66254 1 ig  (Proteintech)

Journal: Neurogastroenterology and Motility

Article Title: Perlecan, CollagenXVIII , and Agrin Expression in Normo‐, Hypo‐, and Aganglionic Segments in Hirschsprung's Disease

doi: 10.1111/nmo.70230

Figure Lengend Snippet: Expression of perlecan, COL18A1, and agrin around enteric ganglia in the normoganglionic segment of a HSCR affected human colon. In the normoganglionic colon perlecan expression was detectable in the matrix (arrowheads) surrounding PGP9.5 positive myenteric ganglia (A) as well as myenteric (B) and submucosal (C) ganglia with HuC/D positive perikaryal. Moreover, we detected pan‐neural markers (p75, D), as well as developmental markers DCX (E) and PHOX2B (G + H) in ganglia of normoganglionic segments. Furthermore, a strong staining for COL18A1 (F + G) and agrin (H) was detectable as a fine layer around myenteric ganglia. In some cases, a weaker, homogeneous immunopositivity of perlecan (B + C), COL18A1 (G) and agrin (H) was found inside the ganglia. Cell nuclei were counterstained with DAPI. Scale A–H: 20 μm.

Article Snippet: PHOX2B , Mice , 66254‐1‐Ig , 1:500 , Proteintech, Rosemont, USA.

Techniques: Expressing, Staining

Journal: Neurogastroenterology and Motility

Article Title: Perlecan, CollagenXVIII , and Agrin Expression in Normo‐, Hypo‐, and Aganglionic Segments in Hirschsprung's Disease

doi: 10.1111/nmo.70230

Figure Lengend Snippet: Expression of perlecan, COL18A1, and agrin in the hypoganglionic segment. The overview of the Tunica muscularis at the transition zone from normo‐ to hypoganglionic gut region in HSCR showed an abrupt vanishing of immunoreactivity for the neuronal marker PGP9.5 (A, arrows), whereas PHOX2B remained unchanged throughout hypoganglionic ganglia stained on a consecutive section of the same patient (B, arrowheads). (C–H) Show high‐power magnification micrographs of myenteric ganglia in the hypoganglionic region. The expression pattern of perlecan remained unchanged in comparison to the normoganglionic segment, while PGP9.5 became undetectable (C). In contrast, PHOX2B (D), as well as p75 (E) and DCX (F) were readily visible in hypoganglionic ganglia. Similar to perlecan, the expression pattern of COL18A1 (G) and agrin (H) was unchanged compared to the normoganglionic segment and surrounded the myenteric ganglia resembling a basal membrane. Cell nuclei were counterstained with DAPI. Scale: A + B: 200 μm, C–H: 20 μm.

Article Snippet: PHOX2B , Mice , 66254‐1‐Ig , 1:500 , Proteintech, Rosemont, USA.

Techniques: Expressing, Marker, Staining, Comparison, Membrane

Journal: Nucleic acids research

Article Title: Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF.

doi: 10.1093/nar/gkad1081

Figure Lengend Snippet: Figure 1. SMARCA4 is a t arget able, tumor-specific vulnerability of adrenergic neuroblastoma (NB). ( A ) Crystal violet staining of adrenergic (ADRN) and mesenchymal (MES) NB or control cell lines treated with serial titration of SWI / SNF inhibitor BRM014. Orange squares indicate MYCN amplification or 1p36 deletion. Quantified in Supplementary Figure 3A. ( B ) R epresentativ e images of NB cells treated with lethal (1 μM) and sub-lethal (1 0–1 00 nM) BRM014 or vehicle control (DMSO). ( C ) Preparation of neural crest cells (NCCs) and NCC-derived peripheral neurons from human embryonic stem cells (hESCs). ( D ) Cell growth over time upon treatment with BRM014 or DMSO. Error bars: mean ± 95% confidence interval ( N = 3 independent samples), abbreviations used as in (C). ( E ) Representative images of IMR-32 cells upon expression of shRNA mediating SMARCA4 or SMARCA2 knock-down, or non-targeting control (NTC). (F) Western blot analysis of SMARCA4 and SMARCA2 levels after SMARCA2 knock-down compared to NTC. ( G ) Western blot analysis of SMARCA4 and SMARCA2 levels in IMR-32 SMARCA4 degron-containing cell line (SMARCA4-EGFP-mAID) after 2 h of vehicle (EtOH) or auxin treatment. ( H ) Quantification of IMR-32 (WT) and SMARCA4-EGFP-mAID (mAID) cell line viability upon treatment with BRM014, auxin or vehicle controls for 96 h. Error bars: mean ± SE. ( I ) Experimental design of animal study. ( J ) Small animal MRI imaging and kidneys extracted from animals comparing BRM014- and vehicle-treated animals ( N = 11 per group). ( K ) Tumor growth time course measured by small-animal MRI. Error bars: mean ± SE. ( L ) Comparison of tumor weights of BRM014 and vehicle control treated animals. Error bars: mean ± SE. ( M ) Representative H&E staining of tumor sections comparing BRM014- and vehicle-treated animals.

Article Snippet: Chromatin was then immunoprecipitated overnight at 4 ◦C with SMARCA4 (Proteintech #21634–1-AP, RRID:AB _ 10858784 ), PHOX2B (Santa Cruz Biotechnology #sc-376997, RRID:AB _ 2813765 ), ISL1 cocktail (DSHB #40.2D6, #39.4D5, #39.3F7, and #40.3A4, RRIDs: AB_528315, AB_2314683, AB_1157901, and AB_528313), HAND2 (Abcam #ab200040, RRID:AB _ 2923502 ), GA T A3 (Thermo Fisher #MA1-028, RRID:AB _ 2536713 ), MYCN (Active Motif #61185 RRID:AB _ 2793543 ), ASCL1 (Abcam #ab74065, RRID:AB _ 1859937 ), EBF3 (Thermo Fisher #PA5-30985, RRID:AB _ 2548459 ), H3K4me1 (Abcam #ab8895, RRID:AB _ 306847 ), or RARA (Abcam #ab41934, RRID:AB _ 777683 ) antibodies bound to Protein A (Thermo Fisher #10001D) or Protein G (Thermo Fisher #10003D) Dynabeads.

Techniques: Staining, Control, Titration, Amplification, Derivative Assay, Expressing, shRNA, Knockdown, Western Blot, Imaging, Comparison

Journal: Nucleic acids research

Article Title: Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF.

doi: 10.1093/nar/gkad1081

Figure Lengend Snippet: Figure 4. Temporal patterns of DNA accessibility re v eal CR C losses are enriched at G1-specific enhancers regulated b y SMAR CA4. ( A ) FACS strategy and gating to analyze genome-wide accessibility of cells in individual cell cycle phases. ( B ) Example browser tracks of cell cycle-phase dependent site sensitive to SWI / SNF inactivation. ( C ) Classification of genome-wide SMARCA4 sites based on their cell cycle-phase dependent accessibility changes, and their response to SWI / SNF inactivation. ( D ) DNA accessibility changes upon BRM014 treatment at SMARCA4-bound clusters shown in panel (C). B o x plot centers indicate median values. ( E ) Enrichment of sites with reduced CRC TF occupancy within clusters shown in panel (C). ( F ) Heatmap of cell cycle phase-specific accessibility changes of CCND1 enhancer, and browser tracks showing reduced accessibility in late G1 phase at CCND1 enhancer. Inset shows rapid loss of CRC TF binding at CCND1 enhancer within 1 h of BRM014 treatment. ( G ) Western blot analysis of CCND1 expression changes upon BRM014 treatment across NB cell lines. ( H ) Expression of CCND1 in NB patient tumors with low SMARCA4 expression compared to those with high SMARCA4 expression. Box plot centers indicate median values.

Article Snippet: Chromatin was then immunoprecipitated overnight at 4 ◦C with SMARCA4 (Proteintech #21634–1-AP, RRID:AB _ 10858784 ), PHOX2B (Santa Cruz Biotechnology #sc-376997, RRID:AB _ 2813765 ), ISL1 cocktail (DSHB #40.2D6, #39.4D5, #39.3F7, and #40.3A4, RRIDs: AB_528315, AB_2314683, AB_1157901, and AB_528313), HAND2 (Abcam #ab200040, RRID:AB _ 2923502 ), GA T A3 (Thermo Fisher #MA1-028, RRID:AB _ 2536713 ), MYCN (Active Motif #61185 RRID:AB _ 2793543 ), ASCL1 (Abcam #ab74065, RRID:AB _ 1859937 ), EBF3 (Thermo Fisher #PA5-30985, RRID:AB _ 2548459 ), H3K4me1 (Abcam #ab8895, RRID:AB _ 306847 ), or RARA (Abcam #ab41934, RRID:AB _ 777683 ) antibodies bound to Protein A (Thermo Fisher #10001D) or Protein G (Thermo Fisher #10003D) Dynabeads.

Techniques: Genome Wide, Binding Assay, Western Blot, Expressing

Journal: Nucleic acids research

Article Title: Reactivation of the G1 enhancer landscape underlies core circuitry addiction to SWI/SNF.

doi: 10.1093/nar/gkad1081

Figure Lengend Snippet: Figure 5. SWI / SNF inhibition synergizes with retinoic acid to promote cell-cycle exit in G1. ( A ) Flow cytometry cell cycle analysis of NB cells upon treatment with 13- cis retinoic acid (13- cis RA), BRM014, or vehicle control (DMSO). ( B ) Model by which SWI / SNF inhibitors sensitize cells to retinoic acid-mediated cell cy cle e xit. ( C ) Metagene plots of MYCN and retinoic acid receptor alpha (RARA) chromatin binding at SMARCA4 sites in the presence of BRM014 or DMSO. ( D ) Example ChIP-seq browser track demonstrating preserved RARA binding despite loss of MYCN binding. ( E ) Experimental design. ( F ) Outgrowth of NB cell lines following cross-titration of BRM014 and 13- cis RA. ( G ) Heatmap of synergy scores for BRM014 and 13- cis RA cross-titration across NB cell lines measured by Loewe additivity ( N ≥3 independent replicates). ( H ) Isobolograms of BRM014 and 13- cis RA treatments demonstrating synergy ( N ≥3 independent replicates).

Article Snippet: Chromatin was then immunoprecipitated overnight at 4 ◦C with SMARCA4 (Proteintech #21634–1-AP, RRID:AB _ 10858784 ), PHOX2B (Santa Cruz Biotechnology #sc-376997, RRID:AB _ 2813765 ), ISL1 cocktail (DSHB #40.2D6, #39.4D5, #39.3F7, and #40.3A4, RRIDs: AB_528315, AB_2314683, AB_1157901, and AB_528313), HAND2 (Abcam #ab200040, RRID:AB _ 2923502 ), GA T A3 (Thermo Fisher #MA1-028, RRID:AB _ 2536713 ), MYCN (Active Motif #61185 RRID:AB _ 2793543 ), ASCL1 (Abcam #ab74065, RRID:AB _ 1859937 ), EBF3 (Thermo Fisher #PA5-30985, RRID:AB _ 2548459 ), H3K4me1 (Abcam #ab8895, RRID:AB _ 306847 ), or RARA (Abcam #ab41934, RRID:AB _ 777683 ) antibodies bound to Protein A (Thermo Fisher #10001D) or Protein G (Thermo Fisher #10003D) Dynabeads.

Techniques: Inhibition, Flow Cytometry, Cell Cycle Assay, Control, Binding Assay, ChIP-sequencing, Titration