Journal: Nature communications

Article Title: Defining human cardiac transcription factor hierarchies using integrated single-cell heterogeneity analysis.

doi: 10.1038/s41467-018-07333-4

Figure Lengend Snippet: Fig. 2 Single-cell RNA-seq identified subpopulations of cardiomyocytes. a Late stage cardiomyocytes (4,689 cells with TNNT2 and ACTC1 expression from day 14 and day 45) were further resolved using ICGS to identify subpopulations of cardiomyocytes. Associated t-SNE cell populations were colored by day of differentiation, as well as by cluster identified (markers within each cluster beside each cluster). b Six cardiomyocyte populations were identified representing subpopulations of cardiomyocytes in early proliferative stages (Cluster 4, 5) expressing cyclins, mid-cardiomyocyte stage (Cluster 2, 3) expressing HAND1, and more mature cardiomyocytes (Cluster 0, 1) expressing sarcomeric (MYH6, MYL2, TNNT2, MYBPC3) and calcium handling genes (RYR2, PLN). c Single cells from day 30 of differentiation were profiled using an independent technology (Fluidigm C1) to resolve coincident mid-to-late state differentiation heterogeneity. Genes associated with distinct observed cell populations identified by the algorithm MarkerFinder are shown. Factors with defining expression in each of the HOPACH clustered populations are shown (SF1, ISL1, JARID2, TBX5, MYL2/HEY2, and HOPX). Two major groups in hiPSC-CMs were observed to express either NR2F2 or MYL2/HEY2. Within each group, identified heart developmental regulators were identified using the software GO-Elite (left panel)

Article Snippet: To overexpress NR2F2, TBX5, and HEY2 in hiPSC-CMs, lentiviral particles were purchased for mGFP-tagged NR2F2 (Cat# RC206753L2V), TBX5 (Cat# RC216520L2V), HEY2 (Cat# RC202544L2V), and control GFP Lent-ORF particles (Cat# PS100071V5) from Origene.

Techniques: RNA Sequencing, Expressing, Software

Journal: Hypertension (Dallas, Tex. : 1979)

Article Title: Mild Hyperuricemia Attenuates Salt-Sensitive Hypertension and Kidney Damage

doi: 10.1161/HYPERTENSIONAHA.125.25550

Figure Lengend Snippet: Oxidative stress in the kidney. A , Differentially expressed (DE) genes known to affect the synthesis of reactive oxygen species (ROS) and oxidative stress in male rats. B , Western blot for AKR1B10 expression in the kidney and its quantification in male and female rats. C , Representative images of paraffin-embedded 8-OxodG-stained kidney and the quantification of the fluorescence intensity of the staining in male age-matched Sprague-Dawley (SD) rats fed a normal diet (healthy control), Dahl SS rats fed a high-salt (HS) diet, or Dahl SS rats fed a HS/oxonic acid diet. D , Representative images of optimal cutting temperature (OCT)-embedded 8-OxodG-stained kidney of the female Dahl SS rats fed a HS diet, or Dahl SS rats fed a HS/oxonic acid diet. Red-8OxodG, blue- nuclei. Values are shown as mean±SEM. An unpaired t test was used. P value **<0.01, ****<0.0001. Parts of the figure are created using BioRender.com.

Article Snippet: The membrane was first incubated with primary antibody for AKR1B10 (Cat# 18252-1-AP, Proteintech, Rosemont, IL) at 1:5000, then with a secondary antibody (Cat# 711-035-152, Peroxidase AffiniPure Donkey Anti-Rabbit IgG, Jackson ImmunoResearch Laboratories Inc., West Grove, PA) at 1:10000 in 2% BSA in Tris-buffered saline with Tween-20 (TBST).

Techniques: Western Blot, Expressing, Staining, Fluorescence, Control

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Huh-7 cells were transfected with A3G and increasing amounts of HBx as indicated. eGFP expression plasmid was used as transfection control. Cells were harvested 48 hours post transfection and cell lysates were analyzed in parallel in immunoblot. ( B ) Relative quantities of A3G in cell lysates as shown in ( A ) were estimated using densitometry scanning and normalized to eGFP quantities, taking values without HBx co-transfection as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Cotransfection

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Time-course study of A3G protein level following cycloheximide (CHX) treatment. Huh-7 cells transfected with A3G and eGFP expression plasmids, with or without HBx plasmid, were treated with CHX at 48 hours post transfection. Cells were collected at indicated time points and analyzed in parallel using immunoblot. ( B ) Relative quantities of A3G (top) and HBx (bottom) in cell lysates as shown in ( A ) were estimated using densitometry scanning and normalized against eGFP quantities, taking values at start of CHX treatment as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Lack of effects of HBx on A3G ubiquitination as detected using immunoprecipitation. Huh-7 cells transfected with indicated plasmids were treated with MG-132 and subjected to cell lysis and purification of His-tagged A3G using Ni 2+ -NTA matrix under denaturing condition. Enriched His-tagged A3G was detected using anti-A3G and A3G modified by HA-tagged ubiquitin (HA-Ub) was detected using anti-HA antibodies. A3G and HBx in 1/10 input were also detected. ( B and C ) Treatment with proteasome inhibitor MG-132 or lysosome inhibitor leupeptin had no marked effect on A3G protein levels, with or without HBx. Huh-7 cells transfected and treated as indicated were lysed and subjected to immunoblot analysis.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Lysis, Purification, Modification, Western Blot

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Detection of A3G protein in cell lysates and exosomes prepared from Huh-7 cells transfected with A3G expression plasmid or vector control. Exosome markers Alix and Rab7, and mitochondria marker VDAC1 were detected in parallel. ( B ) Treatment with GW4869 or transfection of plasmid expressing nSMase2-targeting shRNA (shnSMase2) inhibited exosome secretion by Huh-7 cells. Exosomes were prepared from 1E7 cells treated as indicated and total protein contents were quantified using BCA assay. DMSO vehicle and unrelated shRNA construct (shneg) were used as controls. ( C ) Treatment with GW4869 or shnSMase2 inhibited A3G export via exosome secretion and increased intracellular A3G protein level. Huh-7 cells transfected with A3G expression plasmid or vector control and treated as indicated were used for lysate and exosome preparation. In addition to A3G and exosome/mitochondria markers, nSMase2 was also detected to demonstrate knock-down by shnSMase2.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Marker, shRNA, BIA-KA, Construct, Knockdown

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Effects of HBx on A3G protein levels in cell lysates and exosomes. Huh-7 cells were transfected with indicated plasmids and 48 hours post transfection, cell lysates and exosomes were prepared and analyzed in immunoblot for A3G and HBx levels as well as exosome markers Tsg101 and Rab7. β-actin was used as loading control. ( B ) Relative quantities of A3G and exosome markers Tsg101 and Rab7 in cell lysates and exosomes as shown in ( A ) were respectively estimated using densitometry scanning taking values from vector-transfected cells as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Western Blot, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

doi: 10.1101/678185

Figure Lengend Snippet: A) Wholemount in situ hybridization comparing mmp2, mmp14a and mmp14b expression in WT vs pax2a −/− and DMSO vs DMH4 treated embryos at 32hpf. Mmp2, 14a and 14b signal within the optic fissure (yellow arrowhead) appears decreased in pax2a −/− and DMH4 treated embryos. B) qPCR analysis confirms a decrease in expression of mmp2, 14a and 14b in both pax2a −/− and DMH4 treated embryos. C) Two color wholemount in situ hybridization simultaneously examining mmp2 and kdrl , or mmp2 and rorB expression at 32hpf. DNA was stained with DAPI. Scale bar = 50μm. Clear overlap of signal is observed for mmp2 and kdrl , but not mmp2 and rorB . D) Whole mount Immunohistochemistry was used to visualize laminin (red) in ARP101 treated Tg[ rx3 :GFP] embryos at 48hpf. Central-proximal regions of the optic fissure are displayed. E) Quantification of 48hpf ARP101 treated embryos for fissure fusion (absence of laminin signal), partial fusion (partial retention of laminin) or failure to fuse (retention of laminin throughout the fissure). n= 41 (10mM), 38 (15mM), 42 (20mM) ANOVA p<0.0001. F) Quantification of 48hpf ARP101 treated embryos for fissure fusion, partial fusion or failure to fuse after various treatment initiation times from 24-32hpf. n= 42 (24-48hpf), 29 (26-48hpf), 29 (28-48hpf), 23 (30-48hpf), 37 (32-48hpf). ANOVA p<0.0001.

Article Snippet: For ARP101 (Tocris) treatments embryos were dechorionated and treated with 10, 15, 20, 25 or 30μM ARP101 in fish water from 24 to 48hpf, unless stated otherwise.

Techniques: In Situ Hybridization, Expressing, Staining, Immunohistochemistry

Journal: bioRxiv

Article Title: Pax2a regulates angiogenesis to facilitate mmp2-dependent basement membrane remodeling of the optic fissure

doi: 10.1101/678185

Figure Lengend Snippet: A) Brightfield images of DMSO and ARP treated embryos at 48hpf. Concentrations of ARP101 exceeding 20uM result in toxic effects. B) Whole mount Immunohistochemistry was used to visualize laminin (red) in ARP101 treated Tg[ rx3 :GFP] embryos at 48hpf. Central-proximal and distal regions of the optic fissure are displayed.

Article Snippet: For ARP101 (Tocris) treatments embryos were dechorionated and treated with 10, 15, 20, 25 or 30μM ARP101 in fish water from 24 to 48hpf, unless stated otherwise.

Techniques: Immunohistochemistry

Journal: iScience

Article Title: Conserved role of FOXC1 in TNBC is parallel to FOXA1 in ER+ breast cancer.

doi: 10.1016/j.isci.2024.110500

Figure Lengend Snippet: Figure 4. Identification of FOXC1 cofactors (A) Protein co-interactors of FOXC1 identified in Hs578t and BT-549 via RIME in two biological replicates, after subtracting those identified in negative IgG control. (B) Western blot depicting co-immunoprecipitation of FOXC1 using antibody against NR2F2 with input and IgG negative controls.

Article Snippet: Antibodies FOXC1 (ChIP-seq and RIME) Simeoni et al.19 Recombinant Anti-FOXC1 antibody [EPR20685] (For WB, Co-IP) Abcam ab227977; RRID:AB_2916124 Mouse Anti-Actin, beta Monoclonal Antibody, Unconjugated Abcam ab6276; RRID:AB_2223210 Goat Anti-Rabbit IgG - H&L Polyclonal antibody, Hrp Conjugated Abcam ab6721; RRID:AB_955447 Anti-NR2F2 (ARP-1 (B-2)) (Co-IP) Santa Cruz Biotech sc-271265X; RRID:AB_10608838 Recombinant Anti-NR2F2 antibody [EPR18443] (WB) Abcam ab240387; RRID:AB_2895604 Chicken polyclonal Secondary Antibody to Mouse IgG - H&L Abcam ab6706; RRID:AB_956003 Chemicals, peptides, and recombinant proteins DSG (disuccinimidyl glutarate) Thermoscientific Catalog number: 20593, 50MG PMSF (Phenylmethylsulfonyl fluoride) Roche Catalog number: 10837091001 TrueCut Cas9 Protein v2 Invitrogen Catalog number: A36499 Lipofectamine CRISPRmax ThermoFisher Catalog number: CMAX00003 Propidium Iodide Thermoscientific Catalog number: P3566

Techniques: Control, Western Blot, Immunoprecipitation

Journal: Genes

Article Title: NR2F2 Regulates Cell Proliferation and Immunomodulation in Whartons’ Jelly Stem Cells

doi: 10.3390/genes13081458

Figure Lengend Snippet: NR2F2 knockdown affects key factors of immunity and differentiation in WJ-MSCs. ( A ) Immunocytochemical staining for DAPI (blue) and NR2F2 (green). Images were taken at 40× magnification. Scale bar = 100 μM. ( B ) NR2F2 mRNA levels (relative to β-actin) in WJ-MSCs transfected with control siRNA (control siRNA), or NR2F2 siRNA (NR2F2 RNAi) were analyzed by qPCR. The data were analyzed by the δ-δ-Ct method, and the genes in control siRNA-transfected WJ-MSCs were set to 1. ( C ) Western blot analysis of NR2F2 protein levels in WJ-MSCs. ( D ) Relative gene expression changes of IL6 , ALDH1A1 , IL8 , BMP6 , DSG2 , CD200 , and MMP1 genes upon NR2F2 depletion (control RNAi as control). * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: NR2F2 siRNA (sc-38818, Santa Cruz) and the scrambled control siRNA (sc-37007, Santa Cruz).

Techniques: Knockdown, Staining, Transfection, Control, Western Blot, Gene Expression