Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 expression is lowly expressed in NPC. A Expression profile of circular RNAs (circRNAs) in 5-8F cells. Five types were included: exon-derived circRNAs, intron-derived circRNAs, anti-sense strand circRNAs, overlapping regions of sense strand circRNAs, and intergenic circRNAs, according to their different looping regions. CircRNF13 was ranked 14th among the top 20, and was screened due to its high abundance (RPM = 0.59). B Expression of circRNF13 was examined in NPC ( n = 36) and NPE ( n = 12) tissues using RT-PCR, NPE, non-tumor nasopharyngeal epithelial tissues. Data have been represented as mean ± standard deviation (SD). ****, p < 0.0001. C Schematic structure of circRNF13 . circRNF13 is 716 bp in length and circularly spliced by 2–8 exons of the RNF13 gene (RefSeq: NM_183383.2) on the chromosome 3q25.1 region, as verified using Sanger sequencing. D circRNF13 is primarily localized in the cytoplasm, as identified using nucleoplasmic separation experiments. U6 was selected as a nuclear marker and GAPDH was selected as a cytoplasmic marker. E Intracellular localization of circRNF13 was examined using fluorescence in situ hybridization. Scale bar: 20 μm. F Stability of circRNF13 and RNF13 mRNA was detected in RNase R-treated HNE2 and CNE2 cells using RT-PCR. Data have been represented as mean ± SD. ****, p < 0.0001

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Standard Deviation, Sequencing, Marker, Fluorescence, In Situ Hybridization

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 inhibits the proliferation, migration, and invasion of NPC. A Overexpression of circRNF13 inhibited, while knockdown of circRNF13 promoted proliferation of HNE2 and CNE2 cells, as assessed using MTT assay. Data have been represented as mean ± SD. *, p < 0.05; **, p < 0.05; ***, p < 0.001. B Clone formation assay showed that overexpression of circRNF13 inhibited the colony formation capability of HNE2 and CNE2 cells. Data have been represented as mean ± SD. ***, p < 0.001. C CircRNF13 ’s function in the tumor cell cycle was assessed using flow cytometry. HNE2 and CNE2 cells were stained with PI, after overexpression or knockdown of circRNF13 . D CircRNF13 inhibited the migration ability of HNE2 and CNE2 cells transfected with sicircRNF13 or circRNF13 overexpression vector, as assessed using wound-healing assay. Images were acquired at 0 and 24 h. Data have been represented as mean ± SD. *, p < 0.05; ***, p < 0.001. E CircRNF13 inhibited the invasive ability of HNE2 and CNE2 cells after knockdown or overexpression of circRNF13 , as assessed using Transwell invasion assays. Images were acquired at 24 h, and are representative of three independent experiments. Data have been represented as mean ± SD. **, p < 0.01; ***, p < 0.001

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Migration, Over Expression, Knockdown, MTT Assay, Tube Formation Assay, Flow Cytometry, Staining, Transfection, Plasmid Preparation, Wound Healing Assay

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 inhibits NPC growth and metastasis in vivo. A Images of subcutaneous tumor formation in nude mice at 4 weeks. Mice were injected with 2 × 10 6 CNE2 cells, with knockdown or overexpression of circRNF13 . B , C Tumor volumes (B) and tumor weights (C) were measured for each group ( n = 7 per group). Data have been represented as mean ± SD. *, p < 0.05; ***, p < 0.001. D Representative images of in situ hybridization for circRNF13 and immunohistochemical staining for Ki-67 expression in subcutaneous tumors (200 × , scale bar: 50 μm). E Images of visible nodules on the lung surface. CNE2 cells transfected with scrambled siRNA, empty vector, sicircRNF13, or circRNF13 overexpression vector were injected into each nude mouse tail vein ( n = 7 for each group), and the mice were sacrificed 8 weeks later. F The number of lung metastatic nodules on each lung surface was quantified. Data have been represented as mean ± SD (each point represents one mouse; n = 7 per group; right). ***, p < 0.001. G Representative images of circRNF13 expression, as assessed using in situ hybridization, and lung metastatic tumor foci after H&E staining (200 × , scale bar: 50 μm)

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: In Vivo, Injection, Knockdown, Over Expression, In Situ Hybridization, Immunohistochemical staining, Staining, Expressing, Transfection, Plasmid Preparation

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 inhibits glycolysis in NPC cells. A Glucose consumption and lactate production in HNE2 and CNE2 cells were examined using an automated biochemical analyzer, upon circRNF13 overexpression. Data have been represented as mean ± SD from three independent experiments. *, p < 0.05. B The extracellular acidification rate (ECAR) in HNE2 and CNE2 cells was measured using Seahorse XF assay, in response to circRNF13 overexpression or knockdown. Glycolysis, glycolytic capacity, and glycolytic reserve were analyzed. Data have been represented as mean ± SD. *, p < 0.05; **, p < 0.01. C Expression levels of downstream targets of phosphorylated AMPKα and mTOR, S6K and 4EBP1, were measured using western blotting in HNE2 and CNE2 cells in response to circRNF13 overexpression or knockdown. D Expression levels of downstream targets of phosphorylated AMPKα and mTOR, S6K and 4EBP1, were measured using western blotting in HNE2 and CNE2 cells treated with the glycolysis inhibitor 2-DG, in response to circRNF13 overexpression or knockdown. E The glycolysis inhibitor 2-DG attenuated the effect of circRNF13 on NPC cell migration in HNE2 and CNE2 cells transfected with sicircRNF13, as assessed using wound-healing assay. All experiments were repeated at least three times. Data have been represented as mean ± SD. ***, p < 0.001. F The glycolysis inhibitor 2-DG weakened the function of circRNF13 on NPC cell invasion when HNE2 and CNE2 cells transfected with sicircRNF13 were treated with the glycolysis inhibitor 2-DG, as assessed using Transwell assays. All experiments were repeated at least three times. Data have been represented as mean ± SD. ***, p < 0.001

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Over Expression, XF Assay, Knockdown, Expressing, Western Blot, Migration, Transfection, Wound Healing Assay

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 inhibits glycolysis in NPC cells by promoting GLUT1 ubiquitination. A The mRNA levels of GLUT1, HK2, and LDHA in HNE2 and CNE2 cells were examined using RT-PCR, upon overexpression and knockdown of circRNF13 . Data have been represented as mean ± SD; ns, no significance; ***, p < 0.001. B The expression levels of GLUT1, HK2, and LDHA proteins were examined using western blotting in HNE2 and CNE2 cells, upon overexpression and knockdown of circRNF13 . C Degradation of GLUT1 was detected in HNE2 and CNE2 cells using western blotting, after overexpression and knockdown of circRNF13 and treatment with 50 μg/mL cycloheximide (CHX). D Ubiquitination levels of GLUT1 protein were detected in HNE2 and CNE2 cells using pull-down with an anti-Flag antibody and western blot with an anti-ubiquitin antibody, after overexpression or knockdown of circRNF13

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Reverse Transcription Polymerase Chain Reaction, Over Expression, Knockdown, Expressing, Western Blot

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 directly binds to and stabilizes SUMO2 mRNA, to upregulate its expression A. Expression of SUMO2 protein was examined using western blotting in HNE2 and CNE2 cells, after overexpression and knockdown of circRNF13 . B Expression of SUMO2 mRNA was examined in HNE2 and CNE2 cells using RT-PCR, after overexpression and knockdown of circRNF13 . C Bioinformatics prediction using the RNAhybrid website revealed that circRNF13 has a continuous binding site with the 3′-UTR of SUMO2 mRNA. D circRNF13 was found to bind to the 3′-UTR of SUMO2 mRNA in HNE2 and CNE2 cells using RNA pull-down assay. GADPH was used as a negative control. Data have been represented as mean ± SD. ***, p < 0.001. E circRNF13 enhanced the luciferase activity of the SUMO2 mRNA 3′-UTR in HNE2 and CNE2 cells, as assessed using dual luciferase reporter assay. The 3′-UTR of GADPH was used as a negative control. Data have been represented as mean ± SD. *, p < 0.05; **, p < 0.01. F. Degradation of SUMO2 was detected in HNE2 and CNE2 cells using RT-PCR, after overexpression and knockdown of circRNF13 and treatment with 2 μg/mL actinomycin D. Data have been represented as mean ± SD. ***, p < 0.001, ****, p < 0.0001

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Expressing, Western Blot, Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Pull Down Assay, Negative Control, Luciferase, Activity Assay, Reporter Assay

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 promotes ubiquitination of GLUT1 via SUMO2. A The interaction between GLUT1, SUMO2, and UBC9 was examined using immunoprecipitation and an anti-Flag (GLUT1) antibody in HNE2 and CNE2 cells transfected with Flag-GLUT1 vector, followed by western blotting using the anti-SUMO2 and anti-UBC9 antibodies. B The interaction between SUMO2, GLUT1, and UBC9 proteins was examined using immunoprecipitation with an anti-HA (SUMO2) antibody in HNE2 and CNE2 cells transfected with HA-SUMO2 vector, followed by western blotting using anti-GLUT1 and anti-UBC9 antibodies. C Immunofluorescence experiments performed using anti-Flag (GLUT1) and anti-SUMO2 antibodies in HNE2 and CNE2 cells showed that GLUT1 and SUMO2 were co-localized. DAPI-stained nucleus: blue; anti-Flag (GLUT1): red; anti-SUMO2: green; co-localization of GLUT1 and SUMO2: yellow; the merged image represents the overlap of DAPI, GLUT1, and SUMO2 (scale bar: 20 μm). D Immunoprecipitation experiments using an anti-Flag-GLUT1 antibody, followed by western blotting using an anti-SUMO2 antibody, were performed, to identify the SUMOylation level of the GLUT1 protein in HNE2 and CNE2 cells transfected with sicircRNF13 or circRNF13 overexpression vector. E The ubiquitination level of GLUT1 protein was detected in HNE2 and CNE2 cells using pull-down with an anti-Flag-GLUT1 antibody, followed by western blotting with an anti-ubiquitin antibody, to determine whether SUMO2 affects GLUT1 ubiquitination. F A pull-down experiment using an anti-Flag-GLUT1 antibody, followed by western blot using an anti-ubiquitin antibody, were performed to identify the ubiquitination level of GLUT1 protein in HNE2 and CNE2 cells co-transfected with sicircRNF13 and SUMO2 overexpression vector

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence, Staining, Over Expression

Journal: Molecular Cancer

Article Title: Circular RNA circRNF13 inhibits proliferation and metastasis of nasopharyngeal carcinoma via SUMO2

doi: 10.1186/s12943-021-01409-4

Figure Lengend Snippet: CircRNF13 inhibits the proliferation and metastasis of NPC via SUMO2. A Overexpression of SUMO2 rescued the circRNF13 knockdown-mediated altered proliferative capacity of HNE2 and CNE2 cells, as assessed using MTT assay. Data have been represented as mean ± SD. **, p < 0.01; ***, p < 0.001. B Overexpression of SUMO2 decreased the circRNF13 knockdown-mediated altered migration of HNE2 and CNE2 cells, as assessed using wound-healing assays. All experiments were repeated at least three times (Scale bar: 100 μm). Data have been represented as mean ± SD; ns, no significance; ***, p < 0.001. C. Overexpression of SUMO2 reduced the circRNF13 knockdown-mediated altered invasive ability of HNE2 and CNE2 cells, as assessed using Transwell assays. All experiments were repeated at least three times (Scale bar: 100 μm). Data have been represented as mean ± SD; ns, no significance; ***, p < 0.001. D The extracellular acidification rate (ECAR) was measured using Seahorse XF assay in HNE2 and CNE2 cells co-transfected with sicircRNF13 and SUMO2 overexpression vector. Glycolysis, glycolytic capacity, and glycolytic reserve were analyzed. All experiments were repeated at least three times. Data have been represented as mean ± SD; ns, not significant; **, p < 0.01. E Representative images of SUMO2 and GLUT1 expression in lung tissues of nude mice, as determined using immunohistochemistry ( n = 7 per group, 200 × , scale bar: 50 μm). F Schematic diagram of the molecular mechanism of circRNF13 in NPC. circRNF13 may activate and stabilize the SUMO2 protein by binding to the 3′-UTR of SUMO2 mRNA. Upregulation of SUMO2 promotes GLUT1 degradation through SUMOylation and ubiquitination of GLUT1, which regulates the AMPK pathway by inhibiting glycolysis, ultimately resulting in the proliferation and metastasis of NPC

Article Snippet: After centrifugation, 50 μL of the supernatant was retained as input, and the remaining part was incubated with a circRNF13 -specific probe streptavidin Dynabeads (M-280; Invitrogen) mixture overnight at 4 °C.

Techniques: Over Expression, Knockdown, MTT Assay, Migration, XF Assay, Transfection, Plasmid Preparation, Expressing, Immunohistochemistry, Protein Binding, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: (A) Western blot of whole protein lysate from wildtype +/+ and homozygous mutant rbbp4 Δ4/Δ4 5 dpf larvae probed with a rabbit polyclonal anti-Rbbp4 antibody. The antibody recognizes a single polypeptide band of ~50kDA, close to the Rbbp4 predicted size of 48kDa. The band is absent from the rbbp4Δ4/Δ4 mutant protein extract. Lower panel, loading control blot probed with mouse monoclonal antibody β-actin. (B, C) Rbbp4 and proliferative marker PCNA antibody labeling of transverse sections through the head of 5 dpf wildtype (B) and rbbp4Δ4/Δ4 homozygous mutant (C) zebrafish larvae. Confocal images for each larva were captured at approximately the same gain settings, with a slightly higher gain setting for the homozygote, to confirm the absence of Rbbp4 signal. In wildtype brain (B) high levels of Rbbp4 are present in the nucleus of neurons in the tectum and thalamic regions, but are nearly absent in the rbbp4Δ4/Δ4 homozygote (C). Scale bars: 50 μm. (D) RBBP4 Affymetrix data from 2284 human tumor samples (German Cancer Research Center DKFZ). atrt atypical teratoidIrhabdoid tumor, cns central nervous system; cpc choroid plexus carcinoma; cph, cpp choroid plexus papilloma; dipg diffuse intrinsic pontine glioma; etmr embryonal tumor with multilayered rosettes; ews Ewing’s sarcoma; mb medulloblastoma; ptpr papillary tumor of the pineal.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Western Blot, Mutagenesis, Marker, Antibody Labeling

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: (A) Hematoxylin-labeled cross-section and diagram of adult zebrafish midbrain. Representative transverse cryosections (n=3 independent animals) through adult zebrafish midbrain (B-E) or rb1- embryonal brain tumor (F-I) labeled with antibodies to Rbbp4 (green), proliferation maker PCNA (red), and nuclear stain DAPI (blue). Rbbp4 is detected in the nuclei of cells lining the midbrain ventricle (C, D) and throughout the peri-granular zone (E), and is enriched in PCNA positive cells (D, arrow). Section through rb1- brain tumor shows expansion of the ventricular zone (F, G). The lesion is filled with Rbbp4 positive cells that frequently co-label with PCNA (H, I). Hy, hypothalamus; OT, optic tectum; PGZ, peri-granular zone; v, midbrain ventricle. Scale bars: A, E 400 μm; B, F 100 μm; D, G, H 40 μm; C 20 μm. See also . (J) RBBP4 expression in human embryonal tumors. ATRT, Atypical Teratoid/Rhabdoid Tumors: ATRT_TYR (n=25), ATRT_SHH (n=20), ATRT_MYC (n=17); MB, Medulloblastoma: MB_WNT (n=40), MB_SHH (n=112), MB_GRP3 (n=81), MB_GRP4 (n=135); EWS, Ewing Sarcoma (n=2); CNS EFT_ CIC , CNS Ewing sarcoma family tumor with CIC alteration (n=4); CNS HGNET_ BCOR , CNS high-grade neuroepithelial tumor with BCOR alteration (n=16); CNS HGNET_ MN1 , CNS high-grade neuroepithelial tumor with MN1 alteration (n=7); CNS-NB- FOXR2 , CNS neuroblastoma with FOXR2 activation (n=10); EMTR, embryonal tumors with multilayered rosettes (n=18).

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Staining, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: Lateral and dorsal views of head region in zebrafish 2 dpf embryo (A, B) and 5 dpf larva (C-D) with midbrain optic tectum (OT, arrows) and retina (R) labeled. (E-P) Representative transverse cryosections (n=3) through 2 dpf and 5dpf wildtype zebrafish midbrain labeled with antibodies to Rbbp4 (green), proliferation maker PCNA (red), and nuclear stain DAPI (blue). (E) Diagram of location of stem cells lining the midbrain ventricle and at the ciliary marginal zone (CMZ) of the retina. (F-H) Boxes outline the ventricle at the top of the thalamus in the midbrain (G) and the retina ciliary marginal zone (H, bracket). See also . (I) Diagram of 5 dpf midbrain showing location of stem cells (red) and progenitors (blue) at the ventricle in the dorsal optic tectum and ventral thalamus (Th). (J-L) 5 dpf wildtype zebrafish midbrain. Higher magnification view of the medial tectal proliferative zone (K) and midbrain ventricle (L). (M) Diagram of 5 dpf retina showing location of stem cells and retinal precursors at the ciliary marginal zone and the three nuclear retinal layers. (N-P) 5 dpf retina. Higher magnification view of the ciliary marginal zone (O) and central laminated layers of the retina (P). The highly proliferative, PCNA+ population of cells are outlined; arrows indicate cells co-labeled with Rbbp4 and PCNA. Rbbp4 is enriched in a subset of neurons in the midbrain and vitreal side of the retinal inner nuclear layer. See also . GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 100 μm A, B, F, J, N; 200 μm C, D; 20 μm G, H; 10 μm K, L, O, P.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: (A-A’’’) Representative transverse cryosections (n=3) through 2 dpf wildtype zebrafish midbrain labeled with antibodies to Rbbp4 (green), proliferation maker PCNA (red), and nuclear stain DAPI (blue). Boxes outline the ventricle at the top of the thalamus in the midbrain (B) and the ciliary marginal zone (CMZ) of the retina (C). (B-B’’’) Higher magnification view of the midbrain ventricle at the thalamus. (C-C’’’) Higher magnification view of the ciliary marginal zone of the retina (C-C’’’). Scale bars: 100 μm A-A’’’; 20 μm B-C’’’.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: 2 dpf and 3 dpf wildtype and rbbp4 Δ4/Δ4 larval midbrain sections after tp53 knockdown. (A) Inhibition of Tp53 activity by antisense morpholino injection into embryos from rbbp4 Δ4 /+ adult incross. (B-I) Activated Caspase-3 (green), HuC/D (red) and DAPI (blue) labeling at 2 dpf (B-E) and 3 dpf (F-I). (J) Comparison of Caspase-3 positive cells in the midbrain between un-injected and tp53 MO injected wildtype at 2 dpf (un-injected n=4; tp53 MO injected n=4; p =0.017), rbbp4 Δ4/Δ4 at 2 dpf (un-injected n=2; tp53 MO injected n=4; p =0.0112), wildtype at 3 dpf (un-injected n=3; tp53 MO injected n=3; p =0.3349), and rbbp4 Δ4/Δ4 at 3 dpf (un-injected n=4; tp53 MO injected n=6; p =0.1736). (K) Comparison of Caspase-3 positive cells in the retina between un-injected and tp53 MO injected wildtype at 2 dpf (un-injected n=4; tp53 MO injected n=4; p =0), rbbp4 Δ4/Δ4 at 2 dpf (un-injected n=2; tp53 MO injected n=4; p =0.0032), wildtype at 3 dpf (un-injected n=3; tp53 MO injected n=3; p =0.0285), and rbbp4 Δ4/Δ4 at 3 dpf (un-injected n=4; tp53 MO injected n=6; p =0.0037). (L-S) γ-H2AX labeling (green), and nuclear stain DAPI (blue) at 2 dpf (K-N) and 3 dpf (O-R). (T) Comparison of γ-H2AX positive cells in the midbrain between un-injected and tp53 MO injected wildtype at 2 dpf (un-injected n=4; tp53 MO injected n=3; p =0.2344), rbbp4 Δ4/Δ4 at 2 dpf (un-injected n=2; tp53 MO injected n=4; p =0.0024), wildtype at 3 dpf (un-injected n=3; tp53 MO injected n=5; p =0.1101), and rbbp4 Δ4/Δ4 at 3 dpf (un-injected n=6; tp53 MO injected n=10; p =0.0657). (U) Comparison of γ-H2AX positive cells in the retina between un-injected and tp53 MO injected wildtype at 2 dpf (un-injected n=4; tp53 MO injected n=3; p-value=0), rbbp4 Δ4/Δ4 at 2 dpf (un-injected n=2; tp53 MO injected n=4; p =0.0001), wildtype at 3 dpf (un-injected n=3; tp53 MO injected n=5; p =0.5784), and rbbp4 Δ4/Δ4 at 3 dpf (un-injected n=6; tp53 MO injected n=10; p =0.0128). Data represent mean ± s.d. p values calculated with one-tailed Student’s t-test. Scale bars: 100 μm.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Inhibition, Activity Assay, Injection, Labeling, Staining, One-tailed Test

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: Representative transverse cryosections (n=3) of 5 dpf wildtype zebrafish midbrain (A-A’’’) and retina (D-D’’’) labeled with antibodies to Rbbp4 (green), proliferation maker PCNA (red), nuclear stain DAPI (blue). Higher magnification view of the medial tectal proliferative zone (B-B’’’), brain ventricle (C-C’’’), ciliary marginal zone (E-E’’’), and central laminated layers of the retina (F-F’’’). The highly proliferative, PCNA+ population of cells are outlined; arrows indicate cells co-labeled with Rbbp4 and PCNA. Rbbp4 is enriched in a subset of neurons in the midbrain and vitreal side of the retinal inner nuclear layer. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars: 100 μm A-A’’’, D-D’’’; 10 μm B-B’’’, C-C’’’, E-E’’’, F-F’’’.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Staining

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: (A-K) rbbp4 cDNA rescue of rbbp4 Δ4/Δ4 homozygotes demonstrates requirement for Rbbp4 in brain and neural crest development. (A) Lateral view of 5 dpf wildtype zebrafish indicating location of measurements for head height (black arrow) and eye width (white arrow). (B) Lateral view of 5 dpf rbbp4 Δ4/Δ4 homozygote showing gross defects including microcephaly and microphthalmia. Lateral and ventral views of 5 dpf wildtype (C, E) and rbbp4 Δ4/Δ4 homozygous (D, F) larva stained with alcian blue to reveal cartilage structures. (G) Schematic of Tol2 cDNA rescue construct driving expression of rbbp4–2AGFP by the ubiquitin promoter. (H) 5 dpf transgenic Tg ( Tol2 ) and (I) 5 dpf transgenic rbbp4 Δ4/Δ4 ; Tg ( Tol2 ) larva showing rescue of the rbbp4 Δ4/Δ4 homozygous mutant phenotype. (J) rbbp4 Δ4/Δ4 homozygotes (n=6) have a significantly smaller head than wildtype (n=3, p =0.0015). Transgenic GFP+ rbbp4 Δ4/Δ4 homozygotes (n=4) show no significant difference in head height compared to GFP+ +/+ wildtype (n=4) (p=0.4595). (K) rbbp4 Δ4/Δ4 homozygotes (n=8) have a significantly smaller eye than wildtype (n=5, p =0.0001). Transgenic rbbp4 Δ4/Δ4 GFP+ (n=5) show no significant difference in eye size compared to GFP+ +/+ wildtype (n=8, p =0.6293). Data represent mean ± s.d. p values calculated with one-tailed Student’s t-test. ch, ceratohyal cartilage; m, Meckel’s cartilage; cbs, ceratobranchial cartilage. See also and . (L-S) Transverse sections of midbrain and retina from wild type +/+ (n=5) and homozygous mutant rbbp4 Δ4/Δ4 (n=4) 5 dpf zebrafish larvae labeled with antibodies to proliferative marker PCNA (red) and stem/progenitor marker Sox2 (green) and nuclear stain DAPI (blue). (L, N) In wildtype PCNA and Sox2-positive stem cells line the midbrain ventricle (L, N dashed outline). Sox2-positive progenitors lie adjacent to the ventricle in the tectum (N bracket). (N, O) PCNA and Sox2-positive cells with enlarged morphology line the midbrain ventricles in rbbp4 Δ4/Δ4 mutant (O outline). The dorsal tectum is significantly reduced in height in the rbbp4 Δ4/Δ4 mutant (O bracket). (P, R) In wild type retina PCNA-positive stem cells are restricted to the outer edge of the ciliary marginal zone (R outline). Sox2-positive amacrine and displaced amacrine cells are distributed throughout the inner nuclear and ganglion cell layers. (Q, S) PCNA-positive stem cells with enlarged, hypertrophic morphology at the ciliary marginal zone in rbbp4 Δ4/Δ4 mutant retina (S outline). Arrow shows missing tissue adjacent to the ciliary marginal zone. Sox2-positive cells representing early born amacrine cells are present in the retinal layers. See also . Scale bars: A-F 100 μm; 50 μm L, M, P, Q; 20 μm N, O, R, S.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Staining, Construct, Expressing, Transgenic Assay, Mutagenesis, One-tailed Test, Labeling, Marker

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: Transverse section of midbrain and retina from wild type +/+ (n=3) and rbbp4Δ4/Δ4 homozygous (n=3) 5 dpf larva labeled with antibodies to glial markers Gfap (red) and Blbp (green) and nuclear stain DAPI (blue). (A, C) In wild type midbrain Blbp and Gfap label radial glia cell bodies and projections in the tectum (C bracket) and thalamic regions, respectively. (B, D) The rbbp4Δ4/Δ4 mutant midbrain contains mature radial glia in the thalamic region (B). Intense Blbp labeling is present where hypertrophic stem cells are located at the ventricle (D bracket). (E, G) Blbp and Gfap label Mueller glia cells bodies and projections across the inner plexiform layer of the wild type retina. (F, H) Blbp and Gfap label persists in early born glia in the rbbp4Δ4/Δ4 mutant retina. (I-P) Transverse section of midbrain and retina from wild type +/+ (n=3) and rbbp4Δ4/Δ4 mutant (n=3) 5 dpf larvae labeled with antibodies to the synaptic vesicle marker Sv2 (red) and interneuron and ganglion cell marker Calretinin (green) and nuclear stain DAPI (blue). (I, K) In wildtype brain, Sv2 labels the neuropil, and Calretinin labels the cell bodies of a subset of mature neurons in the thalamus and tectum (K bracket). (J, L) The rbbp4Δ4/Δ4 mutant shows a reduction in the tectal neuropil and number of Calretinin-positive neurons (L bracket). (M, O) Sv2 and Calretinin labeling in wild type retina. (N, P) Early born neurons maintain Calretinin labeling and Sv2 labeled projections across the inner and outer plexiform layers in the rbbp4 Δ4/Δ4 mutant retina. Scale bars: 50 μm A, B, E, F, I, J, M, N; 20 μm C, D, G, H, K, L, O, P.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Staining, Mutagenesis, Marker

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: See also . Lateral (A-D) and dorsal (E-H) images of 2 dpf and 3 dpf larva show the size of the optic tectum (arrows, OT) and midbrain overall (brackets) is reduced in rbbp4 Δ4/Δ4 mutants compared to +/+ wildtype. (I-N) Transverse sections of zebrafish midbrain and retina labeled with antibodies to the apoptosis marker activated-Caspase-3 (green), neural marker HuC/D (red), and nuclear label DAPI (blue). (I, K, M) Few activated Caspase-3 labeled cells are detected in wildtype at 2dpf (n=4) and 3 dpf (n=3). (J, L, N) Extensive activated Caspase-3 labeling is present throughout the midbrain tectum and retina in the rbbp4 Δ4/Δ4 mutant at 2 dpf (n=7) and 3 dpf (n=5). (L) Higher magnification view of nuclear morphology shows pyknosis and nuclear fragmentation in the rbbp4 Δ4/Δ4 mutant 2 dpf retina. (O, P) Counts of activated Caspase-3 positive cells plotted for individual larva show a significant difference between wildtype and rbbp4 Δ4/Δ4 mutant at 2 dpf in the midbrain (O, p =0.0022) and in the retina (P, p =0.0010). At 3 dpf the difference is no longer significant in the midbrain (O, p =0.2569) and only slightly significant in the retina (P, p =0.0358), due to loss of cells by apoptosis. (Q-V) Transverse sections of zebrafish midbrain and retina labeled with an antibody to the DNA damage marker γ-H2AX (green) and nuclear label DAPI (blue). (Q, S, U) Few γ-H2AX labeled cells are present in wildtype midbrain or retina at 2 dpf (n=3) and 3 dpf (n=3). (R, T, V) In the rbbp4 Δ4/Δ4 mutants (n=3) at 2 dpf a significantly greater number of cells with γ-H2AX labeling was present in the brain (W, p =0.0008) and retina (X, p =0.0032). By 3 dpf (n=3) the number was not significantly different than in wildtype (midbrain W, p =0.1211; retina X, p =0.0537), likely due to loss of cells by apoptosis. (T) Higher magnification view of nuclear morphology and γ-H2AX labeling in the rbbp4 Δ4/Δ4 mutant 2dpf retina. Closed arrow shows pannuclear staining; open arrow shows nuclear ring staining; dotted arrow shows foci pattern of staining. Data represent mean ± s.d. p values calculated with one-tailed Student’s t -test. Scale bars: 100 μm (I, J, M, N, Q, R, U, V); 10 μm (K, L, S, T).

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Marker, Mutagenesis, Staining, One-tailed Test

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: (A-F) BrdU pulse-chase labeling to examine the fate of new born neurons in the rbbp4 Δ4/Δ4 mutant retina. (A, B) 2 dpf zebrafish embryos treated with a 2.5 hour BrdU pulse and immediately sacrificed. Transverse sections of retina from wildtype (n=4) and rbbp4 Δ4/Δ4 mutants (n=5) were labeled with antibodies to BrdU and mitotic marker phospho-Histone H3 (pH3). BrdU labels proliferating cells at the retina ciliary marginal zone and cells scattered throughout the laminating retina. (C,D) Pulse-chased larvae at 3dpf. In wildtype retina (n=8) BrdU labeled cells are located in the region adjacent to the cmz where neural precursors and newly differentiated neurons reside (C outline). A small section of BrdU labeled cells remains at the ciliary marginal zone in the rbbp4 Δ4/Δ4 mutant retina (n=6) (D outline). (E,F) Pulse-chased larvae at 5 dpf. In wildtype (n=7) the BrdU labeled cells are now more centrally located in an older region of the growing retina (E outline). rbbp4 Δ4/Δ4 mutant retina (n=7) older born neurons in the central retina maintain BrdU labeling, however, BrdU-labeled newborn neurons are absent. BrdU-negative stem cells persist at the ciliary marginal zone in the rbbp4 Δ4/Δ4 mutant (F arrow). (G) Diagram of pattern of gene expression marking stages of neurogenesis in the developing zebrafish retina. (H-O) Transverse sections of 3 dpf zebrafish retina labeled by in situ hybridization to examine expression of the stem cell marker mz98 (H, L arrows), proliferating progenitor marker ccnD1 (I, M brackets), committed progenitor marker ath5 (J, N brackets) and differentiating precursor marker cdkn1c (K, O arrows). (H-K) Wildtype retina (n=5) shows location of progressively committed precursor cell populations. (L-O) In the rbbp4 Δ4/Δ4 homozygous mutant retina (n=3) the proliferating and committed neural progenitor cell populations are expanded compared to wild type. cdkn1c expression is absent in the rbbp4 Δ4/Δ4 mutant retina (O arrow). Scale bars: 50 μm.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Pulse Chase, Labeling, Mutagenesis, Marker, Expressing, In Situ Hybridization

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: 3 dpf wild type +/+ (n=3), rbbp4 Δ4/Δ4 (n=3), rb1 A7/A7 (n=4), and rbbp4 Δ4/Δ4 ; rb1 A7/A7 (n=2) siblings from a rbbp4 Δ4/+ ; rb1 Δ7/+ incross were sectioned and labeled with antibodies to activated caspase-3 and HuCID (A-H) or γ-H2AX and _hosphor-Histone H3 (I-P). Activated caspase-3 and nuclear fragmentation are present in the region containing neural precursors (dashed outline) adjacent to the ciliary marginal zone in both rbbp4 Δ4/Δ4 and rbbp4 Δ4/Δ4 ; rb1 Δ7/Δ7 mutants. (Q) Diagram modeling requirement for Rbbp4 and Rbl in retinal neurogenesis. Newborn retinal neural precursors adjacent to the ciliary marginal zone will differentiate into neural cell types that populate the retina ganglion cell, inner nuclear, and outer nuclear layers. Both Rbbp4 and Rb1 are required for neural precursors to initiate quiescence and differentiation, however, loss of rbbp4 leads to DNA damage-induced apoptosis, while loss of rb1 allows entry into mitosis. Double mutant precursors undergo apoptosis, indicating rb1 mutant precursor viability is dependent on Rbbp4. CMZ ciliary marginal zone; GCL ganglion cell layer; INL inner nuclear layer; ONL outer nuclear layer. Scale bars: A-D, I-L 100 μm; E-H, M-P 20 μm.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques: Labeling, Mutagenesis

Journal: bioRxiv

Article Title: Retinoblastoma binding protein 4 maintains cycling neural stem cells and prevents DNA damage and Tp53-dependent apoptosis in rb1 mutant neural progenitors

doi: 10.1101/427344

Figure Lengend Snippet: Each lane corresponds to an individual progeny. A Gels of rbbp4 exon 2 PCR amplicons digested with Sml I. The digestion-resistant bands correspond to the rbbp4Δ4 allele. B Gels of rb1 exon 2 PCR amplicons. The 7bp deletion and wildtype alleles can be distinguished based on band size.

Article Snippet: Antibodies used for labeling: rabbit polyclonal anti-RB Binding Protein 4 RBBP4 1:200 (Bethyl A301-206A-T, RRID: AB_890631); rabbit polyclonal anti-SOX2 1:200 (EMD Millipore AB5603, RRID:AB_2286686); mouse monoclonal anti-proliferating cell nuclear antigen PCNA 1:300 (Sigma P8825); mouse monoclonal anti-glial fibrillary acid protein GFAP 1:1000 (Zebrafish International Research Center zrf-1); rabbit polyclonal anti-phospho-Histone H3 PH3 1:1000 (Cell Signaling Technology; 9701); mouse monoclonal anti-phospho-Histone H3 (Ser10), clone 3H10 1:500 (Millipore 05-806); rabbit polyclonal anti-Brain Lipid Binding Protein BLBP 1:200 (Abcam ab32423); mouse monoclonal anti-HuC/D 1:500 (Invitrogen A-21271); rabbit polyclonal anti-gamma H2A histone family, member X γ-H2AX 1:200 (GeneTex GTX127342); rabbit polyclonal anti-CASPASE-3 1:500 (BD Biosciences 559565); mouse monoclonal anti-SV2 1:100 (Developmental Studies Hybridoma Bank AB_2315387); rabbit polyclonal anti-CALRETININ 1:1000 (Millipore AB5054); mouse monoclonal anti-BrdU 1:500 (Bio-Rad MCA2483); Alexa-594 (Invitrogen A-11005) and Alexa-488 (Invitrogen A-11008) conjugated secondary antibodies 1:500.

Techniques:

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Schematic representation of FLNA structure including numbering of the Ig-like domain repeats, and labelling of the actin-binding domain (ABD). The asterisks mark the repeats lacking in the FLNA variant form used in ( B ). ( B ) Pull-down assays showing direct interaction between recombinant DPP9 and recombinant FLAG tagged wt FLNA or a mutated form of FLAG-FLNA (lacking repeats 4, 9, 12, 17, 19, 21, and 23). Shown is a representative result of at least three independent experiments. ( C ) Recombinant DPP9 binds directly to GST- FLNA construct containing repeats 5–7 but not to GST-FLNA construct containing repeats 6–7. Shown is a representative result of at least three independent experiments. ( D ) Co-immunoprecipitation of endogenous FLNA with endogenous DPP9 from HeLa cells treated with different cross-linkers. Binding was observed in the presence of the sulfhydryl cross-linker DPDPB. Shown is a representative result of at least three independent experiments. To control for the specificity of the cross link, we blotted for DPP8, which did not bind to DPP9 in the presence of DPDPB ( E ) Quantification of the proximity ligation assay (in situ PLA) visualizing DPP9-FLNA interaction in HeLa cells treated with FLNA silencing oligos or non-targeting (NT) siRNAs for control shown in ( F ). The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Data are represented as mean ± SEM. Signals of more than 130 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0005). ( F ) PLA showing interaction of DPP9 with FLNA in HeLa cells. Each red dot represents a single FLNA-DPP9 interaction. The number of PLA signals is significantly decreased in cells silenced for FLNA compared to cells treated with NT siRNA. Actin filaments are stained in green, nuclei were visualized by using HOECHST. Shown are representative images of at least three independent PLA experiments. DOI: http://dx.doi.org/10.7554/eLife.16370.003

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Binding Assay, Variant Assay, Recombinant, Construct, Immunoprecipitation, Control, Proximity Ligation Assay, In Situ, Software, Two Tailed Test, Staining

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) In vitro cleavage of a synthetic Syk peptide corresponding to the N-terminus of Syk (1–31) by recombinant DPP9. 50 µM of a synthetic Syk (1–31) peptide was incubated for 6 hr, either alone or with 130 nM DPP9. For control 10 µM allosteric DPP9 inhibitor SLRFLYEG was added in addition to 130 nM DPP9 and (6 hr). An additional control included the peptide and the inactive DPP9 S730G variant. Samples were analysed by high resolution liquid chromatography/tandem mass spectrometry in triplicate. Quantitation was achieved by extracting ion chromatograms and integrating peak areas for the most abundant 3+ charge state of the intact 1–31 ([M+3H] 3+ m/z 1149.8589) and the cleaved 3–31 ([M+3H] 3+ m/z 1082.4997) peptides. The identities and retention times of the peptides were established by accurate mass measurement and product ion spectra (data not shown). ( B – G ) PLA assays showing that the interaction between DPP9 and Syk requires the active site of DPP9. Shown are representative images with the corresponding quantifications of at least three independent PLA experiments. Actin filaments are stained in green, and nuclei were visualized by using HOECHST. The number of PLA signals (red dots) per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 300 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (**p<0.005; ***p<0.0005; n.s = not significant). ( B ) The interaction between DPP9 and Syk is markedly decreased in HeLa cells treated with 10 µM SLRFLYEG compared to control cells treated with DMSO. ( C ) Quantification of the PLA DPP9-Syk shown in ( B ). Data are represented as mean ± SEM. ( D ) The number of PLA signals representing DPP9-Syk interactions per cell is reduced upon treatment of HeLa cells with the competitive DPP8/9 inhibitor 1G244 (10 µM, for 5 min) compared to control cells treated with DMSO. ( E ) Quantification of the PLA DPP9-Syk shown in ( D ). Data are represented as mean ± SEM. ( F ) The interaction of DPP9 with FLNA is not significantly altered upon treatment of HeLa cells with 1G244 (10 µM, 30 min) compared to control cells treated with DMSO. ( G ) Quantification of the PLA DPP9- FLNA shown in ( F ). Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.008

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: In Vitro, Recombinant, Incubation, Control, Variant Assay, Liquid Chromatography, Mass Spectrometry, Quantitation Assay, Mass Measurement, Staining, Software, Two Tailed Test

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: HeLa cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cells were lysed and extracts (5 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. Experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.16370.009

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Activity Assay, Fluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Total cell lysates (10 µg per lane) of DG-75 cells stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (0, 1 and 4 min) were analyzed for DPP9 protein levels by Western blotting. Tubulin was used as loading control. Shown is a representative blot, an experiment was performed more than five times. ( B ) DG-75 cells were treated with 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (0, 5 and 30 min). Cell lysates (10 µg) of were analysed for DPP activity in the presence of the artificial DPP substrate GP-AMC (250 µM) or the unrelated substrate R-AMC (50 µM). Fluorescence was measured over time. An experiment was performed at least three times, each time in triplicates. Shown is a representative, data are represented as mean ± SEM. ( C ) Indirect immunofluorescence images of DG-75 cells decorated with antibodies against DPP9, DPP8 and DPPIV. DOI: http://dx.doi.org/10.7554/eLife.16370.013

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Western Blot, Control, Activity Assay, Fluorescence, Immunofluorescence

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A and B ) PLA showing that the interactions of FLNA with Syk and DPP9 are conserved in human DG-75 B cells. Each PLA interaction is shown here as a white dot, nuclei were visualized by using HOECHST. Control reactions (NCtrl) were performed with only one primary antibody (αSyk, αFLNA or αDPP9). Shown are representative images and quantifications of at least three independent PLA experiments. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 80 cells were quantified for each condition respectively. Data are represented as mean ± SEM. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001). ( C and D ) PLA in DG-75 cells showing that Syk interacts specifically with DPP9 but not with its homologs DPP8 and DPPIV. Control reactions (NCtrl) cells were treated with one primary antibody only: αDPP9, αDPP8 or αDPPIV. Shown are quantifications of the PLA DPP9-Syk, DPP8-Syk and DPPIV-Syk in DG-75 cells from three independent experiments. Data are represented as mean ± SEM. The number of PLA signals per cell were quantified in a blinded manner using the Duolink ImageTool software (SIGMA). Signals of more than 100 cells were quantified for each condition respectively. Statistical analysis was carried out by an unpaired two-tailed t test (***p<0.0001; n.s = not significant). ( E ) CHX chase experiment showing reduced stability of endogenous Syk upon stimulation of the BCR. Human DG-75 cells were stimulated with 12 µg/ml F(ab’) 2 fragment goat-anti-human IgG+IgM (+ stim), or left untreated (- stim), and simultaneously subjected to CHX chase. DPP9 was analysed as a loading control. Shown is one representative result of at least three independent experiments. ( F ) CHX chase experiments showing that the stability of endogenous Syk in stimulated DG-75 cells, is determined by the proteasome and by DPP9. DG-75 cells were treated either with the DPP8/9 inhibitor 1G244 (10 µM), with the proteasome inhibitor MG132 (100 µM) or with DMSO for control (MOCK). Cell lysates were analysed for protein levels of Syk and of DPP9 for loading control by Western blotting. Shown is one representative result of at least three independent experiments. ( G ) Quantification of the Western blot results shown in ( F ). The ratio of Syk/DPP9 at time 0 hr was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( H ) CHX chase experiment assaying the stability of endogenous phosphorylated Syk (p-Y323) in stimulated DG-75 cells upon treatment with the DPP8/9 inhibitor 1G244 (10 µM) or with DMSO for control (MOCK). Tubulin was assayed as loading control. Shown is one representative result of at least three independent experiments. ( I ) Quantification of the Western blot results shown in ( H ). The ratio of Syk p-Y323/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. DOI: http://dx.doi.org/10.7554/eLife.16370.012

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Software, Two Tailed Test, Western Blot

Journal: eLife

Article Title: DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk

doi: 10.7554/eLife.16370

Figure Lengend Snippet: ( A ) Higher levels of endogenous active Syk (phosphorylated on Y352) in stimulated DG-75 cells treated with the DPP8/9 inhibitor 1G244 compared to the mock (DMSO) treated cells. 1G244 (10 µM) was added at the same time of BCR stimulation (time 0). Tubulin was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( B ) Quantification of the Western blot results shown in ( A ). The ratio of Syk p-Y352/tubulin at time 10 min was normalized to 100%. For signal quantification GelQuant.NET software provided by biochemlabsolutions.com was used. ( C ) Inhibition of DPP9 in DG-75 cells leads to increased Ca 2+ mobilization, which is not dependant on BCR stimulation. Shown are flow cytometric Ca 2+ profiles after the addition of 10 µM DPP8/9 inhibitor 1G244 or DMSO for control (marked by an arrow). To monitor Ca 2+ mobilization upon BCR stimulation in either 1G244-treated or control cells, cells were treated with 10 µg/ml F(ab) 2 goat-anti-human IgM. ( D ) Same as in ( C ) using Ramos cells as a second B cell line. ( E – F ) In the absence of BCR stimulation, DPP9 inhibition leads to higher basal levels of phosphorylated Syk and its down stream effector protein PLCγ2. ( E ) Western blotting analysis of DG-75 cells treated with 1G244 in the absence of BCR stimulation. Lysates were analysed with antibodies specific against phosphorylated Syk (p-Y352) or phosphorylated PLCγ2. For loading control lysates were analysed with antibodies recognizing unmodified Syk and PLCγ2. ( F ) Cells were treated for 20 min with 1G244 (10 µM) or DMSO for control. Alternatively, cells were treated for 30 min with the allosteric DPP9 inhibitor SLRFLYEG peptide complexed with the carrier peptide (pep-1). Control cells were treated with the carrier peptide only. Following inhibitor treatment, cells were lysed and subjected to immunoprecipitation assays against Phospho-Y. Eluted proteins were analysed for Syk and PLCγ2 levels by Western blotting. Total protein levels in cell lysates were monitored for control. ( G ) Lower levels of phosphorylated ERK1/2 (both bands) are detected in the 1G244 treated DG-75 cells compared to mock (DMSO) treated cells. 1G244 (10 µM) was added prior to BCR stimulation (time 0). DPP9 was assayed for loading control. Shown is a representative result of at least three independent pulse chase experiments. ( H ) Quantification of the Western blot results shown in ( G ) as described in ( B ). DOI: http://dx.doi.org/10.7554/eLife.16370.015

Article Snippet: In immunofluorescence studies and in situ Proximity ligation assays (PLAs) the following antibodies were used: self-generated goat anti-DPP9 (1:10–20), mouse anti DPPIV (1:50–100; Santa Cruz Biotechnology, #sc-19607), mouse anti DPP8 (1:50–100, Santa Cruz Biotechnology, #sc-37699), rabbit anti-Syk (1:100–1:200), goat anti-Cbl (C-15) (1:60; Santa Cruz Biotechnology, #sc-170-G), mouse anti-FLNA (1:100), rabbit anti-FLNA (1:100, NB100-58812; Novus Biologicals, Germany) and mouse anti-HA (1:400, , MMS-101P; Covance Germany).

Techniques: Control, Pulse Chase, Western Blot, Software, Inhibition, Immunoprecipitation