Journal: bioRxiv

Article Title: Immune Checkpoint Molecules as Biomarkers of Staphylococcus aureus Bone Infection and Clinical Outcome

doi: 10.1101/2024.12.30.630837

Figure Lengend Snippet: (A) Immunofluorescent histochemistry analyses of tibia sections from uninfected and MRSA-infected humanized BLT mice 14 days post-op were performed with labeled antibodies against CD3, LAG-3, TIM-3, and PD-1 with DAPI counter stain, and representative images are shown at 4x. Note the increased numbers of T cells near the SAC (dashed yellow line) in the infected tibiae. (B) A multichromatic spectral flow cytometry analyses were performed on tibial bone marrow cells from uninfected and MRSA-infected BLT mice. Live human CD45+/CD3+/T cells and their subpopulations (CD4+, CD8+, Tregs) were analysed for immune checkpoint expression (LAG3, TIM-3, and PD-1) and proliferation (Ki67), and representative histograms are shown. Note the frequency of human CD3 + /CD4 + T cells expressing TIM-3, LAG3 & PD-1) in the cells from MRSA-infected bone marrow (n=4-8 mice, *p<0.05, t-test).

Article Snippet: Primary antibodies: The following antibodies were utilized for immunostaining: goat anti-CD3ε (clone M-20, sc-1127, RRID:AB_631128, Santa Cruz Biotechnology), mouse anti-PD-1 (10377-MM23, RRID:AB_2936309, Sino Biologicals), Rabbit anti-LAG3 (clone BLR027F, NBP2-76402, RRID:AB_3403543, Novus Biologicals), Mouse anti-TIM3/HAVCR2 (clone TIM3/4031, V8754-20UG, NSJ Bioreagents), Rabbit anti- S. aureus (PA1-7246, RRID:AB_561546, Thermo Fisher Scientific), and Mouse anti-CD66b (G10F5, NBP2-80664, RRID:AB_3096017, Novus Biologicals).

Techniques: Infection, Labeling, Staining, Flow Cytometry, Expressing

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Quantitative increase in mRNA expression for m2 muscarinic receptors, Gαo G-proteins, and the splice variant Kir3.2c. A, B, Bar graphs show the average of several (3–4 separate RNA preparations) experiments in which the levels of mRNA for the indicated Kir genes in control and NGF-differentiated PC12 cells were quantified using qRT-PCR. Control undifferentiated and NGF-differentiated PC12 cells (A) and rat brain (B). Kir3.2a, Kir3.2b, Kir3.3, and Kir3.4 mRNAs were detected in rat brain but were not evident in either control or NGF-differentiated PC12 cells. Kir3.1 and Kir2.1 mRNA levels were unchanged by NGF treatment. Levels of RNA were normalized to β-actin [arbitrary units (AU)]. C, Bar graph shows the fold change in mRNA produced by NGF for indicated genes. Genes required for Kir3 signaling, including m2 muscarinic receptor, Gαo G-protein and Kir3.2c were significantly increased with NGF treatment, whereas mRNA for PTX-insensitive Gαq and the m1R were significantly decreased. Fold change in gene expression was determined by dividing the mean expression for each gene analyzed for NGF-treated cells by non-NGF-treated cells. Relative gene expression changes between control and NGF-treated groups were analyzed using the mean ± SEM for three to four separate RNA preparations (*p < 0.05).

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Expressing, Variant Assay, Quantitative RT-PCR, Produced

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: NGF-mediated increase in protein for Kir3.2 channels, Gαo subunits, and m2/m4 muscarinic receptors. Levels of protein for the indicated signaling molecules were studied by Western blot analysis and immunostaining in undifferentiated and NGF-differentiated PC12 cells. A, Western blot analysis demonstrates the NGF-mediated increase in Kir3.2 channel protein in NGF-differentiated PC12 cells. Kir3.1 channel expression was not significantly effected by NGF treatment (middle). β-Actin immunoblot (IB) (bottom) shows equal protein for the PC12 cell lysates. Mouse brain lysate (brain) was loaded onto each gel to demonstrate the correct size protein band for each respective protein. B–M, Immunostaining and confocal imaging demonstrates that Kir3.1 protein shows little or no increase after NGF treatment (B, C); in contrast, Kir3.2 protein shows a significant increase in expression (D, E). To a lesser extent, PTX-sensitive Gαo G-protein are increased after NGF treatment (F, G). Both m2 (H, I) and m4 (J, K) muscarinic receptors are increased with NGF treatment. Both m2 and m4 muscarinic receptor expression appear strikingly cytoplasmic and perinuclear. L, M, m1 muscarinic receptor immunoreactivity modestly decreases after NGF treatment. Scale bars, 20 μm.

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Western Blot, Immunostaining, Expressing, Imaging

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Upregulated m2 receptor/Kir3 channel complex is functionally silent in NGF-differentiated PC12 cells. A, Differential interference contrast (DIC) picture of a typical NGF-differentiated PC12 cell. B, Representative example of an NGF-differentiated PC12 cell in which muscarinic receptor agonist Oxo-M (100 nm) or ethanol (200 mm) did not elicit a Kir3 channel response. The Kir3 channel inhibitor Ba2+ (1 mm) demonstrated no detectable Kir3.2 basal currents (n = 30). Current was measured at −120 mV in voltage ramps and plotted as a function of time. C, Ectopic expression of m2 receptor did not rescue muscarinic receptor/Kir3 signaling complex. Five day NGF-treated PC12 cells were transfected with m2 muscarinic receptor cDNA (1 μg) and whole-cell recordings made 2 d later. Oxo-M (100 nm)-activated or Ba2+-inhibited Kir3 currents were not observed. D, Schematic shows topology and placement of extracellular HA epitope into the p-loop of Kir3.2c. E, Immunostaining for HA demonstrates that HA–Kir3.2c is undetectable on the plasma membrane of NGF-differentiated cells. F, Detergent permeabilized cells exhibit strong cytoplasmic anti-HA immunoreactivity, demonstrating predominantly intracellular expression of HA–Kir3.2c.

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Expressing, Transfection, Immunostaining

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Summary of Kir3 currents in PC12 cells

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Muscarinic receptor antagonists rescue Kir3 signaling in NGF-differentiated PC12 cells. A, NGF-differentiated PC12 cells transfected with HA–Kir3.2c were treated with atropine (100 μm for 2 h) and immunostained with anti-HA antibodies without permeabilization. Note prominent surface expression of HA–Kir3.2c (arrows). B, NGF-differentiated PC12 cells were treated with atropine (100 μm for 2 h), permeabilized, and immunostained with anti-m2 muscarinic receptor antibodies. Immunostaining shows both surface and intracellular expression for the m2 muscarinic receptor, in contrast to m2 receptor expression in untreated NGF-differentiated cells (Fig. 2H). C, Immunostaining with anti-m4 antibody after treatment with atropine. m4 muscarinic receptors are expressed on processes in atropine-treated cells. Cells were permeabilized. D, Representative whole-cell current recording from NGF-differentiated PC12 cell treated with atropine. Atropine was removed from treated cells just before recording. Plot shows current at −120 mV in extracellular 20K (basal), 20K plus Oxo-M (100 nm; Oxo), or 20K plus Oxo (100 nm) plus atropine (1 μm). Note large muscarinic receptor-activated endogenous Kir3 current with Oxo-M. E, Current response elicited by voltage steps from −120 to +30 mV (holding potential was 0 mV). Trace shows the difference between with Oxo-M (100 nm) and basal (20K). The current–voltage plot shows the current level at the beginning (■) and end (□) of the voltage step. Note the strong inward rectification, presence of fast activation kinetics, and zero-current potential near EK (−50 mV). F, Muscarinic-activated currents are mediated by endogenous Kir3 channels. Representative whole-cell recording from NGF-differentiated PC12 cells treated with atropine. Plot shows current at −120 mV with Oxo (100 nm)-activated currents in the absence or presence of SCH-23390 (30 μm), a selective inhibitor of Kir3 channels (Kuzhikandathil and Oxford, 2002).

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Transfection, Expressing, Immunostaining, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Summary of oxotremorine-M responses in NGF-differentiated PC12 cells. Cumulative plot shows current density for Oxo-M-activated currents in control and atropine-treated NGF-differentiated PC12 cells for each cell. Functional coupling was observed in ∼50% of NGF-differentiated PC12 cells (n = 43). Control cells did not demonstrate a detectable muscarinic receptor-mediated Kir3 current (n = 26). No detectable Oxo-M-activated (100 nm) Kir3 currents were observed in control (n = 19) and atropine-treated (n = 19) undifferentiated PC12 cells (Table 1). Thus, surface expression of the GPCR/Kir3 channel complex occurs only in atropine-treated NGF-differentiated cells.

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Functional Assay, Expressing

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Muscarinic receptor antagonist atropine or endocytosis inhibitor wortmannin promotes surface expression of m2 muscarinic receptors. A, NGF-differentiated PC12 cells transfected with m2R–CFP cDNA or m2R–CFP and Kir3.2c cDNAs were either untreated or exposed to atropine (100 μm) for 2 h at 37°C. The same cell was imaged under epifluorescence and TIRF to visualize the expression of m2R–CFP in the cytoplasm and membrane surface, respectively. Images adjusted to the same fluorescence intensity scale. B, NGF-differentiated PC12 cells transfected with m2R–CFP cDNA were either untreated or exposed to wortmannin (10 μm) for 2 h at 37°C. C, Mean fluorescence measured under TIRF is shown for two different experiments: control (n = 23), plus atropine (n = 20) and plus Kir3.2c (n = 10), and control (n = 17) or plus wortmannin (n = 14). Both atropine and wortmannin treatment led to significant increases in m2R–CFP expression on the plasma membrane (*p < 0.05, Student's t test; **p < 0.05, one-way ANOVA with Dunnett's post hoc). Fluorescence measured for m2R-CFP alone [∼5 arbitrary units (AU)] could be from cytoplasmic or membrane receptors. In addition to TIRF microscopy, mean CFP fluorescence was measured using epifluorescence illumination [atropine experiment: 8.6 ± 1.9 AU for control, 15.2 ± 2.3 AU for plus atropine, and 7.3 ± 1.9 AU for plus Kir3.2c cDNA; wortmannin experiment: 4.2 ± 0.6 AU for control and 12.8 ± 3.2 AU for plus wortmannin].

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Expressing, Transfection, Fluorescence, Microscopy

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Selective inhibition of m2 or m4 muscarinic receptors rescues Kir3 signaling in NGF-differentiated PC12 cells. Immunostaining studies revealed that 2 h pretreatment with either the m2 muscarinic-receptor antagonist (100 nm) AF-DX116 (A) or the m4 muscarinic-receptor antagonist tropicamide (200 nm) (B) rescued m2 and m4 muscarinic receptor plasma membrane expression, respectively. C–E, NGF-differentiated PC12 cells transfected with HA–Kir3.2c and incubated for 2 h in the m2 muscarinic antagonist (AF-DX116) or the m4 muscarinic antagonist (tropicamide). Each muscarinic receptor antagonist pretreatment resulted in surface expression of HA–Kir3.2c. F, Cumulative plot shows current density for Oxo-M-activated currents in m2-antagonist-treated, m4 antagonist-treated, or m1/m3 (pirenzipine, 4-DAMP)-treated NGF-differentiated PC12 cells. Both the m2 muscarinic receptor antagonist and the m4 muscarinic receptor antagonist treatments led to significant muscarinic receptor-activated Kir3 currents compared with m1/m3 antagonists treatment (p < 0.05, one-way ANOVA on ranks followed by Tukey's post hoc test). Conversely, m1/m3 muscarinic receptor antagonist did not rescue muscarinic-activated Kir3 currents. Current densities for m2 antagonist treatment were −31.4 ± 13.8 pA/pF (n = 16), for m4 antagonist treatment −22.3 ± 7.7 pA/pF (n = 16), and for m1/m3 antagonists treatment −0.69 ± 0.24 pA/pF (n = 16).

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques: Inhibition, Immunostaining, Expressing, Transfection, Incubation

Journal: The Journal of Neuroscience

Article Title: Coregulation of Natively Expressed Pertussis Toxin-Sensitive Muscarinic Receptors with G-Protein-Activated Potassium Channels

doi: 10.1523/JNEUROSCI.1190-07.2007

Figure Lengend Snippet: Both m2 and m4 muscarinic receptors couple with endogenous Kir3 channels. A, Whole-cell current recording from NGF-differentiated PC12 cells pretreated with atropine (100 μm for 2 h). Oxo-M (100 nm) activates endogenous Kir3 current and is partially inhibited by (∼40%) the selective m2 muscarinic receptor antagonist AF-DX116 (100 nm) and completely inhibited with coapplication of both m2 (AF-DX116, 100 nm) and m4 (tropicamide, 200 nm) muscarinic receptor antagonists. B, Bar graph shows the percentage of Oxo-M (100 nm)-mediated response current remaining after either two consecutive Oxo-M pulses (which shows some desensitization), a selective m2 muscarinic receptor antagonist AF-DX116 (100 nm), or coapplication of the both m2 and m4 muscarinic receptor antagonists (n = 8).

Article Snippet: For Western blot analysis, proteins were transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK) and blocked with SuperBlock buffer (Pierce) for 1 h. Subsequently, blots were exposed to primary anti-Kir3.1 (1:200; AB5198; Millipore, Billerica, MA) or anti-Kir3.2 (1:200; APC-006; Alomone Labs, Jerusalem, Israel) antibodies for 1 h, followed by 1 h in chicken anti-rabbit secondary antibody (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA) at room temperature.

Techniques:

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) tSNE plot of scRNA-seq results from sorted P14 lung ECs with each cell population color-coded and the corresponding cell number shown in parenthesis. Lower panels: gene expression showing that Car4 marks Car4 ECs; Plvap marks Plvap ECs, Vwf ECs, and lymphatic ECs. Epithelial and mesenchymal populations are minor contaminants from sorting. (B) Violin plots showing markers used to identify the six cell populations. (C) Heat map showing top 5 genes of each EC population. Plvap is expressed by all non-Car4 ECs, as shown in (A), and thus not among the top genes for Plvap ECs.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Expressing

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Representative en face view of immunostaining images from at least 5 mice. Boxed region is magnified as a section view in the first three images of the bottom row. CAR4 staining covers, whereas PLVAP staining surrounds, alveolar islands (dash). Perinuclear CAR4 and PLVAP staining allows assignment of ERG nuclei to CAR4 (cyan arrowhead) versus PLVAP (magenta arrowhead) ECs, which is automatically identified, as shown in the lower rightmost image (grey nuclei are ambiguous). Scale: 10 um. (B) Wholemount immunostaining of lungs with sparsely-labeled ECs, representative of at least 5 mice, viewed as a stack (40 um), a slab (top 20 um), or a section (1 um). Accumulation of tdT to ERG nuclei allows cell numeration (1 through 5). Cell #1 is a Car4 EC and cells #2-5 are non-Car4 ECs. Line profile analysis shows aligned versus shifted peaks for Car4 versus non-Car4 ECs, respectively. For shifted peaks, Car4 ECs are closer to the airspace than non-Car4 ECs (e.g. cell #3). Asterisk, avascular tissue surrounded by a single net-like Car4 EC. Open arrow, CDH5 junction overlapping with a single Car4 EC. Cell perimeter is measured by connecting protrusions that are visible in a projection view. Tam, 0.25 ug tamoxifen. Scale: 10 um. (C) Quantification of cell perimeter and comparison using Student’s t-test.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Staining, Labeling

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: Wholemount immunostaining of lungs (A) and retinas (B) with sparsely-labeled ECs, representative of at least 5 mice. Tam, 0.25 ug tamoxifen. Scale: 10 um. (A) Cells #1-2 are Car4 ECs and cells #3-6 are non-Car4 ECs. ECs in non-capillary vessels (#4-6) are more elongated along the direction of blood flow. (B) Retinal macrophages (m), but not ECs, express CAR4. Tip ECs display characteristic filopodia (bracket) without a discernable lumen (weak ICAM2). Non-capillary ECs are similarly elongated, but none of the retinal ECs feature the net-like morphology of lung Car4 ECs.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Labeling

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Violin plots of lung EC scRNA-seq data from showing retinal tip and stalk EC genes. Apln and Kdr are enriched in Car4 ECs; Aplnr , Tek (also known as Tie2 ), and Nrarp are enriched in Plvap ECs. (B) tSNE plot showing Esm1 expression in sporadic Plvap ECs (arrowhead in insert). (C) Dot plot of 25 recently identified tip EC genes with those enriched in Car4 ECs highlighted in red. (D) Representative wholemount immunostaining images of at least 2 mice. ESM1 is expressed by retinal tip ECs (filled arrowhead), as well as lung ECs near lobe edge in embryos (filled arrowhead), but in transitional ECs between capillaries and non-capillaries (open arrowhead). CAR4 expression initiates at E19 (open arrowhead), concomitant with AT1 cell differentiation. DLL4 staining is stronger in retinal arteries (a) than veins (v), distinguishable by vessel diameter, and is wide-spread and cord-like (dash) in the lung. Scale: 10 um.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Expressing, Immunostaining, Cell Differentiation, Staining

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Representative wholemount immunostaining images of at least 2 mice showing Apln CreER labels ECs but not specifically Car4 ECs. The specificity is quantified for 2 lungs and found to be variable. Filled arrowhead, CAR4 and GFP double-positive ECs; open arrowhead, GFP single positive ECs. Tam, 250 ug tamoxifen. Scale: 10 um. (B) Apln undergoes X-inactivation. FACS purification of P14 lung ECs (0.5 mg tamoxifen at P13) that are labeled (red box) or unlabeled (blue box) with native fluorescence from Rosa tdT , which are subjected to RT-PCR analysis. Labeled ECs, which are expected to express CreER, do not express Apln despite the presence of the wildtype allele, indicating X-inactivation. The same is true for P35 lung ECs (2 mg tamoxifen at P34). (C) tSNE plots of scRNA-seq results from sorted lung ECs from littermate Apln mutant and control males showing deletion of Apln but no change in the cell number and gene expression of Car4 and Plvap ECs. Asterisk, contaminating immune, epithelial, and mesenchymal cells (non-EC).

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Immunostaining, Purification, Labeling, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Left panels: tSNE plot of scRNA-seq results from sorted ECs from littermate lungs showing a specific, complete loss of Car4 ECs in the mutant with the percentages out of all ECs in parenthesis. Plvap ECs and proliferating ( Mki67 ) ECs are unaffected. Cell populations are color-coded as in . Mes, mesenchyme; Epi, epithelium; Lym, lymphatic EC; Vwf, Vwf EC. Compared to P14 , P7 lungs have more proliferating ( Mki67 ) ECs; their percentage is calculated with a UMI cutoff of 1. Right panels: en face view of immunostained littermate lungs, representative of at least 3 littermate pairs, showing rare Car4 staining in the remaining vessels in the mutant (open arrowhead). m, macrophage. Scale: 10 um. (B) Section immunostaining images of littermate lungs representative of at least 3 littermate pairs. As diagrammed, Car4 vessels (filled green arrowhead) abut the epithelium (AQP5), separated with a thin basement membrane (weak COL4 staining; dash open arrowhead) with no intervening pericytes (CSPG4; dash open arrowhead), but their sides away from the air space have a thicker basement membrane (strong COL4 staining; solid open arrowhead) and pericytes (solid open arrowhead). Non-Car4 vessels (filled white arrowhead; a subset of vessels in the control and all vessels in the mutant) do not abut the epithelium and are surrounded by a thick basement membrane and pericytes (solid open arrowhead). Scale: 10 um.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Mutagenesis, Staining, Immunostaining

Journal: bioRxiv

Article Title: Epithelial Vegfa specifies a distinct endothelial population in the mouse lung

doi: 10.1101/840033

Figure Lengend Snippet: (A) Left panels: en face view of immunostained littermate lungs, representative of at least 3 littermate pairs, showing a smoother surface (RAGE) of alveolar islands that are not subdivided by Car4 vessels in the mutant. Scale: 10 um. Right panels: H&E section images of littermate lungs with the corresponding mean linear intercept (MLI) and D 2 measurements. Scale: 100 um. Each symbol represents one mouse and is the average of 3 regions (882 um x 664 um; Student’s t-test). (B) En face view of immunostained littermate lungs, representative of at least 3 littermate pairs, showing fewer pericytes (PDGFRB; open arrowhead) but normal myofibroblasts (SMA/TAGLN/PDGFRA triple positive, although variable in staining intensity; filled arrowhead) in the mutant. Boxed regions are magnified in merged views showing costaining of SMA, TAGLN, and PDGFRA that is distinct from PDGFRB. Scale: 10 um. (C) Schematics with color-coded cell types showing AT1 derived Vegfa signals to Car4 ECs, which, together with myofibroblasts, promote secondary septation and persist in the resulting septae even after disappearance of myofibroblasts in the mature lung. The Vegfa mutant fails to form Car4 ECs; non-Car4 ECs and myofibroblasts are insufficient for secondary septation, resulting in alveolar enlargement. Note that Car4 vessels may consist of Car4 ECs and non-Car4 ECs, as diagrammed in . Alv, alveolus.

Article Snippet: The following antibodies were used: rabbit anti-Aquaporin 5 (AQP5, 1:2500, ab78486, Abcam), goat anti-Carbonic anhydrase IV (CAR4, 1:500, AF2414, R&D), BV786 rat anti-CD31 (1:250, 740870, BD Biosciences), PE/Cy7 rat anti-CD45 (1:250, 103114, BioLegend), mouse anti-Claudin 5 (Cldn5, 1:500, Invitrogen, 352588), rabbit anti-collagen IV (COL4, 1:2500, LSL-LB-1403, CosmoBioUSA), goat anti-Delta like canonical Notch ligand 4 (DLL4, 1:250, AF1389, R&D), Alexa Fluor 488 rat anti-CD324 (ECAD, 1:500, 53-3249-80, eBioscience), goat anti-Endothelial cell specific molecule 1 (ESM1, 1:500, AF1999, R&D), rabbit anti-Avian erythroblastosis virus E-26 (v-ets) oncogene related (ERG, 1:5000, ab92513, Abcam), goat anti-Vegfr3/Flt4 (1:1000, R&D, AF743), chicken anti-beta Galactosidase (LacZ, 1:500, Ab9361, Abcam), chicken anti-Green fluorescent protein (GFP, 1:5000, AB13970, Abcam), Alexa Fluor 647 rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, A15452, ThermoFisher), rat anti-Intercellular adhesion molecule 2 (ICAM2, 1:2500, 16-1021-82, eBioscience), goat anti-Intercellular adhesion molecule 2 (ICAM2, 1:500, AF774, R&D systems), eFluor 570 rat anti-Ki67 (1:500, 41-5698-82, eBioscience), rabbit anti-Ki67 (1:1000, RM9106S0, ThermoFisher), rabbit anti-Chondroitin sulfate proteoglycan 4 (CSPG4, 1:1000, AB5320, Millipore), rabbit anti-NK2 Homeobox 1 (NKX2.1, 1:1000, sc-13040, Santa Cruz), rat anti-Platelet derived growth factor receptor alpha (PDGFRA, 1:1000, 14-1401-82, eBioscience), goat anti-Platelet derived growth factor receptor beta (PDGFRB, 1:1000, AF1042, R&D systems), rat anti-Plasmalemma vesicle associated protein (PLVAP, 1:125, 553849, BD Biosciences), rabbit anti-Prospero Homeobox 1 (PROX1, 1:250, 11-002, AngioBio), rat anti-Advanced glycosylation end-product specific receptor (RAGE, 1:1000, MAB1179, R&D systems), rabbit anti-Red fluorescent protein (RFP, 1:1000, 600-401-379, Rockland), Cy3-conjugated mouse anti-alpha-Smooth muscle actin (SMA, 1:1000, C6198, Sigma), rabbit anti-SM22 (TAGLN, 1:2500, Abcam, ab14106), Alexa Fluor 647 rat anti-Vascular endothelial cadherin (VECAD/CDH5, 1:250, 562242, BD Biosciences), rabbit anti-Von Willebrand Factor (VWF, 1:2500, Abcam, ab6994).

Techniques: Mutagenesis, Staining, Derivative Assay

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scheme of ARTR-seq. Specific speckle scaffold protein is immunostained by primary and secondary antibodies sequentially. pAG-RTase is then allowed to bind to the antibody to initiate reverse transcription in situ. The generated biotinylated cDNAs are collected and prepared for sequencing. (B) Representative image showing colocalization of pAG-RTase labeled with Alexa Fluor 647 (AF647) (red), secondary antibody labeled with Alexa Fluor 568 (AF568, yellow) against anti-SON primary antibody, and generated biotinylated-cDNA detected by Alexa Fluor 488 (AF488) labeled antibody against biotin (green). Scale bar: 3 µm. (C) Venn diagram of overlapped speckle-enriched genes identified through targeting SON and SRRM2 in HeLa cells using Method 1 or Method 2. Speckle-enriched transcripts are defined by I NSE >2 and adjusted p-value < 0.05. (D) Genome tracks showing ARTR-seq reads generated from targeting SON or SRRM2 proteins, and from control samples without primary antibody, mapped to gene locus encoding lncRNA MALAT1 . (E) RNA FISH images showing speckle-enriched LAMA5 transcript in comparison with speckle non-enriched P4HB transcript. RNA FISH probes are labeled with AF647 (red). Nucleus was stained with DAPI. Scale bar: 10 μm. (F) Correlation between speckle partition coefficient (R NS/NU ) measured by RNA FISH imaging and I NSE values determined by ARTR-seq using Method 1.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: Reverse Transcription, In Situ, Generated, Sequencing, Labeling, Control, Comparison, Staining, Imaging

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Fraction of unspliced introns (EI/(EI+EE)) calculated from the number of reads mapping to exon-intron boundary (EI) and the number of reads mapping to exon-exon junction (EE) in polyA RNA-seq, nuclear RNA-seq, ARTR-seq without antibody and ARTR-seq with SON antibody. (B) Alternative estimation of the fraction of unspliced introns (IN/(IN+EE)) calculated using reads mapping to intronic positions within 100 nucleotides of splice sites (IN) instead of EI. (C) Ratio of IN reads to EI reads in polyA RNA-seq, nuclear RNA-seq, ARTR-seq without antibody and ARTR-seq with SON antibody. The two replicates of RNA-seq were calculated individually. Each bar with error bars in (A)-(C) reports mean and standard deviation of the two replicates. (D) Violin plot showing the speckle size immunostained with SRRM2 antibody under NT Plad B treatment. “N” indicates the total number of speckles in each data set. (E-F) 2D histogram showing the correlation of I NSE(intron) (E) or I NSE(exon) (F) between NT and Plad B treatment conditions in HeLa cells. Genes were categorized into three Groups (Group A, B, C) and depicted in (F). (G-H) Violin plot comparing I NSE(exon) (G) or I NSE(intron) (H) values among Group A, B, and C genes under NT and Plad B treatment conditions. P-values calculated with unpaired t-test are reported above each violin plot. “N” reports the total number of genes in each comparison. (I) GO analysis for speckle-enriched Group A and Group B genes, and non-speckle-enriched Group C gene. The analysis is performed using g:Profiler , using the background consisting of all Group A, B and C genes. GO terms in biological processes (BP) and cellular compartment (CC) were identified.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: RNA Sequencing Assay, Standard Deviation, Comparison

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) Scatter plot showing randomly selected genes from Group A, B and C genes, and corresponding I NSE(exon) under NT and Plad B treatment conditions in HeLa cells. (B) Genome tracks showing polyA RNA-seq (pink) and nuclear RNA-seq (blue) under NT and Plad B treatment conditions for selected genes: THOC6 in Group A gene, TUBB4B in Group B gene, and NCL in Group C gene. Selected efficiently spliced or inefficiently spliced introns for RT-PCR assay are highlighted in cyan and red boxes respectively. Genome tracks of other selected genes for RT-PCR assays are shown in Figure S8. (C) Schematic description of the RT-PCR assay. After reverse transcription of extracted total RNA, primers located on two adjacent exons of selected introns were used for amplification and the PCR products were analyzed by electrophoresis. (D) Representative immunofluorescence images showing nuclear speckles upon SON / SRRM2 double knockdown (KD) and treated with control siRNA (siC). Nuclear speckles were stained with AF488 labeled antibody against SRRM2 antibody (blue); and nuclei were stained with DAPI (grey). Scale bar: 10 μm. (E) Histogram of SRRM2 immunofluorescence intensity distribution of cells with double knockdown (KD) or treatment with control siRNA (siC). (F) Violin plot showing the number of speckles per cell for KD and siC treatment. Total number of cells included in each data set is indicated by “N” in (E) and (F). P-values calculated with unpaired t-tests are reported above each violin plot. (G) Representative electrophoresis analysis of RT-PCR products from THOC6 , TUBB4B and NCL upon KD and siC treatment. Gels of other selected genes for RT-PCR assays are shown in Figure S8. Gels were imaged with Chemidoc Imaging System. (H) Apparent unspliced fractions of selected introns were calculated by ratios of the intensity of the unspliced band and the sum of the unspliced band and spliced band. The intensity of bands was quantified using Fiji. Error bars report standard deviation from two biological replicates.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription, Amplification, Electrophoresis, Immunofluorescence, Knockdown, Control, Staining, Labeling, Imaging, Standard Deviation

Journal: bioRxiv

Article Title: Dynamics of RNA localization to nuclear speckles are connected to splicing efficiency

doi: 10.1101/2024.02.29.581881

Figure Lengend Snippet: (A) IRFinder analysis showing heat shock-induced upregulation of intron retention. The number of intron retention events with more than 15% increase (ΔIR >15% ) or decrease (ΔIR <-15% ) are labeled. (B) Violin plot showing the speckle size change up heat shock compared to NT. “N” indicates the total number of speckles in each data set. (C) Viability of HeLa cells with nuclear speckle disrupted using SON/SRRM2 double knockdown (KD) or cells treated with control siRNA (siC) upon heat shock stress or NT. Hoechst staining reflected the whole cell population (with cell number denoted as N Total ), whereas Trypan blue stained the dead cell (with number denoted as N Dead ). Cell viability was calculated by 1-N Dead /N Total . P-values calculated with unpaired t-test are reported above each violin plot and box plot. Error bars report standard deviation from 3 biological replicates (in black dots). (D) 2D histogram showing the correlation between I NSE(exon) values under heat shock and NT. (E) Percentage of Group A genes and Group B and C genes without and with taking the subset of genes containing ΔIR >15% introns. P-value: Fisher’s exact test. (F) Violin plot showing the Type I sequence feature associated with three groups of introns (ΔIR >15% , ΔIR (–15%, 15%) , ΔIR <-15% ). The GC content, intron length, splice site strength and intronic ML score are compared for three groups of introns.

Article Snippet: Cells were immunostained with rabbit anti-SON antibody (1:200 dilution, Novus) or mouse anti-SRRM2 antibody (1:200 dilution, Sigma Aldrich) for 1h at room temperature followed by 3 times wash with DPBS.

Techniques: Labeling, Knockdown, Control, Staining, Standard Deviation, Sequencing

Journal: British Journal of Pharmacology

Article Title: Ca 2+ paradox injury mediated through TRPC channels in mouse ventricular myocytes

doi: 10.1111/j.1476-5381.2010.00986.x

Figure Lengend Snippet: Functional expression of transient receptor potential canonical (TRPC) channels in mouse ventricular myocytes. (A–C) Activation of TRPC current by thapsigargin recorded under conditions where Na+, Ca2+ and K+ channel currents were minimized. (A) Time course of changes in membrane current measured at +50 and −110 mV during the voltage-ramp protocol (from +50 to −110 mV), before and during exposure to thapsigargin (Thap, 2 µmol·L−1), without and then with 2-APB (20 µmol·L−1). (B) I-V relationships measured at time points (a, b, c) indicated in (A). (C) Difference currents obtained by digital subtraction as indicated (b-a: thapsigargin-activated current; b-c: 2-APB-sensitive current). (D) Immunostaining of TRPC1, TRPC3 and TRPC4. Scale bar in all panels, 25 µm.

Article Snippet: Rabbit anti-TRPC1 antibody directed against an extracellular epitope of human TRPC1 (ACC-010), rabbit anti-TRPC3 antibody directed against an intracellular C-terminal epitope of mouse TRPC3 (ACC-016), rabbit anti-TRPC4 antibody directed against an intracellular C-terminal epitope of mouse TRPC4 (ACC-018), rabbit anti-TRPC5 antibody directed against an intracellular epitope of human TRPC5 (ACC-020) were from Alomone Laboratories (Jerusalem, Israel), and normal rabbit IgG was from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Functional Assay, Expressing, Activation Assay, Immunostaining

Journal:

Article Title: Immunolocalization of the Voltage-gated Potassium Channel Kv2.2 in GABAergic Neurons in the Basal Forebrain of Rats and Mice

doi: 10.1002/cne.22457

Figure Lengend Snippet: Expression of Kv2.2 in the neurons of the magnocellular preoptic nucleus (MCPO) and the horizontal band of Broca (HDB). A–C: Expression of Kv2.2 in the NeuN-positive neuronal population in the basal forebrain. Scale bar, 50 μm. D–E: Nickel enhanced 3-3′diaminobenzidine (DAB) immunostaining was used to determine specific localization of Kv2.2-expressing neurons. Rat coronal sections (8.9 and 9.2mm anterior to the interaural line) were immunostained with the anti-Kv2.2 antibody. Anatomical landmarks such as the anterior commissure (indicated by asterisk) were used to locate the expression of Kv2.2 in the MCPO/HDB nuclei.

Article Snippet: For DAB staining, sections were incubated with rabbit anti-Kv2.2 antibody (0.3 μg/ml, Alomone Labs, Jerusalem, Israel) overnight, washed, and probed with a biotinylated anti-rabbit secondary antibody (1:250, Vector Labs, Burlingame, CA).

Techniques: Expressing, Immunostaining

Journal:

Article Title: Immunolocalization of the Voltage-gated Potassium Channel Kv2.2 in GABAergic Neurons in the Basal Forebrain of Rats and Mice

doi: 10.1002/cne.22457

Figure Lengend Snippet: Enriched expression of Kv2.2 in non-cholinergic neurons in the MCPO/HDB of the mouse brain. A: Nickel enhanced DAB immunostaining of Kv2.2-immunoreactive neurons in the mouse brain. A coronal section (3.94 mm anterior to the interaural line) was immunostained with the anti-Kv2.2 antibody. B: Corresponding mouse brain atlas to A. VLPO, ventrolateral preoptic nucleus; LPO, lateral preoptic nucleus; VP, ventral pallidum; AC, anterior commissure; SIB, substantia innominata basal; Tu, olfactory tubercle. C–E: Confirmation of the enrichment of Kv2.2 in the MCPO in immunofluorescence staining. Mouse coronal sections were double immunostained with K89 anti-Kv2.1 and anti-Kv2.2 antibodies. F–H: Reciprocal expression of Kv2.2 and ChAT in the MCPO of mouse. Mouse coronal sections were double immunostained with anti-Kv2.2 and anti-ChAT antibodies. Scale bars, 100 μm.

Article Snippet: For DAB staining, sections were incubated with rabbit anti-Kv2.2 antibody (0.3 μg/ml, Alomone Labs, Jerusalem, Israel) overnight, washed, and probed with a biotinylated anti-rabbit secondary antibody (1:250, Vector Labs, Burlingame, CA).

Techniques: Expressing, Immunostaining, Immunofluorescence, Staining