Journal: BMC Cancer

Article Title: PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment

doi: 10.1186/s12885-022-09651-9

Figure Lengend Snippet: Tumors originated from PARG- overexpressing 3 T3 cells (‘PARG overexpression’) exhibit lower endothelial count than tumors derived from non-induced cells (‘Control’). Tumors were fixed, cryosectioned, and stained with rabbit antibodies to endothelial marker CD31 (BBA7, R&D Systems). The bar represent 100 μm

Article Snippet: Fixed cells were blocked in 5% normal goat serum (Thermo Fisher) on 0.1% TritonX100 and PBS for 1 h at room temperature and then stained with primary rabbit monoclonal anti-PARG antibodies (1:500, D4E6X, Cell Signaling) and mouse anti-pADPr antibodies (1:800, sc-56,198, Santa Cruz) on blocking solution at 4 °C overnight.

Techniques: Over Expression, Derivative Assay, Staining, Marker

Journal: BMC Cancer

Article Title: PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment

doi: 10.1186/s12885-022-09651-9

Figure Lengend Snippet: The ectopic expression of hPARG in 3 T3 cells does not affect their viability in vitro. A Schematic of the 13.5 kb lentiviral cassette pLVX-TetONE-hPARG-IRES-mClover3-NLS-Puro co-expressing hPARG and nucleus-targeted fluorescent protein mClover3 under control of the TetON promoter. The cassette was used to generate a 3 T3 cell line which stably expresses hPARG in the presence of doxycycline (3 T3-hPARG). The Puromycin-resistant gene (Puro) was used as a selection marker. The cells were split into two test groups, one of which received 500 ng/ml doxycycline (+) and the other receiving no doxycycline (−). These cells were then subjected to the assays indicated. B Rabbit monoclonal anti-hPARG and mouse anti-pADPr antibodies were used to detect the hPARG polypeptide and pADPr moieties, respectively, in 3 T3-hPARG cells that were cultured either in the absence (−) or in the presence (+) of doxycycline. Staining with anti-tubulin antibodies was used as a loading control (Tubulin). The uniform expression of hPARG in cultured cells and the corresponding reduction in pADPr was confirmed by immunofluorescence (Supplemental Fig. S ). Uncropped version of Western Blot is presented at Supplemental Fig. S . C Doubling time assay comparing 3 T3-hPARG cell cultures grown either in the presence (‘With Dox’; red line) or in the absence (‘No Dox’; blue line) of doxycycline with regard to the number of cells produced over the indicated time periods. The two treatment conditions showed a similar doubling time (26 ± 7 hours). D Clonogenic assay comparing # of clones (≥50 cells/each) formed by single 3 T3-hPARG cells over 14 days either after doxycycline induction (+DOX) or in the absence of doxycycline (−DOX) ( n = 3 for each group; error bars represent SEM). E Wound healing assay shows the ability of cultured 3 T3-hPARG cells to restore the damaged monolayer in the presence (DOX) and in the absence of doxycycline (No DOX). F 3 T3-hPARG wound healing assay quantified as % of wound closure ( n = 5; the difference is not statistically significant (t-test p value = 0.2076). Error bars represent SEM

Article Snippet: All blots were blocked in 5% dry milk resuspended in 1X PBS (PBS Santa Cruz, CAT# sc-24,947), 0.1% Tween 20 (Sigma, CAT# P2287), then were probed by incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-hPARG (1:2000) (Cell Signaling Technology, CAT# D4E6X), mouse monoclonal anti-pADPr (1:150) (Santa Cruz Biotechnology, CAT# 10H), mouse monoclonal anti-Tubulin (1:20000) (Sigma, CAT# B512).

Techniques: Expressing, In Vitro, Stable Transfection, Selection, Marker, Cell Culture, Staining, Immunofluorescence, Western Blot, Produced, Clonogenic Assay, Clone Assay, Wound Healing Assay

Journal: BMC Cancer

Article Title: PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment

doi: 10.1186/s12885-022-09651-9

Figure Lengend Snippet: The proposed role of poly(ADP-ribosyl) ation pathway in tumorigenesis. hPARG overexpression in 3 T3 cells cause reduction of pADPr level, transcriptional changes and diminish 3 T3 originated tumor vascularization and growth

Article Snippet: All blots were blocked in 5% dry milk resuspended in 1X PBS (PBS Santa Cruz, CAT# sc-24,947), 0.1% Tween 20 (Sigma, CAT# P2287), then were probed by incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-hPARG (1:2000) (Cell Signaling Technology, CAT# D4E6X), mouse monoclonal anti-pADPr (1:150) (Santa Cruz Biotechnology, CAT# 10H), mouse monoclonal anti-Tubulin (1:20000) (Sigma, CAT# B512).

Techniques: Over Expression

Journal: eLife

Article Title: Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

doi: 10.7554/eLife.69815

Figure Lengend Snippet: ( A ) Images of coronal sections of PND25 mouse brains taken through the anterior and middle portions of the corpus callosum (CC) and stained for neurofilament to label axons and DAPI to stain nuclei. An asterisk indicates the position of the brain midline. Scale bar, 200 μm. ( B ) Quantification of thickness of middle regions of the CC at different points from the brain midline from control (n = 4) and AnkB440 KO (n = 5) brains. ( C ) Images of PND0 brains stained for Satb2, Ctip2, and Tbr1 to label neocortical layers and DAPI to stain nuclei. Scale bar, 200 μm. ( D ) Quantification of total, Sabt2-, Ctip2-, and Tbr1-positive cortical layer thickness from PND0 control (n = 5–6) and AnkB440 KO (n = 4) brains. Data in B and D represent mean ± SEM. Unpaired t test. *p < 0.05, **p < 0.01.

Article Snippet: Commercial antibodies used for immunofluorescence included mouse anti-neurofilament (1:200, clone SMI-312, #837904) from Biolegend; mouse anti-βIII-tubulin (1:100, clone TU-20, #MAB1637), rat anti-L1CAM (1:200, clone 324, #MAB5272), guinea pig anti-NeuN (1:1,000, clone A60, #ABN90P) from Millipore-Sigma; sheep anti-alpha/beta tubulin (1:200, #ATN02) from Cytoskeleton; chicken anti-GFP (1:1000, #GFP-1020) from Aves; rabbit anti-Olig2 (1:200, # 13999–1-AP) from Proteintech, and mouse anti-Satb2 (1:200, clone SATBA4B10, # ab51502), rat anti-Ctip2 (1:500, clone 25B6, # ab18465), rat anti-MBP (1:100, #ab7349) and rabbit anti-Tbr1 (1:200, # ab31940), all from Abcam.

Techniques: Staining

Journal: eLife

Article Title: Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

doi: 10.7554/eLife.69815

Figure Lengend Snippet:

Article Snippet: Commercial antibodies used for immunofluorescence included mouse anti-neurofilament (1:200, clone SMI-312, #837904) from Biolegend; mouse anti-βIII-tubulin (1:100, clone TU-20, #MAB1637), rat anti-L1CAM (1:200, clone 324, #MAB5272), guinea pig anti-NeuN (1:1,000, clone A60, #ABN90P) from Millipore-Sigma; sheep anti-alpha/beta tubulin (1:200, #ATN02) from Cytoskeleton; chicken anti-GFP (1:1000, #GFP-1020) from Aves; rabbit anti-Olig2 (1:200, # 13999–1-AP) from Proteintech, and mouse anti-Satb2 (1:200, clone SATBA4B10, # ab51502), rat anti-Ctip2 (1:500, clone 25B6, # ab18465), rat anti-MBP (1:100, #ab7349) and rabbit anti-Tbr1 (1:200, # ab31940), all from Abcam.

Techniques: Knock-Out, Recombinant, Mutagenesis, In Situ