Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Dll4-Asp 8x recombinant proteins. From left to right, protein molecular mass marker (Marker), extract of untransfected HEK293 cells (Cells (-)) OR pcDNA3.1-Dll4-Asp 8x -transfected cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Dll4-Asp 8 -transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elute (Elute), elute after dialysis (Elute (AD)), and Opti-MEM culture medium (Medium) were loaded. ( B, C ) RT-qPCR analysis of DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) stimulated with soluble ( B ) or in sub-confluent HUVECs stimulated with immobilized Dll4-Asp 8x or BSA with poly-L-lysine coating ( C ). Data represent mean ± s.e.m. (n = 4 for ( B ) and n = 6 for ( C )) (p-values determined by multiple unpaired t-test with Welch’s correction). Figure 1—figure supplement 1—source data 1. Source data for . Figure 1—figure supplement 1—source data 2. Images of uncropped blots and gels with the relevant bands labeled for , , and . Figure 1—figure supplement 1—source data 3. ZIP file containing the full raw unedited blots and gels for , , and .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Quantitative RT-PCR, Labeling

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Diagram showing the amino acid sequence of mouse and rat Dll4 extracellular domain (ECD). Amino acid replacements shown in red were introduced into the murine sequence for the generation of Dll4 (E12) with increased Notch-binding affinity. ( B ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Dll4 (E12) recombinant protein. From left to right, protein molecular mass marker (Marker), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Dll4 (E12) -transfected HEK293 cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elute (Elute), elute after dialysis (Elute (AD)), and Opti-MEM culture medium (Medium) were loaded. ( C, D ) RT-qPCR analysis of DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) stimulated with soluble ( C ) or in sub-confluent HUVECs stimulated with immobilized Dll4 (E12) , Dll4-Asp 8x , and BSA by poly-L-lysine coating ( D ). Data represent mean ± s.e.m. (n = 3 for ( C ) and n = 4 for ( D )) (p-values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test). ( E ) Western blot analysis of Dll4 (E12) protein expression in liver lysate of pLIVE-Dll4 (E12) and control-injected mice. GAPDH is used as loading control. Figure 1—figure supplement 4—source data 1. Source data for .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Sequencing, Binding Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Quantitative RT-PCR, Comparison, Western Blot, Expressing, Control, Injection

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet: ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified Jag1-Asp 8x recombinant protein. From left to right, protein molecular mass marker (Marker), extract of untransfected HEK293 cells (Cells (-)) OR pcDNA3.1-Jag1-Asp 8x -transfected cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) OR pcDNA3.1-Jag1-Asp 8x -transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elution of Jag1-Asp 8x (Elute), and Opti-MEM culture medium (Medium) were loaded. ( A, B ) SDS-PAGE analysis of purified Jag1-Asp 8x ( A ) and Jag1 (JV1) ( B ) recombinant proteins. From left to right, protein molecular mass marker (Marker), extract of untransfected (Cells (-)) or transfected HEK293 cells (Cells (+)), supernatant after culture of untransfected (Supernatant (-)) or transfected cells (Supernatant (+)), flow-through after loading of Supernatant (+) on His-tag protein purification column (Flow-through), wash buffer (Wash), elution of recombinant Jag1 (Elute) and Opti-MEM culture medium (Medium) were loaded. ( C ) Western blot analysis of eluted Jag1-Asp 8x and Jag1 (JV1) recombinant proteins used for in vitro stimulation experiments ( D, E ). Proteins detected at the expected molecular weight with anti-Jag1 and anti-His1 antibodies. ( D, E ) RT-qPCR analysis of human ( HS) DLL4 , HEY1 , HEY2 , HES1 , EFNB2 , and NRARP transcripts in confluent human umbilical vascular endothelial cells (HUVECs) after stimulation with soluble ( D ) or immobilized (poly-L-lysine coating) Jag1-Asp 8x , Jag1 (JV1) , and BSA ( E ). Data represent mean ± s.e.m. (n = 3) (p-values determined by ordinary one-way ANOVA with Tukey’s multiple comparison test). Figure 5—figure supplement 1—source data 1. Source data for .

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Polyacrylamide Gel Electrophoresis, SDS Page, Purification, Recombinant, Marker, Transfection, Protein Purification, Western Blot, In Vitro, Molecular Weight, Quantitative RT-PCR, Comparison

Journal: eLife

Article Title: Induction of osteogenesis by bone-targeted Notch activation

doi: 10.7554/eLife.60183

Figure Lengend Snippet:

Article Snippet: The following FAM-conjugated TaqMan gene expression probes (ThermoFisher) were used in combination with SsoAdvanced Universal Probes Supermix (BIO-RAD): DLL4 (Hs00184092_m1), HEY1 (Hs00232618_m1), HEY2 (Hs00232622_m1), HES1 (Hs00172878_m1), EFNB2 (Hs00187950_m1), NRARP (Hs01104102_s1).

Techniques: Recombinant, Variant Assay, Plasmid Preparation, Sequencing, Control, Gene Expression, Bicinchoninic Acid Protein Assay, cDNA Synthesis, Amplification, Magnetic Beads, Protease Inhibitor, Western Blot, Software, Staining

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Proteins from the co-immunoprecipitation (co-IP) assay using anti-HA magnetic beads in 293T with ectopic expression of 3×HA-tagged KRAS were separated by polyacrylamide gel electrophoresis and visualized using silver staining. (B) The workflow for identifying KRAS-interacting proteins was shown, including co-IP assay, sample preparation, LC-MS/MS analysis, protein quantification, and further computational analyses for differential proteins. (C) Volcano plot of proteins identified by LC-MS/MS analysis in HA-vector and 3×HA-KRAS groups. The ratio (KRAS/HA) and the mean of protein spectra counts (MeanSP) in two groups were calculated. The x axis shows the log2 ratio (KRAS/HA) and the y axis shows the log2 MeanSP. The proteins distributed outside the dark-red boundary line (y = log 2 2.5 / (x-1)) were labeled as significant differential proteins in KRAS group. TOP 10 differential proteins from each group were labeled. (D) Western blotting (WB) analysis of whole-cell lysates and immunoprecipitation (IP) of SETD7 in 293T cells transfected with the indicated plasmids. (E) Immunofluorescence staining of SETD7 (green) and KRAS (red) in 293T cells co-transfected with SETD7 and KRAS. DAPI was used to label the nuclei. Scale bar, 10 µm. (F and G) WB analysis of the co-IP assay using anti-SETD7 (F) or anti-RAS (G) antibodies in H358-SETD7-O/E stable cells. ( H ) WB analysis of the co-IP assay with anti-SETD7 antibody in non-small-cell lung cancer (NSCLC) cell line A549.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Co-Immunoprecipitation Assay, Magnetic Beads, Expressing, Polyacrylamide Gel Electrophoresis, Silver Staining, Sample Prep, Liquid Chromatography with Mass Spectroscopy, Plasmid Preparation, Labeling, Western Blot, Immunoprecipitation, Transfection, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Two representative MS2 spectra of SETD7 peptides (116-143 and 324-344) from 3×HA-KRAS group. (B) Western blotting (WB) analysis of SETD7 expression in NSCLC cell lines from the American Type Culture Collection (ATCC). Staging information is shown above each cell line in red font.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Western Blot, Expressing

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A-B) Cell proliferation (A) and two-dimensional (2-D) colony formation (B) of A549-vector and A549-SETD7-OE stable cells was measured separately. (C) WB analysis of SETD7 and RAS-related signaling pathways in A549 stable cells. (D-E) Cell proliferation (D), 2-D colony formation (E) of H358-control and two SETD7-KD stable cells were measured separately. (F-G) 3-D spheroid growth of H1437 (F) and H358 (G) controls and two SETD7-KD stable cells was measured separately. (H) WB analysis of SETD7 and RAS-related signaling pathways in H358 stable cells. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Plasmid Preparation, Protein-Protein interactions, Control

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Measurement of volumes of xenograft tumors derived from A549-vector or A549-SETD7-O/E stable cells (n = 8). (B) A photograph of the xenograft tumors and the tumor weights, 7 weeks after cell injection, are shown (upper and lower panels, respectively). (C) Representative images of immunohistochemical (IHC) staining of SETD7, RAS, and Ki67 in xenograft tumor tissues. Scale bar, 100 μm. (D) Measurement of volumes of xenograft tumors derived from H1437-control and two SETD7-KD stable cells (n = 10). (E) A photograph of the xenograft tumors and the tumor weights, 4 weeks after cell injection, are shown (upper and lower panels, respectively; scale bar, 10 mm). (F) Representative images of IHC staining of SETD7, RAS, and Ki67 in xenograft tumor tissues of each group. Scale bar, 100 μm. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Derivative Assay, Plasmid Preparation, Injection, Immunohistochemical staining, Immunohistochemistry, Control

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A) Tumor volumes of xenografts derived from A549 vector and SETD7-OE stable pools were measured 7 weeks after tumor cell injection. ** P <0.01. (B) Tumor volumes of xenografts derived from H1437 vector and two SETD7-KD stable pools were measured 4 weeks after tumor cell injection. *** P <0.001.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Derivative Assay, Plasmid Preparation, Injection

Journal: bioRxiv

Article Title: Methylation of KRAS at lysine 182 and 184 by SETD7 promotes KRAS degradation

doi: 10.1101/2021.02.10.429309

Figure Lengend Snippet: (A-C) KRAS mRNA expression was detected by qPCR in A549 (A) and H358 (B) stable cells with SETD7 overexpression, or H1437 (C) stable cells with SETD7 knockdown. (D) WB analysis of A549-vector and A549-SETD7-O/E stable cells with CHX (100 μg/mL) treatment, as indicated. The relative RAS protein level is labeled in red. (E) Cell proliferation of A549 vector, SETD7 WT , and SETD7 H297A stable cells was measured using a CCK-8 assay. (F-G) Two-dimensional (2-D) colony formation (F) and 3-D spheroid growth (G) of A549 vector, SETD7 WT , and SETD7 H297A stable cells were measured separately. The bar graphs represent the quantification of numbers of colonies on plates or spheroids in dishes with a cell-repellent surface, from three independent experiments. (H) WB analysis of SETD7 and RAS-related signaling pathways in A549 stable cells. * P <0.05, *** P <0.001. ns, not significant.

Article Snippet: The human non-small-cell lung cancer (NSCLC) cell lines A549, NCI-H358, NCI-H1437, and NCI-H522 were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI1640 (Life Technologies) containing 10% FBS (PAN Seratech) and 100 U/mL penicillin-streptomycin (Life Technologies).

Techniques: Expressing, Over Expression, Knockdown, Plasmid Preparation, Labeling, CCK-8 Assay, Protein-Protein interactions

Journal:

Article Title: The function of Xenopus Bloom's syndrome protein homolog (xBLM) in DNA replication

doi:

Figure Lengend Snippet: Characterization of xBLM protein. (A) Coomassie blue staining of the purified His-tagged recombinant xBLM protein. (B) Helicase activity of the recombinant xBLM. The substrate is a 5′-32P-labeled 48mer oligonucleotide hybridized to m13mp18 single-stranded DNA. The purified xBLM protein was incubated with the substrate in the presence of either buffer alone or nucleotides (ATP or dATP). The right lane contains the heat-denatured substrate. The dissociated oligonucleotide was separated from m13 on 12% polyacrylamide gels. (C) Western blot of interphase egg extracts (2 μL) probed with the affinity-purified rabbit anti-xBLM antibody and detected by ECL (Amersham). The 160-kD band is xBLM. The small band is not always detected, suggesting that it is a degradation product. (D) Immunodepletion of xBLM from egg extracts. Supernatants (2 μL) from the extracts that had been depleted with either anti-xBLM or control antibodies were analyzed by Western blot with the affinity-purified rabbit anti-xBLM antibody. Molecular markers are labeled as kilodaltons on the right side of panels A, C, and D.

Article Snippet: Immunodepletion was performed by incubating extracts (diluted twofold with egg lysis buffer) with Affi-gel Protein A beads (Bio-Rad) that had been precoated with either the affinity-purified rabbit anti-xBLM antibody or rabbit IgG.

Techniques: Staining, Purification, Recombinant, Activity Assay, Labeling, Incubation, Western Blot, Affinity Purification

Journal: Cell

Article Title: CBFβ-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia

doi: 10.1016/j.cell.2018.05.048

Figure Lengend Snippet: (A) Analysis of ATAC-seq and K3K27ac and RUNX1 ChIP-seq profiles in a 2 Mb genomic region downstream of MYC. The three enhancer regions (ME1, ME2 and BDME) are depicted in green. (B-D) ChIP-qPCR analysis for RUNX1 in DMSO or AI-10–49 treated cells (B), in DMSO or AI-10–49 treated human primary CD34+ inv(16) AML cells (C), and for p300 in DMSO or AI-10–49 treated ME1 (D). (E) 5C interaction matrices for the MYC locus (−1Mb to +3Mb) in DMSO (left panel) and AI-10–49 (middle panel) treated ME-1 cells. The right panel shows the log2(AI-10–49/DMSO) ratio of the interaction matrices (blue color scheme: higher interaction frequencies in DMSO treated cells; orange color scheme: higher interaction frequencies in AI-10–49 treated cells). Arrows indicate TAD boundaries, arrowhead points to an example of a CTCF-CTCF looping interaction. Significance was calculated using unpaired t-test, *P < 0.05, or **P < 0.005. See also Figures S5 and S6.

Article Snippet: For ChIP qPCR in human primary AML sample with inv(16), CD34 + cells were enriched using CD34 MicroBead Kit (Miltenyi Biotec) and cultured overnight followed by dead cell removal by dead cell removal kit (Miltenyi Biotec).

Techniques: ChIP-sequencing, ChIP-qPCR

Journal: Cell

Article Title: CBFβ-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia

doi: 10.1016/j.cell.2018.05.048

Figure Lengend Snippet: (A) Heat map representation of differentially expressed genes in RNA-seq analysis between DMSO and AI-10–49 treated ME-1 cells. A total of 591 genes (in red) are up- and 696 genes (in blue) down-regulated in AI-10–49 treated cells (>2fold change, FDR<0.01). (B) Gene set enrichment analysis showing biological processes and signaling pathways correlated with AI-10–49-treatment. (FDR) false discovery rate; (NES) normalized enrichment score. (C) MYC transcript levels in ME-1 cells treated with DMSO (D) or AI-10–49 (49), estimated by RT-PCR. (D) MYC, ERK and LMO1 nascent transcript levels in treated ME-1 cells, estimated by transient transcriptome qRT-PCR. (E) MYC protein levels in treated ME-1 cells, by immunoblotting. (F, G) MYC transcriptional levels in mouse lineage negative wild type bone marrow (mu wt Lin- BM) and lineage negative CBFβ+/MYH11 leukemic cells (F), and human CD34+ cord blood (HPCs) and human primary CD34+ inv(16) AML cells (G); each symbol represents the average of a triplicate experiment, and the average value of three samples is shown in red. Experiments in A, C and D were performed in triplicate treatments. Error bars represent the SD. Significance was calculated using unpaired t-test (C, D, F and G), *P < 0.05, or **P < 0.005. See also Figure S1.

Article Snippet: For ChIP qPCR in human primary AML sample with inv(16), CD34 + cells were enriched using CD34 MicroBead Kit (Miltenyi Biotec) and cultured overnight followed by dead cell removal by dead cell removal kit (Miltenyi Biotec).

Techniques: RNA Sequencing, Protein-Protein interactions, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Cell

Article Title: CBFβ-SMMHC inhibition triggers apoptosis by disrupting MYC chromatin dynamics in acute myeloid leukemia

doi: 10.1016/j.cell.2018.05.048

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For ChIP qPCR in human primary AML sample with inv(16), CD34 + cells were enriched using CD34 MicroBead Kit (Miltenyi Biotec) and cultured overnight followed by dead cell removal by dead cell removal kit (Miltenyi Biotec).

Techniques: Recombinant, SYBR Green Assay, Extraction, Proliferation Assay, Sample Prep, shRNA, Plasmid Preparation, Cloning, Software, Red Blood Cell Lysis, Transfection, Protease Inhibitor, Gel Extraction

Journal: eLife

Article Title: Co-regulation and function of FOXM1 / RHNO1 bidirectional genes in cancer

doi: 10.7554/eLife.55070

Figure Lengend Snippet:

Article Snippet: Antibody , HUS1 (rabbit monoclonal) , Cell Signaling Technology , 16416 , (1:1000).

Techniques: Magnetic Beads, Purification, Single Cell Gel Electrophoresis, Bicinchoninic Acid Protein Assay, DNA Methylation Assay, Gel Extraction, TA Cloning, Reporter Assay, Reporter Gene Assay, Plasmid Preparation, Software

Journal: Cell reports

Article Title: β-Amyloid Clustering around ASC Fibrils Boosts Its Toxicity in Microglia

doi: 10.1016/j.celrep.2020.02.025

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Thereafter, cells were blocked using 5% normal goat serum (Vector Laboratories) in PTX for 20 min and primary antibodies were added for another 30 min. To check for ASC speck formation, the rabbit anti-ASC (1:250; clone AL177, AdipoGen) or mouse-specific rabbit anti-ASC/TMS1 (1:250, D2W8U, Cell Signaling Technology®) and rat anti-CD11b (1:250; Serotec by Bio-Rad) were used.

Techniques: Control, Recombinant, Magnetic Beads, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Quantitation Assay, Gel Extraction, Purification, Isolation, Software, Transmission Assay, Microscopy, Spectrophotometry, Fluorescence, Imaging