western blotting. Tanshinone-1 (40 μ M, Tan-1) enhanced phosphorylation of p38, AKT, and ERK in MDR KB/VCR and parental KB cells ( b ) and in MDR K562/A02 and parental K562 cells ( c ). ( d ) The impact of p38, AKT, and ERK inhibitors on Tan-1-mediated enhancement of cellular p38, AKT, and ERK phosphorylation. KB/VCR cells were pretreated with PI103 (10 μ M, AKT) or AZD6244 (5 μ M, ERK) for 30 min or SB203580 (50 μ M, p38) for 10 min. The cells were then exposed to 20 μ M of Tan-1 for 15 min (p-p38 and p-ERK) or 4 h (p-AKT and p-705-Stat3) followed by western blotting analyses. ( e ) The impact of siStat3-induced Stat3 reduction on the Tan-1-mediated phosphorylation of p38, AKT, and ERK. KB/VCR cells were pretreated with siCtrl or siStat3 for 48 h. The cells were then exposed to 40 μ M of Tan-1 for the indicated time and were analyzed by western blotting

Article Snippet: It is reported that residue substitutions of the mouse Stat3 at A661 and A663 with cysteine result in constitutive activation of Stat3., We amplified the human wild-type stat 3 expression plasmid from the HG10034-M-H clone (Sino Biological Inc., Beijing, China), and constructed an expression plasmid containing constitutively activated human Stat3 (pCMV-H3C) by site-directed mutagenesis as reported previously; , the mouse constitutively activated Stat3 plasmids were gifts from Drs Hua Yu and Jie-Hui Deng (Beckman Research Institute, USA) and Dr. James Darnell (The Rockefeller University, USA).

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Ellagic Acid Attenuates BLM-Induced Pulmonary Fibrosis via Inhibiting Wnt Signaling Pathway

doi: 10.3389/fphar.2021.639574

Figure Lengend Snippet: Ellagic acid promotes pulmonary fibroblast autophagy mainly via inhibiting Wnt-mTOR signaling pathway (A , B) Mlg cells were exposed to CQ (20 µM) and Baf A1 (100 nM) with or without Ellagic acid (5 µM, 10 µM) to analyze the p62 expression level by using western blot. Densitometric analyses were shown beside (C , D) The plasmids of GFP-LC3B and mCherry-GFP-LC3B were transfected to NIH3T3 cells with PEI, and these cells were subsequently exposed to Ellagic acid (5 µM, 10 µM) and/or Wnt3a (100 ng/ml) for 12 h. DNA was counterstained with DAPI (blue). Quantitative analyses are showed beside. Scale bars: 50 μm (E) BLM-PPF cells were treated with Ellagic acid (5 µM, 10 µM) for 24 h, and the Atg16L1, Beclin1 and LC3-II/I expression levels were detected by Western blot. Densitometric analyses were shown below (F) BLM-PPF cells were treated with Ellagic acid (5 µM, 10 µM) for 12 h, and protein expression levels of mTOR, S6RP and their phosphorylation were detected by Western blot. Densitometric analyses were shown below. Data are means ± Standard Error of Mean, n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001, and NS: nonsignificant (one-way ANOVA). β-tubulin or GAPDH were used as a loading control.

Article Snippet: GFP-LC3B (mouse) and Cherry-GFP-LC3B (mouse) plasmids were transfected into NIH-3T3 cells using PEI according to the supplier’s protocol (Sino Biological Inc., Beijing, China).

Techniques: Expressing, Western Blot, Transfection