Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Cas9/sgRNA/Oligo-targeting site near the SOX7 stop codon (underlined in red). The SgRNA sequence is highlighted in red (this contains the stop codon), while the protospacer-adjacent motif (PAM) sequence is shown in blue. The oligo donor containing the V5 tag (green box), is flanked by 60 bp homology arms. The red arrows represent PCR sequencing primers for genotyping (SF, V5R and SR). (B) PCR genotyping of CRISPR Sox7 -V5 injected live-born pups. Left: PCR genotyping with primer pair SF and V5R showed the desired band at the correct size in postnatal (P) 3, P4, P6, P7 and P10. Right: PCR genotyping with primer pair SF and SR produced slightly larger products in P4, P6 and P10 compared to P3 and P7, indicating successful 42 bp V5 tag integration. At least 3 bands were discovered in P3 and P7, but not in P4, P6 and P10 when separated by 3.5% gel electrophoresis (not shown). Red arrows indicate the expected PCR product sizes. (C) Sequencing of PCR product using SF primer (for both SF/V5R and SF/SR) confirmed the correct fusion of V5 tagged to the last codon in both Sox7 alleles of the Sox7 -V5 founders.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Sequencing, CRISPR, Injection, Produced, Nucleic Acid Electrophoresis

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At E10.5, SOX7 (white) is expressed in intersomitic vessels (ISVs), the dorsal aorta (DA) (delineated by arterial marker, SOX17), cardinal vein (CV), and the surrounding migrating endothelial cells (red arrowheads) (B) SOX7 was not detected in PROX1+ migrating lymphatic endothelial cells (LECs) (asterisks). (C) At E16.5, SOX7 is downregulated in the SOX17+ main arterial branch (boxed area with dotted line), but continues to be expressed in the less mature arteries near the midline (box area with solid line). Arteries are delineated by arterial marker SOX17; major veins and vascular plexus are delineated by endomucin (EMCN). (D) SOX7/ β -galactosidase ( β -gal) (green) also stains the SOX17 (white) positive major arteries in the skin plexus of E14.5 Sox7 +/- lac-Z reporter mice. Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Representative images of E10.5 Sox7 iECKO mutants and sibling controls, injected with tamoxifen at E9.5. The endothelium is delineated by endomucin (EMCN) (blue) and endothelial cells by ETS-related gene (ERG) (red). (Top right panel) The percentage of SOX7 positive endothelial cells over the total number of endothelial cells quantified from 10 sequential transverse sections in 2 control and 3 Sox7 iECKO mutants. (Bottom right panel) Graph indicating the levels of Sox7 transcript in sibling controls and Sox7 iECKO mutants. Mean ± SEM; scored sibling control, n=9; Sox7 iECKO mutants, n=7; Mann-Whitney U- test. P <0.0005 (***). (B) Bright-field images of Sox7 iECKO mutants and sibling controls at E14.5, after pulsing with tamoxifen at E9.5 and E10.5. Scored sibling controls, n=8; Sox7 iECKO mutants, n=5. (C) Brightfield images and whole-mount immunostaining of Sox7 iECKO mutant and sibling control skin at E13.5, after Cre induction at E9.5 and E10.5. Dermal lymphatic structures are marked by Neuropilin 2 (NRP2) (membranous white), lymphatic endothelial cells by Prospero-related homeobox 1 (PROX1) (red), and blood vessels by EMCN (green). Dash line represents the midline of the embryo. Scored sibling controls, n=7; Sox7 iECKO mutants, n=7 (D-H) Quantification of (D) lymphatic sprout migration distance from the midline (E) lymphatic branch points/area (F) lymphatic vessel width (μm) (G) PROX1+ nuclei/area and (H) PROX1+ nuclei/lymphatic vessels across the whole skin. Total area = 4200 x 1500 μm on both sides of the midline, in sibling controls (n=3) and Sox7 iECKO mutants (n=5-6). (F,H) Average of width and PROX1+ nuclei was obtained from 7 random representative leading lymphatic vessels, of fixed length at 200 μm, from both sides of the midline in each skin. (I,J) Quantification of (I) disconnected lymphatic endothelial cell (LEC) clusters (<100 μm) and vessel branches (>100μm)/area and (J) PROX1+ nuclei in each LEC cluster. Total area quantified = 4200 x 2200 μm on both side of midline in sibling controls (n=3) and Sox7 iECKO mutants (n=5). PROX1+ nuclei were quantified from n=29 and n=122 LEC clusters, respectively. (D-J) Skins were from the cervico-thoracic regions of E13.5 embryos, defloxed at E9.5, E10.5. Mean ± SEM, Mann-Whitney U –test. P <0.05 (*). LV, lymphatic vessel. Scale bars = 100 μm (immunofluorescence images A,C), 1 mm (bright-field images B,C).

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, MANN-WHITNEY, Immunostaining, Mutagenesis, Migration, Immunofluorescence

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, following injection with tamoxifen at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), blood vessels by EMCN (green) and arterioles by SOX7 (white nuclei). Dashed line represents the midline of the embryo. (B) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control for EMCN (grey), showing dermal blood vessels at E13.5, after injection with tamoxifen at E9.5 and E10.5. Endothelial cells are marked by ERG (red), proliferative cells are stained by phospho-histone 3, H3 (green). (Right panel) Quantitation of EMCN+ (blood) vessel front density (μm 3 ), number of H3+ proliferative and ERG+ endothelial cells in this region. Scored sibling control, n=5; Sox7 iECKO mutants, n=4; Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. (C) Whole-mount immunostaining of Sox7 iECKO ; mT/mG mutant and Cre+; mT/mG control embryonic skin at E14.5, after injection with tamoxifen at E12.5. Blood vessels are stained with EMCN (red), cells after Cre excision are shown in green, and lymphatic endothelial cells are marked by PROX1 (white). (D-F) Graphs showing quantitation of the Cre activity in (D) blood vessels, (E) lymphatic vessels and (F) EMCN+ (blood) vessel density (μm 3 ), from n=4 Cre+; mT/mG controls and n=3 Sox7 iECKO ; mT/mG mutants. Mean ± SEM; Mann-Whitney U -test; ns = not significant. (G) Whole-mount immunostaining of Sox7 iECKO mutant and sibling control embryonic skin at E14.5, after injection with tamoxifen at E11.5 and E12.5. Dermal lymphatic structures are marked by the NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green). Dashed line represents the midline of the embryo. (H) Immunostaining of Sox7 iECKO mutant and sibling control coronal sections, at E11.5, after injection with tamoxifen at E9.5, E10.5. Lymphatic progenitor cells in the cardinal veins (CVs) are PROX1+ (white). Yellow arrowheads highlight the presence of LEC progenitors in the region near the dorsal aorta (DA) in the Sox7 iECKO mutant, which is typically absent in the sibling control. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Injection, Staining, Quantitation Assay, MANN-WHITNEY, Activity Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing the expression of Sox7 and Vegfc. Both Sox7 and Vegfc are found expressed in BECs (red arrowheads), but not LECs (empty red arrowheads). (C) Vegfc (red) fluorescent RNA in situ hybridisation on mouse cross-sections at E11.5. The endothelium is delineated by PECAM (white) and LECs by PROX1 (green nuclei). BEC-specific endogenous levels of Vegfc (red arrowheads) are higher than LECs (asterisks). (D) qPCR on FACs-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (E-F) qPCR on human arterial endothelial cells (HUAECs) transfected with SiSOX7 or SiCTRL for (E) 30 h and (F) 17 h. (G) qPCR on human venous endothelial cells (HUVECS) transfected with SiSOX7 or SiCTRL for 17 h. (E-G) Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. (D–G) Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). BEC, blood endothelial cell; LEC, lymphatic endothelial cell; DA, dorsal aorta; CV, cardinal vein; Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Expressing, In Situ, Hybridization, Injection, Marker, Transfection

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) Nebulosa plots from single-nuclei RNA-Seq on E14.5 embryonic skins, showing markers for BECs ( Emcn ), LECs ( Prox1 ), SMCs ( Acta2 ) and neuronal cells ( Npas3 ). (E) Microarray analysis of dermal BEC and LEC populations sorted from different embryonic stages reveals that Sox7 and Vegfc mRNA are preferentially expressed by BECs. BECs, blood endothelial cells; LECs, lymphatic endothelial cells; and SMC, smooth muscle cells.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: RNA Sequencing Assay, Microarray

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-D) SOX7 transcriptionally activates Notch effector, HEY1, to repress Vegfc . (A) qPCR on FAC-sorted PECAM+CD45- endothelial cells of Sox7 iECKO mutants and sibling controls at E14.5, injected with tamoxifen at E11.5 and E12.5. Expression is normalised to the endothelial marker Pecam and Cdh5 . Scored sibling controls, n=9; Sox7 iECKO mutants, n=8. (B-C) qPCR on (B) human arterial endothelial cells (HUAECs) and (C) human venous endothelial cells (HUVECs) transfected with SiSOX7 or SiCTRL for 17 h. In addition to HEY1, DLL4 levels were also downregulated in the human cell line experiments. Expression is relative to HPRT and GAPDH. Data from one siRNA experiment performed in triplicates. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); P <0.0005 (***). (D) HEY1 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc and either EV (empty vector) or HEY1 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3 independent repeats of the same experiment. Mean ± SEM; t -test. P <0.05 (*). (E-G) SOX7 physically interacts with transcription repressor, HEY1. (E) Amplified Luminescent Proximity Homogenous Assay (ALPHAScreen) shows the heatmap of SOX7 pairwise protein-protein interaction tested, where red indicates strong interaction and light blue indicates an absence of interaction. (F) Single molecule spectroscopy reveals that SOX7 is able to directly interact with HEY1. SOX7 and the target transcription factors were tagged with GFP or Cherry. GFP-SOX7 and HEY1-Cherry show co-incidence at 0.66, suggesting a 1:2 interaction. Peaks centered on 0 (green) or 1 (red) correspond to the GFP or Cherry-tagged proteins only. (G) Co-immnunoprecipitation analysis of SOX7 and HEY1. HEK293 cells were transfected with indicated plasmids and harvested 24 h after transfection, immunoprecipitated by anti-GFP or Ig control before immunoblot analysis to determine the presence of bait/prey (top) and the input (bottom). N=2.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Injection, Expressing, Marker, Transfection, Activity Assay, Plasmid Preparation, Construct, Luciferase, Amplification, Amplified Luminescent Proximity Homogenous Assay, Spectroscopy, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) SOX7 represses human VEGFC promoter activity. HeLa cells were co-transfected with human or mouse VEGFC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*). (B) Schematic representation of the human VEGFC locus 255 kb upstream from the transcription start site (TSS) (denoted as VEGFC-255 region) from the UCSC Genome browser. The H3K27Ac is denoted in light blue, DNAseI hypersensitive hotspots are indicated by black/grey boxes, where the darkness is proportional to the maximum signal strength observed in any cell line. The chromatin state in HUVECs is shown in purple (indicates insulator), green (poised enhancer) and pink (a Polycomb-repressed region). The HUVECs CCCTC-binding factor (CTCF) binding location is shown in orange. Multiple species conservation is shown in blue peaks and alignments with black stripes. (C) The human luciferase VEGFC-255 BCR transgene. B (black), binding region; C (blue), conserved region and R (pink), repressive region. (D) Schematic representation of the mouse Vegfc locus 152kb upstream from the TSS (denoted as Vegfc- 152). The chromatin state in mouse at E11 from ENCODE is denoted in pink (indicates a heterochromatin region enriched for H3K9me3 repressive marker). The ATAC-Seq (in black) indicates accessible DNA regions in E11 mouse hearts. Multiple species conservation is shown in blue peaks and alignments with black stripes. (E) The mouse luciferase Vegfc-152 RBC transgene. R (pink), repressive region; B (black), binding region and C (blue), conserved region. (F) SOX7 represses VEGFC promoter activity through VEGFC-255 and Vegfc-152 . HeLa cells were co-transfected with human or mouse VEGFC-luc, VEGFC-255 BC-luc, VEGFC-255 BCR-luc, Vegfc-152 BC-luc, Vegfc-152 RBC-luc, EV (empty vector), or SOX7 expression constructs as indicated. VEGFC luciferase activity was measured and normalised to Renillla luciferase activity, which was then made relative to the promoter-less vector, pGL3-basic, which was set to 1. Biological replicates, n=3. Mean ± SEM; t -test. P <0.05 (*); P <0.005 (**); ns = not significant.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Construct, Luciferase, Binding Assay, Marker

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) MSigDB pathway analysis on the top 2k peaks from human SOX7 HUVEC ChIP-Seq identified enrichment for genes within the VEGF/VEGFR gene set. These include VEGFR1 , VEGFR2 , NRP1 , NRP2 , PDGFC , VEGFA and VEGFC . (B-F) Binding profiles and motif analysis of mouse SOX7-V5 ChIP-Seq (B) Image from Integrative Genome Viewer (IGV) illustrating ChIP-Seq tracks at E9.5 (pink) and E10.5 (light blue) mapped to the mouse reference genome GRCm38/mm10 (top track, dark blue). The height of a graph peak indicates the number of “positive bindings” on a given chromosomal location. Total number of binding events is indicated on the left under each track name. Top DNA-binding motifs in SOX7 - V5 ChIP-Seq by MEME-ChIP in (C) E9.5 and (D) E10.5 embryos. The different height of the letters in the position weight matrix reveals information content of each position (in bits), correlated by the degree of certainty of the nucleotide at a given position. (E) Spaced motif analysis (SPAMO) found a SOX7 primary motif amongst the topmost enriched motif next to a MEF2A/MEF2C binding motif found in E9.5 SOX7 - V5 ChIP-Seq, with 2093 total occurrences. SOX7 is best gapped at 134 bp from the MEF2A/MEF2C binding motif (24/2093). (F) ChIP-PCR on both bound fraction (left) and corresponding assumed unbound controls (right), as predicted by the peak location from SOX7-V5 ChIP-Seq. (G) A total of 712 genes were found overlapping between E9.5 and E10.5 SOX7-V5 mouse ChIP-Seq. (H-J) KEGG pathway analysis (H) and gene ontological analysis by DAVID bioinformatics database, showing the top biological processes (I) and molecular functions (J) associated to the 712 overlapping genes from the SOX7-V5 mouse ChIP-Seq.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) At 2 days post Cre induction, E11.5 Sox7 iECKO mutants showed ectopic expression of PROX1+ (red) in medial ventral regions of the cardinal veins (CVs), close to dorsal aorta (DA) (yellow arrowheads). (B) Rose diagram indicates the % of PROX1+ cells over total number of EMCN+ endothelial cells, within each indicated region. The medial region closest to the DA is indicated by 0/360°; most lateral region, 180°; dorsal, 90° and ventral, 270°. Scored sibling controls, n=5; Sox7 iECKO mutants, n=4. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*). (C) Graph indicates the % of total PROX1+ cells over the total number of EMCN+ endothelial cells within each embryo analysed. A total of 2907 endothelial cells quantified, from n=5 sibling controls and 1780 endothelial cells from n=4 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P =0.0635. (D) Whole-mount immunostaining of control and Sox7 iECKO embryonic skin at E12.5 after Cre induction at E9.5, E10.5. Dermal lymphatic structures are stained with NRP2 (membranous white), lymphatic endothelial cells, PROX1 (red), and blood vessels, both EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), and arrow shows EMCN + PROX1 + NRP2 low/- LEC within the vessel wall (type II). (E) Graph shows the total events of PROX1+ single LECs or LEC clusters cells associated with EMCN+ blood vessel plexus within each embryo. PROX1+ cells were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P<0.05 (*). Dors., dorsal; Med., medial. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Expressing, MANN-WHITNEY, Immunostaining, Staining

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A-B) Whole-mount immunostaining of Sox7 iECKO mutant or control embryonic skin, showing the different types of LECs emerging from the blood capillary plexus. Skins were harvested from embryos at E12.5, after Cre-induction at E9.5 and E10.5. Dermal lymphatic structures are marked by NRP2 (membranous white), lymphatic endothelial cells by PROX1 (red), and blood vessels are stained by EMCN (green) and SOX7 (white nuclei). Arrowheads indicate EMCN low/- PROX1 + NRP2 +/low individual LECs on blood vessels (type I), arrows show the single EMCN + PROX1 + NRP2 low/- LEC within the blood vessel wall (type II) and empty arrowheads mark the EMCN +/- PROX1 + NRP2 +/low LEC clusters (2-4 cells) exiting from the blood vessel network (type III). (C-E) Graph shows the quantitation of different types of PROX1+ LEC progenitors, comparing between control and Sox7 iECKO mutant skins. PROX1+ LEC progenitors were quantified from n=4 controls and n=5 Sox7 iECKO mutants. Mean ± SEM; Mann-Whitney U -test. P <0.05 (*); ns = not significant. Scale bars = 100 μm.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Immunostaining, Mutagenesis, Staining, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A) Table from HOMER motif database showing the top 10 most enriched transcription motifs associated to SOX7 binding sites (B, G) Graphs showing intersections between top 2k SOX7 ChIP-Seq peak locations and HUVEC or mouse histone markers, respectively from the ENCODE consortium. Only peaks with at least 50% overlapping region with the histone markers are considered. Intersection was performed using the EpiExplorer online resource tool. (C, H, J) Venn diagrams showing intersections of different active histone markers and (D, I, K) repressive histone markers, both displayed at least 50% overlapped, with the top 2k of SOX7 ChIP-Seq binding regions. Diagram was generated using online resource stool, Venny 2.1.0. (E, L) Graphs showing the overall proportion of top 2k SOX7 ChIP-Seq peaks, with at least an active or a repressive histone mark. (F) Graphs showing intersections between top 2k SOX7 HUVEC ChIP-Seq peak locations and chromatin states in HUVECs from the ENCODE consortium. Only peaks with at least 50% overlapping region with each corresponding chromatin state are considered. Intersection was performed using the EpiExplorer online resource tool.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Binding Assay, ChIP-sequencing, Generated

Journal: bioRxiv

Article Title: The blood vasculature instructs lymphatics patterning in a SOX7 dependent manner

doi: 10.1101/2021.07.02.450967

Figure Lengend Snippet: (A and B) LEC progenitor organisation is impaired in both early stage and organ-specific lymphangiogenesis. ( A ) In physiological conditions, LEC progenitors emerge in the dorsal lateral part of the CVs and migrate towards the dorso-lateral aspects of the embryo. In the absence of functional SOX7, blood vascular endothelial cells from the arterial compartment increase the local expression of endothelial VEGFC, perturbing its tissue distribution. This induces and/or expands the number of LEC progenitors in ventro-medial aspects of the CVs. ( B ) Dermal lymphatics are dysmorphic in the absence of SOX7 function in blood vascular endothelial cells. An increase in VEGFC in the dermal blood endothelial cells causes hyperproliferation of local LEC progenitors. This is shown by an increase in the emergence of local LEC progenitors from the endomucin-positive blood capillary plexus at E12.5 (red asterisks). Changes in expressions of other SOX7-dependent lymphangiocrines also contribute to the migration defects in the dermal lymphatics. ( C ) Model showing the molecular mechanisms of SOX7-dependent repression of VEGFC transcription identified in this study: 1) SOX7 binds to distal regulatory elements (silencing regions) to suppress VEGFC transcription. 2) SOX7 represses VEGFC promoter activity. 3) SOX7 acts upstream of Notch effector and repressor, HEY1. SOX7-induced expression of HEY1 causes VEGFC downregulation and 4) Protein-protein interaction between SOX7 and HEY1 forms a complex to repress VEGFC transcription. LEC, lymphatic endothelial cells; AEC, arterial endothelial cells; VEC, venous endothelial cells; Lat., lateral; Med, medial.

Article Snippet: To generate GFP-SOX7, we used the following primers (restriction sites were underlined): GFP-SOX7_F (HindIII): 5’-CGTA AAGCTT CGATGGCTTCGCTGCTGGG-3’ GFP-SOX7_R (BamH1): 5’-GATC GGATCC CTATGACACACTGTAGCTGTTGTAGT-3’ To generate SOX7-GFP, primers used were: SOX7-GFP_F (HindIII): 5’-CGTA AAGCTT ATGGCTTCGCTGCTGGGAGC-3’ SOX7-GFP_R (BamHI): 5’-GATC GGATCC CGTGACACACTGTAGCTGTTGTAGT-3’ To generate Cherry-HEY1, primers used were: Cherry-HEY1_F (HindIII): 5’-CGTA AAGCTT CGATGAAGCGAGCTCACCCC-3’ Cherry-HEY1_R (BamHI): 5’-GATC GGATCC AAAAGCTCCGATCTCCGTCC-3’ To generate HEY1-Cherry, primers used were: HEY1-Cherry_F (Xho1): 5’-GATC CTCGAG ATGAAGCGAGCTCACCCC-3’ HEY1-Cherry_R (HindIII): 5’-CGTA AAGCTT AAAAGCTCCGATCTCCGTCC-3’ Full-length SOX7 was sub-cloned into linearised pEGFP-N1 and pEGFP-C1 (Clontech), respectively to generate SOX7-GFP and GFP-SOX7.

Techniques: Functional Assay, Expressing, Migration, Activity Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) E17.5-E18.5 cortices were dissociated and plated on poly-D-lysine. After 10d, cultures were stained for pan neuronal (MAP2), astrocyte (GFAP) and microglia (IBA1) markers, and cell type composition was determined by quantitative analysis of immunofluorescence images. Based on 6 Rad21 +/+ Nex Cre and 8 Rad21 lox/lox Nex Cre different samples analysed in 4 independent experiments. b) Immunofluorescence staining of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre neuronal explant cultures for RAD21 and MAP2 (left) and distribution of RAD21 expression by MAP + neurons (right). Note the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neurons. Three independent experiments per genotype. DAPI marks nuclei. Scale bar = 60 μm. c) Immunofluorescence staining for RAD21, MAP2, and the marker of GABAergic inhibitory neurons, GAD67 (left). Distribution of RAD21 expression in GAD67 + and GAD67 - neurons (right). Note that the discontinuous distribution of RAD21 expression in Rad21 lox/lox Nex Cre neuronal explant cultures is due to GAD67 + GABAergic inhibitory neurons. Three independent experiments for Rad21 +/+ Nex Cre and 6 independent experiments for Rad21 lox/lox Nex Cre . DAPI marks nuclei. Scale bar = 20 μm. d) Quantitative RT-PCR analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (mean ± SEM, n=18). Hprt and Ubc were used for normalization (left). RAD21 protein expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures was quantified by fluorescent immunoblots (mean ± SEM, n=6) and normalised to LaminB (center). Nex Cre RiboTag RNA-seq of analysis of Rad21 mRNA expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures (right, 3 independent biological replicates). e) 5C heat maps of a 1.72 Mb region on chromosome 2, comparing Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical explant cultures. CTCF ChIP-seq (Ref. ) and mm9 coordinates are shown for reference. Arrowheads mark the position of CTCF-based loops. Results were consistent across two replicates and 3 chromosomal regions Histograms below show the quantification of representative CTCF-based loops (arrowheads) in two independent biological replicates for control and Rad21 lox/lox Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Staining, Immunofluorescence, Expressing, Marker, Quantitative RT-PCR, Western Blot, RNA Sequencing Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Nex Cre -dependent Rpl22-HA (RiboTag) expression is restricted to RAD21-negative cells in Rad21 lox/lox Nex Cre neurons. Immunofluorescence staining for RAD21, the pan-neuronal marker MAP2 and HA (RiboTag) in explant culture. DAPI marks nuclei. Scale bar = 40 μm. b) Nex Cre RiboTag captures excitatory neuron-specific transcripts such as Slc17a7 and Camk2a and depletes cell type-specific transcripts expressed in inhibitory neurons ( Gad1, Gad2, Slc32a1 ), astrocytes ( Gfap, Aqp4, Mlc1 ), and microglia ( Aif ). Transcript enrichment (or depletion) was calculated using the normalized counts from Nex Cre RiboTag versus standard RNA-seq in Rad21 +/+ Nex Cre neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Immunofluorescence, Staining, Marker, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Volcano plot representing log2 fold-change (FC) versus significance (-log10 of adjusted P values) of downregulated genes (1028) and upregulated genes (572) in RiboTag RNA-seq of Rad21 lox/lox Nex Cre versus Rad21 +/+ Nex Cre neurons. Red marks Rad21 . b) Analysis of gene ontology of biological functions of deregulated genes in Rad21 lox/lox Nex Cre neurons. Enrichment is calculated relative to expressed genes. c) The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RiboTag RNA-seq. The P -value (Fisher Exact Test) and Odds ratio indicate that activity-dependent genes are more frequently deregulated than constitutive genes.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Examples of deregulated genes in Rad21 lox/lox Nex Cre neurons. Genes associated with autism spectrum disorders are highlighted in red. b) GSEA for downregulated genes in Nex Cre/+ Rad21 lox/lox neurons using gene sets derived from (i) KEGG pathway database, (ii) GO Biological Process Ontology, (iii) GO Molecular Function Ontology, (iv) GO Cellular Component Ontology in the Molecular Signatures Database (MSigDB). c) Overlap between human genes associated with autism spectrum disorders from the SFARI database and differentially expressed genes (left), downregulated genes (middle) and upregulated genes (right) in Rad21 lox/lox Nex Cre cortical neurons.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expected Mendelian ratios and observed percentages of live Rad21 +/+ Nex Cre , Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at the indicated developmental stages, n = 217. b) Immunofluorescence analysis shows neither the apoptosis marker activated caspase 3 (CC3) nor the DNA damage marker γH2AX in E16.5 (top) and E18.5 Rad21 lox/lox Nex Cre (bottom, white lines demarcate the cortex). Wild type E16.5 thymi are shown as positive controls for CC3 and γH2AX. Two biological replicates. Scale bar = 100 μm. Photomicrographs of coronal brain sections at gestational age E16 modified from the Atlas of the prenatal mouse brain are shown for orientation. c) Quantitative RT-PCR analysis of gene expression in Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre E17.5/18.5 cortical explant cultures 10 d after plating. Hprt and Ubc were used for normalization. Mean ± SEM of 3 cultures per genotype. d) Brain weights of Rad21 +/+ Nex Cre and Rad21 lox/+ Nex Cre , Rad21 lox/lox Nex Cre mice at birth (P0). Mean ± SEM of between 3 and 13 mice per genotype. e) Embryonic cortices from wild-type and Rad21 lox/lox Nex Cre mice were dissected at E17.5 and E18.5 and dissociated. Cortical cell numbers were determined by counting in Neubauer chambers. Each symbol denotes an independent experiment. Mean ± SEM are also shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Marker, Modification, Quantitative RT-PCR, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Schema of cortical layers showing subplate (SP), layer 6 (VI), layer 5 (V), the cortical plate (CP), and the marginal zone (MZ). Immunofluorescence analysis of the neuronal transcription factors CUX1, TBR1, and CTIP2 at E16.5. Representative of 3 biological replicates. Scale bar = 100 μm. b) Morphology of E18.5 neurons after 1 d in explant culture. Immunofluorescence staining for the pan-neuronal marker MAP2, tubulin beta 3 (TUBB3), and DAPI. Scale bar = 20 μm. c) Morphology of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant culture on rat glia . Cultures were sparsely labeled with GFP to visualize individual cells and their processes, and stained for GAD67 to exclude GABAergic neurons. Dendritic traces of GFP + neurons. Scale bar = 50 μm. d) Sholl analysis of Rad21 +/+ Nex Cre and Rad21 lox/lox Nex Cre cortical neurons in explant cultures shown in c). Shown is the number of crossings, dendritic length, terminal points, branch points and spines per 10 μm. Three independent experiments, 32 Rad21 lox/lox Nex Cre and 28 Rad21 +/+ Nex Cre neurons except for the number of spines (two independent experiments, 10 Rad21 lox/lox Nex Cre and 10 Rad21 +/+ Nex Cre neurons). * adj. P <0.05, ** adj. P <0.01, *** adj. P <0.001, **** adj. P <0.0001. Scale bar = 10 μm.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Immunofluorescence, Staining, Marker, Labeling

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) GSEA of the gene set downregulated (DEseq2, adj. P < 0.05) in RAD21-TEV neurons in Rad21 lox/lox Nex Cre neurons (left) and GSEA of genes downregulated in Rad21 lox/lox Nex Cre neurons (DEseq2, adj. P < 0.05) in RAD21-TEV neurons. b) Scatter plots of gene expression within aggregate GO terms, comparing RAD21-TEV with Rad21 lox/lox Nex Cre neurons. Genes that were found deregulated in at least one of the genotypes are shown. P -values and odds ratios refer to the probability of finding the observed patterns of co-regulation by chance. R S : Spearman’s rank coefficient. c) Deregulation of constitutive and activity-dependent genes 24h after acute cohesin depletion by inducible proteolytic cleavage of RAD21-TEV; adj. P <0.05 based on DEseq2 analysis of 3 RNA-seq replicates per experiment. Two independent experiments are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Preferential deregulation in Rad21 lox/lox Nex Cre neurons of genes near constitutive and KCl-inducible neuronal enhancers . Based on 3 RiboTag RNA-seq replicates per genotype. b) Enrichment of inducible activity-dependent genes near constitutive and KCl-inducible neuronal enhancers . c) CTCF binding at neuronal genes and enhancers. All genes: all expressed genes in total RNA-seq; Activity-dependent genes: Previously defined activity-dependent genes (Kim et al., 2010) that are inducible by KCl in our experiments (KCl minus TTX adj. P < 0.05); Constitutive genes: Expressed genes that are not inducible by KCl in our experiments (KCl minus TTX adj. P > 0.05); Enhancers: Previously defined forebrain enhancers ; CTCF binding: Previously defined CTCF binding peaks within 1kb of TSS or enhancer. d) Only a minority of activity-dependent gene promoters are directly bound by CTCF, and most activity-dependent genes that lack CTCF promoter binding are nevertheless deregulated in cohesin-deficient neurons. e) Models of gene regulation by direct (left) versus domain-wide chromatin contacts (right). CD: contact domain.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: RNA Sequencing Assay, Activity Assay, Binding Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: The percentage of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in explant culture at baseline as determined by RNA-seq. Analysis of previously defined activity-dependent genes . Middle: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons in the presence of TTX and D-AP5 (TTX). Bottom: Fraction of constitutive and activity-dependent genes deregulated in Rad21 lox/lox Nex Cre neurons after 6h stimulation with KCl. b) Expression of activity-dependent genes in explant cultures of Rad21 +/+ and Rad21 lox/lox Nex Cre neurons under baseline conditions that allow for cell-cell communication versus TTX/D-AP5 (TTX). Comparison by two-sample Kolmogorov-Smirnov test showed that Rad21 +/+ Nex Cre neurons showed stronger expression of activity-dependent genes than Rad21 lox/lox Nex Cre neurons ( P = 5.89e-11). c) Fraction of activity-dependent genes significantly induced by KCl in Rad21 lox/lox Nex Cre neurons at 1 and 6h. In wild-type neurons, 117 and 810 genes were induced ≥2-fold at 1 and 6h of KCl treatment, respectively. d) Fraction of activity-dependent genes that were significantly induced by BDNF at 30 and 120min in RAD21-TEV neurons 24h after RAD21 cleavage. In control RAD21-TEV neurons, 16 and 261 activity-dependent genes were induced ≥2-fold 30 and 120min after BDNF treatment, respectively. Most of these remained inducible 24h after RAD21-TEV cleavage. Dark orange: Strongly induced: log2 FC>1, adj. P <0.05; light orange: moderately induced (log2 FC > 0.5, adj. P <0.05); grey: weakly induced (adj. P <0.05); white: not induced (adj. P >0.05).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Activity Assay, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of the activity-dependent Fos gene at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (left, mean log2-transformed counts from 3 biological replicates, * adj. P < 0.05). b) Enhancer transcripts in control and Rad21 lox/lox Nex Cre neurons were quantified based on normalized RNA-seq reads within 1kb of the eRNA transcription start site. An intergenic region on chr11 was used as a negative control (71.177.622-71.177.792). c) H3K27ac ChIP normalized to H3 in control and Rad21 lox/lox Nex Cre neurons at a control site, Fos enhancer 1 and Fos enhancer 2 after TTX/D-AP5 (TTX) or 1h KCl (KCl). d) Interaction score heatmaps of the 65 kb region immediately surrounding Fos obtained by 5C. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. Previously published CTCF-ChIP-seq is shown for orientation and H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . RNA-seq in TTX-treated and 1h KCl-activated control and Rad21 lox/lox Nex Cre neurons shows KCl-inducible transcription of Fos enhancers in wild-type and cohesin-deficient neurons. Two independent biological replicates are shown in . e) Quantification of 5C contacts between the Fos promoter and Fos enhancer 1 (top), the Fos promoter and Fos enhancer 2 (middle), and CTCF-marked boundaries of the sub-TAD containing Fos (bottom). Two replicates per genotype and condition. f) Model for how cohesin-mediated domain-wide contacts alter the probability of enhancer-promoter contacts, and in this way fine-tune the transcription of activity-dependent genes at baseline and in response to activation. In the absence of cohesin, many activity-dependent genes are expressed at inappropriate levels, but most remain responsive to inducing activation signals. At the Fos and Arc loci, cohesin is not required for chromatin contacts between inducible enhancers and their target promoters. See text for details.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Activity Assay, Transformation Assay, RNA Sequencing Assay, Negative Control, ChIP-sequencing, Activation Assay

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Expression of Arc mRNA at baseline, after TTX/D-AP5 (TTX), and KCl-stimulation (top, mean log2-transformed counts from 3 biological RNA-seq replicates, * adj. P < 0.05) and at the indicated time after BDNF stimulation (bottom, data points represent biological RT-PCR replicates). P- values refer to induction relative to 0 min. * P < 0.05, *** P <0.001. b) Interaction score heatmaps of the ∼40 kb region immediately surrounding Arc obtained by 5C for resting (TTX) and 1h KCl-activated wild-type (top) and Rad21 lox/lox Nex Cre neurons (bottom). Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). Previously published CTCF-ChIP-seq is shown . H3K27ac ChIP-seq in inactive (TTX-treated) and activated neurons is shown to annotate enhancer regions . Two independent biological replicates are shown in . c) Quantification of 5C data. Arc enhancer-promoter loop (top). A CTCF-based loop that braces the Arc locus (arrowhead marked with * in panel b) is quantified for comparison (bottom).

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Expressing, Transformation Assay, RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, ChIP-sequencing

Journal: bioRxiv

Article Title: Activity-induced gene expression and long-range enhancer-promoter contacts in cohesin-deficient neurons

doi: 10.1101/2021.02.24.432639

Figure Lengend Snippet: a) Top: Interaction frequency zoom-in heatmaps of 250 kb region surrounding the Fos gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of the domain that contains Fos . Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the 65 kb region immediately surrounding the Fos gene. Black frames highlight interactions between the Fos gene and upstream enhancers 1 and 2. H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown. b) Top: Interaction frequency zoom-in heatmaps of ∼200 kb region surrounding the Arc gene. Dashed lines and arrow heads mark major CTCF binding sites at the boundaries of domains that contain the Arc locus. Note the weakening of these contacts in Rad21 lox/lox Nex Cre neurons. Bottom: Interaction score heatmaps of the ∼40 kb region immediately surrounding the Arc gene. Black frames highlight interaction between the Arc gene (y-axis) and a nearby downstream enhancer (x-axis). H3K27ac ChIP-seq data from Bicuculline-treated (active) and TTX-treated (inactive) neurons annotate enhancer regions. Two independent biological replicates are shown.

Article Snippet: Primary antibodies used were specific to RAD21 (1:500; rabbit polyclonal ab154769, Abcam), MAP2 (1:5000; chicken polyclonal ab611203, Abcam), GAD67 (1:500; mouse monoclonal MAB5406, Millipore), HA (1:1000; mouse monoclonal MMS-101R, Covance), GFAP (1:500; rabbit polyclonal Z0334, Dako) or IBA1 (1:250; rabbit polyclonal 019-19741, Wako).

Techniques: Binding Assay, ChIP-sequencing

Journal: Scientific Reports

Article Title: The pro-angiogenic role of hypoxia inducible factor stabilizer FG-4592 and its application in an in vivo tissue engineering chamber model

doi: 10.1038/s41598-019-41924-5

Figure Lengend Snippet: Representative images and morphometric analysis of CD31 immunohistochemical (IHC) staining sections. Morphometric analysis (B,C,D) performed based on CD31 staining images ( A ). Although the amount of neovasculature in the HD group was lower than in NC group ( B ), the neovasculature of HD group had a larger average vessel area ( C ). Furthermore, FG-4592 reduced vessel shape index in a dose-dependent manner, suggesting that neovasculature tended to grow upwards after drug treatment ( D ). *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar = 100 um.

Article Snippet: Primary antibodies used are as follow: goat anti-CD31 (santa, sc-1506, 1:50), rabbit anti-Ki67 (abcam, ab16667, 1:200), rabbit anti-HIF-1α (GeneTex, 1:200), rabbit anti VEGF (ptg, 19003-1-ap, 1:200), goat anti-mac2 (rd, af1154, 1:200).

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: The pro-angiogenic role of hypoxia inducible factor stabilizer FG-4592 and its application in an in vivo tissue engineering chamber model

doi: 10.1038/s41598-019-41924-5

Figure Lengend Snippet: The attenuated endothelial cell proliferation after FG-4592 administration. The CD31 and Ki67 double staining immunofluorescence images showed that after FG-4592 administration, the number of CD31(+) Ki67(+) proliferating active endothelial cells was declined, which were observed as orange fluorescent cells around the vascular lumen in merged images. Scale bar: 50 um.

Article Snippet: Primary antibodies used are as follow: goat anti-CD31 (santa, sc-1506, 1:50), rabbit anti-Ki67 (abcam, ab16667, 1:200), rabbit anti-HIF-1α (GeneTex, 1:200), rabbit anti VEGF (ptg, 19003-1-ap, 1:200), goat anti-mac2 (rd, af1154, 1:200).

Techniques: Double Immunofluorescence Staining

Journal: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: Immunofluorescence analysis for IL-13Rα2 ( A ) and IL-13Rα1 ( B ) proteins. Non-scratched control or scratched confluent keratinocyte sheets were immunostained with anti-IL-13Rα2 or anti-IL-13Rα1 antibodies. Data is shown as the mean ± SEM. * p < 0.05. Scale bar: 50 µm.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: Immunohistochemical analysis for IL-13Rα2 expression. Control normal skin and lichenified lesional AD skin were immunostained with control IgG and anti-IL-13Rα2 antibody ( A ). The percentage of IL-13Rα2 positive keratinocytes was calculated in 11 normal skin and 11 AD skin samples ( B ). Data is shown as the mean ± SEM. *** p < 0.001. Scale bar: 50 µm.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Scratching Counteracts IL-13 Signaling by Upregulating the Decoy Receptor IL-13Rα2 in Keratinocytes

doi: 10.3390/ijms20133324

Figure Lengend Snippet: IL13RA2 expression was upregulated in the IL-13Rα2-Tg-HaCaT cells more than in control Moc-HaCaT cells ( A ). Upregulated IL-13Rα2 protein expression was observed in the IL-13Rα2-Tg-HaCaT cells, compared to that in Moc-HaCaT cells ( B ). IL-13-induced IVL downregulation was partially restored in IL-13Rα2-Tg-HaCaT cells ( C ). ** p < 0.01, *** p < 0.001.

Article Snippet: The antibodies for immunofluorescence and immunohistochemistry staining were used as follows: Anti-IL-13Rα2 mouse monoclonal antibody (Abcam, Cambridge, UK), normal mouse IgG (Santa Cruz Biotechnology, Dallas, TX, USA), and goat anti-mouse IgG conjugated with Alexa Fluor 488 dye (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Expressing

Journal: Frontiers in Microbiology

Article Title: β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum

doi: 10.3389/fmicb.2019.00066

Figure Lengend Snippet: The expression profile of hepatic β-actin in S. japonicum infected mice. (A) Total liver lysates were subjected to detect the expression levels of β-actin, GAPDH, and β-Tubulin by western blot. (B) Densitometric analysis of β-actin, GAPDH, and β-Tubulin using Image J2x software. The fold changes were calculated by comparing to the control group. (C) Absolute quantification of β-actin by real-time quantitative PCR. Data represent the mean ± SE. ∗ P < 0.05, infected groups vs control group; ∗∗ P < 0.01, infected groups vs control group; ∗∗∗ P < 0.001, infected groups vs control group. && P < 0.01.

Article Snippet: Equal amounts (60 μg) of protein were loaded and separated on a 10% polyacrylamide gel with 200 V for 2 h and then transferred to a 0.22 μm PVDF membrane (Millipore, MA, United States) with 300 mA for 3 h. After blocking in PBS containing 5% milk and 0.1% Tween-20 for 2 h at room temperature, the membranes were incubated with primary antibodies mouse anti-β-actin (58169, Cell Signaling Technology, Boston, MA, United States, used at 1:2000 dilution), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), and mouse anti-β-Tubulin (T0198, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), rabbit anti-α-SMA (19245, Cell Signaling Technology, Boston, MA, United States, used at 1:1000 dilution), rabbit anti-Collagen I (COL1A1) (BA0325, Boster, Wuhan, China, used at 1:1000 dilution), rabbit anti-Collagen III (COL3A1) (BM1625, Boster, Wuhan, China, used at 1:1000 dilution) at 4°C overnight.

Techniques: Expressing, Infection, Western Blot, Software, Control, Quantitative Proteomics, Real-time Polymerase Chain Reaction

Journal: Frontiers in Microbiology

Article Title: β-Actin: Not a Suitable Internal Control of Hepatic Fibrosis Caused by Schistosoma japonicum

doi: 10.3389/fmicb.2019.00066

Figure Lengend Snippet: Co-localization of β-actin and α-SMA in S. japonicum infected mice liver. (A) Co-immunofluorescence staining for β-actin (green) and α-SMA (red). (B) The integrated optical densities (IODs) of β-actin and α-SMA were analyzed by Image Pro Plus 6.0 software. The correlation between the IOD of β-actin and α-SMA was analyzed by Spearman’s correlation analysis. (C) The correlation between the concentration of hepatic hydroxyproline and the level of β-actin mRNA in mice was analyzed by Spearman’s correlation analysis.

Article Snippet: Equal amounts (60 μg) of protein were loaded and separated on a 10% polyacrylamide gel with 200 V for 2 h and then transferred to a 0.22 μm PVDF membrane (Millipore, MA, United States) with 300 mA for 3 h. After blocking in PBS containing 5% milk and 0.1% Tween-20 for 2 h at room temperature, the membranes were incubated with primary antibodies mouse anti-β-actin (58169, Cell Signaling Technology, Boston, MA, United States, used at 1:2000 dilution), mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), and mouse anti-β-Tubulin (T0198, Sigma-Aldrich, St. Louis, MO, United States, used at 1:2000 dilution), rabbit anti-α-SMA (19245, Cell Signaling Technology, Boston, MA, United States, used at 1:1000 dilution), rabbit anti-Collagen I (COL1A1) (BA0325, Boster, Wuhan, China, used at 1:1000 dilution), rabbit anti-Collagen III (COL3A1) (BM1625, Boster, Wuhan, China, used at 1:1000 dilution) at 4°C overnight.

Techniques: Infection, Immunofluorescence, Staining, Software, Concentration Assay

Journal: Nature protocols

Article Title: DERIVATION OF ENTERIC NEURON LINEAGES FROM HUMAN PLURIPOTENT STEM CELLS

doi: 10.1038/s41596-019-0141-y

Figure Lengend Snippet: a) Protocol for neuronal differentiation and maturation of ENC precursors, b) Flow cytometry analysis of CD49D positive ENC cells from hESC line UCSF4 and hiPSC line WTC11 on day 12. c) Flow cytometry analysis of CD49D positive ENC cells from hESC line UCSF4 and hiPSC line WTC11 after ENC spheroid enrichment on day 15. d) Immunofluorescence staining of TUJ1/TRKC on day 30 of EN induction. e) Flow cytometry analysis of TUJ1 and TRKC expression in EN cells on day 20, day 40 and day 55. f) Immunofluorescence images of CHAT, 5HT, NOS1, and GABA stained ENs on day 50. g) Flow cytometry analyses of CHAT, 5HT, NOS1, and GABA on ENS at day 75. AA, ascorbic acid; GDNF, Recombinant Human Glial Derived Neurotrophic Factor, F647, Alexa Fluor™ 647. Scale bars = 100 μm in c, f and 20 μm in e. o

Article Snippet: The cell lines used in this manuscript are human ES cell line H9 (WA-09) H9-derivative SOX10::GFP (WiCell Research Institute, Memorial Sloan Kettering Cancer Center), human ES cell line UCSF4 (UCSF) and human iPS cell line WTC11 (Coriell Institute, UCSF).

Techniques: Flow Cytometry, Immunofluorescence, Staining, Expressing, Recombinant, Derivative Assay

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: Secreted protein acidic and rich in cysteine like 1 (SPARCL1) interacts with integrin β1 (ITGB1). ( A ) Co-immunoprecipitation with an anti-SPARCL1 antibody followed by Western blotting with an anti-ITGB1 antibody. ( B ) Co-immunoprecipitation with an anti-ITGB1 antibody followed by Western blotting with an anti-SPARCL1 antibody. Input, IgG, and IP represent the positive control, negative control, and target experimental group, respectively.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Immunoprecipitation, Western Blot, Positive Control, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: SPARCL1 influences MDSC migration and differentiation. ( A , C ) Cell scratch assay images at 20 h after SPARCL1 activation or inhibition. ( B , D ) Quantification of the cell migration rate at 20 h after SPARCL1 activation or inhibition according to ( A , C ). ( E , G ) Desmin immunofluorescence staining in MDSCs after SPARCL1 activation or inhibition. ( F , H ) Quantification of the myotube rate based on ( E , G ). ( I , K ) Western blotting images of MyoD expression after SPARCL1 activation or inhibition. ( J , L ) Quantification of MyoD from Western blotting results presented in ( I , K ). Scale bar = 100 μm. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Migration, Wound Healing Assay, Activation Assay, Inhibition, Immunofluorescence, Staining, Western Blot, Expressing

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: SPARCL1 affects an ITGB1-mediated signaling pathway. ( A , I ) Western blotting results for ITGB1, p-FAK, FAK, p-paxillin, paxillin, vinculin, Cdc42, and Arp2/3 after SPARCL1 activation or inhibition, respectively. ( B – H ) Quantification of the Western blotting results presented in ( A ). ( J – P ) Quantification of the Western blotting results presented in ( I ). ** p < 0.01.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Western Blot, Activation Assay, Inhibition

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: SPARCL1 influences cell migration and differentiation through ITGB1. ( A ) Cell scratch assay images after simultaneous SPARCL1 activation and ITGB1 inhibition. ( B ) Quantification of the cell migration rate based on ( A ). ( C ) Desmin immunofluorescence staining of MDSCs after SPARCL1 activation and ITGB1 inhibition. ( D ) Quantification of the myotube rate represented in ( C ). ( E ) Western blotting results of MyoD expression after SPARCL1 activation and ITGB1 inhibition. ( F ) Quantification of the MyoD Western blotting results presented in ( E ). pSPgRNA is the blank control for the SPARCL1 activation group, NC is the negative control for the ITGB1 siRNA interference group, VPR-S represents the SPARCL1 activation group, and siRNA-I represents the ITGB1 siRNA interference group. * p < 0.05, ** p < 0.01, *** p < 0.001; scale bar = 100 μm.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Migration, Wound Healing Assay, Activation Assay, Inhibition, Immunofluorescence, Staining, Western Blot, Expressing, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: SPARCL1 regulates an ITGB1-mediated signaling pathway through ITGB1. ( A ) Western blotting results for ITGB1, p-FAK, FAK, p-paxillin, paxillin, vinculin, Cdc42, and Arp2/3 expression after SPARCL1 activation and ITGB1 inhibition. ( B – H ) Quantification of the Western blotting results presented in ( A ). pSPgRNA is the blank control for the SPARCL1 activation group, NC is the negative control for the ITGB1 siRNA interference group, VPR-S represents the SPARCL1 activation group, and siRNA-I represents the ITGB1 siRNA interference group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Western Blot, Expressing, Activation Assay, Inhibition, Negative Control

Journal: Animals : an Open Access Journal from MDPI

Article Title: SPARCL1 Influences Bovine Skeletal Muscle-Derived Satellite Cell Migration and Differentiation through an ITGB1-Mediated Signaling Pathway

doi: 10.3390/ani10081361

Figure Lengend Snippet: Proposed model for SPARCL1 regulation of bovine MDSC migration and differentiation through ITGB1. As an extracellular matrix protein, SPARCL1 interacts with the transmembrane receptor ITGB1 and regulates changes in the expression of adhesion-plaque-associated proteins, such as ITGB1, p-FAK, FAK, p-paxillin, paxillin, and vinculin to affect the formation of focal adhesions. The Cdc42 and Arp2/3 complex was also affected by SPARCL1 through ITGB1. Further, bovine MDSC migration was stimulated by SPARCL1, and cells gathered and apparently expressed MyoD to begin an early-stage differentiation.

Article Snippet: The following primary antibodies were used: anti-SPARCL1, anti-ITGB1, anti-p-FAK, anti-FAK, anti-p-paxillin, anti-paxillin, anti-vinculin, anti-Arp2/3, anti-Cdc42, anti-MyoD, and anti-GAPDH (all at dilution 1:500; Bioss Antibodies, China).

Techniques: Migration, Expressing