Journal: eLife

Article Title: Massively parallel reporter assay for mapping gene-specific regulatory regions at single-nucleotide resolution

doi: 10.7554/eLife.107565

Figure Lengend Snippet: ( A ) TF binding motifs predicted using HOMER identified within the Olig2 -NR2 CRM candidate, aligned with the average d-massively parallel reporter assay (MPRA) plot for this region (from ). ( B–D ) Position frequency matrices of TF binding sites aligned to motifs predicted by HOMER in Olig2 -NR2: Mybl1 to Motif 1 ( B ), Foxn4 and Pax6 to Motif 3 ( C ), and Otx2 to Motif 14 ( D ). ( E ) UMAP visualization of scRNA profiles from E14 mouse retinas, with cell types previously identified by marker genes by . ( F ) Expression of Olig2 on UMAP. ( G ) Co-expression (pink) of Olig2 (blue) with Mybl1, Foxn4, Pax6 , or Otx2 (red) on UMAP. Percentages of respective populations that co-express Olig2 are indicated (* denotes significance using Fisher’s exact test, p ≤0.05). ( H ) TF occupancy track aligned to (i) Olig2 locus-specific massively parallel reporter assay (LS-MPRA) barcode enrichment after 12 hr LY411575 treatment (replicate of ) and (ii) gene models. Occupancy peaks for Mybl1, Foxn4, and Pax6 align with the Olig2-NR2 CRM candidate (blue box).

Article Snippet: Mouse monoclonal antibodies raised against Pax6 (Developmental Studies Hybridoma Bank, cat. #AB_528427) and against Foxn4 (Santa Cruz Biotechnologies, cat. #sc-377166) as well as rabbit polyclonal antibodies raised against Mybl1 (Millipore-Sigma, cat. #HPA008791) were used, adding 500 ng of the respective antibody to each sample during the antibody binding stage.

Techniques: Binding Assay, Reporter Assay, Marker, Expressing

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: Primers for amplification of target- and off-target genomic DNA

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Amplification

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: PAX6 is knocked out by CRISPR-Cas9 technology in U251 N glioblastoma cells and reintroduced by an EGFP-PAX6 expressing plasmid ( a ) Western blot on cell lysates originating from single cell clones after CRISPR-Cas9 treatment. PAX6 Control cells have undergone CRISPR-Cas9 treatment and single cell sorting but still express PAX6. Detection by anti-PAX6 antibody. Actin staining was used for loading control. KO, knock out; WT, wild type U251 N; L, Molecular weight marker. b Immunocytochemistry (ICC) on WT U251 N and pooled PAX6 KO cell lines (2A.3, 2A.28 and 2.10). Anti-PAX6 antibody detect endogenous PAX6 protein, DAPI staining identifies the nucleus. Scale bars indicate 10 μm. c Western blot on the individual PAX6 KO cell lines transduced with a Dox inducible EGFP-PAX6 expressing plasmid (2A.3R, 2A.28R and 2.10R). The right panel show cells treated with 10 ng/ml Dox for 24 h. Anti-PAX6 and anti-Actin antibodies were used for detection. d ICC of Dox treated pooled Rescue cells expressing EGFP-PAX6. EGFP is detected as green fluorescence, anti-PAX6 antibody stain PAX6 protein, and the nucleus is stained by DAPI in blue. Scale bar indicates 20 μm. Western blots and ICCs were performed on more than three samples of each cell line and showed the same results

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: CRISPR, Expressing, Plasmid Preparation, Western Blot, Clone Assay, Control, FACS, Staining, Knock-Out, Molecular Weight, Marker, Immunocytochemistry, Transduction, Fluorescence

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: The PAX6 KO cell lines show morphological variations. Cells were micro-photographed at 20×. a KO cells and controls. b Pooled PAX6 KO-, PAX6 Control, and PAX6 Rescue cells

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Control

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: Knock out of PAX6 alters morphology of colonies and increases colony-formation ability. Five hundred cells (WT, Control or KO) per well in a 6-well plate was seeded and left for ( a ) 16 days or ( b ) 12 days to study colony forming abilities. c Five hundred cells of the Dox inducible EGFP-PAX6 pooled rescue cell lines and the pooled PAX6 KO cells were seeded per well in 6-well plates and left for 12 days. Colonies were micro-photographed. d Colonies were stained with 0.005% crystal violet before counting. Arrows indicate WT U251 N colonies containing less than 50 cells, and PAX6 KO colonies containing over 50 cells. e Five hundred cells were seeded in three wells in 6-well plates and left to form colonies for 12 days for each of the PAX6 KO cell lines (2.10 KO, 2A.3 KO and 2A.28 KO), and their Rescued counterparts (2.10 R, 2A.3 R and 2A.28 R) as well as the WT cell line U251 N. Pooled versions containing equal amounts of each of the three individual cell lines within each group were also included (3xKO, 3xR and 3xR + Dox). The term “+DOX” indicate Rescue cells treated with Dox 18–24 h before FACS sorting. The day after FACS sorting they were seeded for the colony formation assay. Rescue cells without Dox treatment were also included. Colony-forming abilities were measured by counting colonies consisting of over 50 cells. The figure is representative of three experiments done in triplicate. For each experiment the colonies were counted by two persons.

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Knock-Out, Control, Staining, Colony Assay

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: Percentage of colony formation ability in various PAX6 KO and Rescue cell lines

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques:

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: PAX6 KO cells migrate faster than WT and control cells. a WT U251 N cells, pooled PAX6 control cells and pooled PAX6 KO cells were seeded in Ibdi 2-well silicone inserts and left for 24 h. Cells were Mitomycin C treated for 2 h before inserts were removed, creating a cell free area (gap) at the start of the assay. Cells were micro-photographed at 0, 20, 48, and 72 h after well inserts were removed. The white lines demark migration edges. b The average migration distance was calculated after 20 h as described in methods. Students t-test showed that the difference in migration distance observed between pooled PAX6 KO cells and WT U251 N was significant ( p < 0.002). The difference between pooled PAX6 Control cells and WT U251 N cells was not. Migration assays were repeated three times. Each assay showed the same result

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Control, Migration

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: PAX6 restricts cell proliferation and regulates cell cycle. a Fifty thousand WT U251 N cells, PAX6 1.7 Control cells and PAX6 2.10 KO cells were seeded at day 0 and counted at day 2, and 3. Proliferation assays were repeated three times. b Fifty thousand WT U251 N cells, pooled PAX6 Control cells and pooled PAX6 KO cells were seeded at day 0 and counted at day 1, 2, and 3. c The Cell Titer Glo assay was used to compare proliferation (viability) in the WT U251 N cell line with the Rescue cell line treated with and without Dox. Dox treated Rescue cells were FACS sorted so that all cells expressed EGFP-PAX6. Values obtained at day 1 was set to 1, and the fold increase in proliferation at day 2 and 3 was calculated. Three independent experiments show similar results. d The cell cycle distributions of WT U251 N cells, pooled PAX6 control cells and pooled PAX6 KO cells at 70–90% confluency were examined by PI staining and FACS (upper panel). WT U251 N cells, pooled PAX6 KO cells and pooled PAX6 Rescue cells were Dox treated, and the cell cycle distributions were investigated at 70–90% confluency by PI staining and FACS (lower panel). e Average cell-cycle distribution from three experiments of cells mentioned in D. Student’s t-test, p < 0.05

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Control, Glo Assay, Staining

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: PAX6 KO cells are less sensitive to oxidative stress. Cells were exposed to 300 μM H 2 O 2 for 24 h, and Annexin V FITC /PI stained before FACS analysis. Percentage of viable cells, cells in early apoptosis and cells in late apoptosis/necrosis were determined. a Cells without Doxycycline treatment. b Doxycycline treated cells. Figure shows representative results from three to four experiments

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Staining

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: PAX6 KO cells respond differently to treatment with the chemotherapeutic drugs TMZ, WA and SFA compared to WT cells. U251 N PAX6 KO and WT cells were treated with 250 μM Temozolomide (TMZ), 1.5 μM Withaferin A (WA) and 10 μM Sulforophane (SFA) for 48 (WA and SFA) or 72 (TMZ) hours and analyzed for apoptosis by anti-Annexin/PI staining and FACS. The experiment shown is representative of three independent experiments

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Staining

Journal: BMC Cancer

Article Title: Generation of a PAX6 knockout glioblastoma cell line with changes in cell cycle distribution and sensitivity to oxidative stress

doi: 10.1186/s12885-018-4394-6

Figure Lengend Snippet: RT qPCR shows negative fold change of gene expression in PAX6 KO cells compared to WT. ∆∆Ct method was used for analysis

Article Snippet: Rabbit anti-PAX6 antibody (cat#AB2237, Millipore) at 1:1000 and mouse anti-Pax6 antibody(Cat# pax6, RRID:AB_528427, Developmental Studies Hybridoma Bank, University of Iowa) at a 1:37 dilution was used for screening single cell clones, and rabbit anti-Actin (cat#A2066, Sigma-Aldrich) at a 1:1000 dilution as a loading control.

Techniques: Quantitative RT-PCR, Gene Expression