Journal: eLife

Article Title: FABP4-mediated lipid accumulation and lipolysis in tumor-associated macrophages promote breast cancer metastasis

doi: 10.7554/eLife.101221

Figure Lengend Snippet:

Article Snippet: Antibody , Alexa Fluor 594-conjugated AffiniPure mouse anti-goat IgG polyclonal , Jackson ImmunoResearch , Cat#205-605-108 , 1:2000.

Techniques: Concentration Assay, Staining, Saline, SYBR Green Assay, Red Blood Cell Lysis, Cell Culture, XF Assay, Reverse Transcription, Polymer, Plasmid Preparation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Transfection, Recombinant, Microarray, Gene Expression, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Software, Sterility, Migration, Pore Size, Flow Cytometry, Quantitative RT-PCR, Imaging, Microscopy, Transmission Assay, Electron Microscopy

Journal: Cell chemical biology

Article Title: RUVBL1/RUVBL2 ATPase Activity Drives PAQosome Maturation, DNA Replication and Radioresistance in Lung Cancer

doi: 10.1016/j.chembiol.2019.12.005

Figure Lengend Snippet: (A) Compound B treatment lowers, then increases, the amount of ssDNA. H2009 cells were analyzed for ssDNA and total DNA (PI) by flow cytometry after 100 nM of compound B or compound C treatment for indicated times. Top graphs: numbers in upper gate represent the percentage of cells with high levels of ssDNA and bottom gates capture cells in S/G2/M. Bottom graphs: amount of ssDNA in cells in S/G2/M phase (from top graph). MFI, median fluorescent intensity; HU + ATRi, 2 mM hydroxyurea (HU) for 4 h followed by 500 nM VE-822 (ATR inhibitor) for 2 h. Numbers are average ±SD of three biological replicates. (B) RPA loading onto chromatin parallels ssDNA. H2009 cells were pre-extracted and analyzed for chromatin-bound RPA2 and DNA content (PI) by flow cytometry after 100 nM compound B or compound C treatment for indicated times. Top graphs: numbers in upper gate represent the percentage of cells with hyperloaded RPA and bottom gates capture cells in S/G2/M. Bottom graphs: amount of chromatin-bound RPA2 in cells in S/G2/M phase (from top graph). Hydroxyurea, 2 mM for 2 h. Numbers are average ±SD of three biological replicates. (C) Complete loss of ATR causes replication catastrophe after RUVBL1/2 inhibition. H2009 cells were pre-extracted and analyzed for chromatin-bound RPA2 and DNA content (PI) by flow cytometry after being treated with 100 nM compound B or compound C ± 80 nM VE-822. Numbers are average ±SD of three biological replicates. See Figure S5B for the same experiment with a CHEK1 inhibitor. (D) Compound B treatment increases the speed of individual replication forks. H2009 cells were treated with 100 nM compound C or compound B, labeled with IdU and CldU, and the length of IdU and CldU tracks were measured. Only the total length of dual IdU/CldU-positive tracks were measured. Data from two biological replicates in which >100 tracks were quantified for each condition. Statistical significance from one-way ANOVA with a post-hoc Sidak’s multiple comparisons test. (E) The abundance of core replication factors at replisomes does not change after compound B treatment. H2009 was treated with 100 nM compound C or compound B for 12 h, active replisomes were purified by iPOND, and eluted proteins were analyzed by mass spectrometry. Values are averages ± SEM from three biological replicates. See also Table S5 for the complete list of identified proteins. (F) POLE abundance increases, whereas histone abundance decreases, at active replisomes after compound B treatment. Data are from same experiment as in. (E). Values are averages ± SEM from three biological replicates. See also Table S5. (G) iPOND immunoblot confirms that compound B increases the abundance of POLE2 but decreases the abundance of histones at active replisomes. No click samples lack the biotin conjugation step. Chase samples contain only chromatin-bound proteins. Asterisk, nonspecific protein; arrow, specific protein.

Article Snippet: Mouse monoclonal CHEK1 (Immunoblot 1:1000) , Santa Cruz , RRID: AB_627257 Cat# sc-8408.

Techniques: Flow Cytometry, Inhibition, Labeling, Purification, Mass Spectrometry, Western Blot, Conjugation Assay

Journal: Cell chemical biology

Article Title: RUVBL1/RUVBL2 ATPase Activity Drives PAQosome Maturation, DNA Replication and Radioresistance in Lung Cancer

doi: 10.1016/j.chembiol.2019.12.005

Figure Lengend Snippet: TAGTGAAGCCACAGATGTA

Article Snippet: Mouse monoclonal CHEK1 (Immunoblot 1:1000) , Santa Cruz , RRID: AB_627257 Cat# sc-8408.

Techniques: Western Blot, Immunofluorescence, Flow Cytometry, Immunoprecipitation, Control, Polymer, Plasmid Preparation, Virus, Microarray, Recombinant, Synthesized, Proliferation Assay, Electron Microscopy, Amplification, TUNEL Assay, cDNA Synthesis, SYBR Green Assay, Purification, Sequencing, Software, Imaging, Light Microscopy

Journal: Journal of visualized experiments : JoVE

Article Title: Sequential Immunofluorescence and Immunohistochemistry on Cryosectioned Zebrafish Embryos

doi: 10.3791/59344

Figure Lengend Snippet: Materials

Article Snippet: anti-Ser10 phosphorylated Histone H3 antibody , Santa Cruz , sc-8656-R , used at 1:500 dilution for IF.

Techniques: Blocking Assay, Imaging, Software, Light Microscopy, Fluorescence, Microscopy, Polymer, Plasmid Preparation, Modification, Saline, Electron Microscopy

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: Human DLL4 , Origene , Cat#RC212628.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: HCC2429 cells were plated in wells with or without DLL4 ligand by first coating wells for 1 hour at room temperature with 2 μg/mL of recombinant human DLL4 Fc chimera protein (R&D Systems, # 10185-D4-050) or left uncoated and cultured overnight in complete growth medium according to ATCC instructions.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: HCC2429 cells were plated in wells with or without DLL4 ligand by first coating wells for 1 hour at room temperature with 2 μg/mL of recombinant human DLL4 Fc chimera protein (R&D Systems, # 10185-D4-050) or left uncoated and cultured overnight in complete growth medium according to ATCC instructions.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: HCC2429 cells were plated in wells with or without DLL4 ligand by first coating wells for 1 hour at room temperature with 2 μg/mL of recombinant human DLL4 Fc chimera protein (R&D Systems, # 10185-D4-050) or left uncoated and cultured overnight in complete growth medium according to ATCC instructions.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: HCC2429 cells were plated in wells with or without DLL4 ligand by first coating wells for 1 hour at room temperature with 2 μg/mL of recombinant human DLL4 Fc chimera protein (R&D Systems, # 10185-D4-050) or left uncoated and cultured overnight in complete growth medium according to ATCC instructions.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: To generate the ligand-expressing cells, HEK293 cells were transfected with a vector for expression of human DLL4 in the pCMV6-AC-HA-His backbone (Origene, Rockville, MD) and termed HEK-DLL4.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3 is overexpressed in multiple human tumors (A) Expression of NOTCH3 mRNA in primary human tumors from TCGA compared to normal tissues. Box and whiskers plots are drawn with individual points below 10 th and above 90 th percentiles. Median values are drawn as a line in the middle of the box. TPM, transcripts per kilobase million, ns, non-significant. (B) NOTCH3 mRNA fold change in primary human lung, breast, and ovarian tumors and xenografts compared to normal tissues (baseline, dashed line). Data represent mean (n = 2–4). (C) Quantitation of NOTCH3 ISH staining as a measure of percentage of area stained on breast (ER + , TNBC), lung (LUAD, LUSC), ovarian, and normal (breast, lung, and ovary) tissue sections.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Expressing, Quantitation Assay, Staining

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Generation and characterization of therapeutic anti-NOTCH3 mAbs (A) NOTCH3-dependent report gene assay with NOTCH3-targeted and control mAbs. Data represent mean ± SEM from 3 biological replicates (n = 4 per replicate), ns, non-significant. (B) qRT-PCR of HES1 gene expression in treated cells. Data represent mean ± SD from 2 biological replicates (n = 3 per replicate), ns, non-significant. (C) NOTCH3 immunoblot from mAb-treated cells and xenografts. GAPDH is shown as a loading control. Schematic diagram of NOTCH3 cleavage events and protein fragments. M, mouse number. (D and E) Immunoblot using N- or C-terminal domain antibodies to detect NOTCH3 fragments after DLL4 activation in treated cells. β-actin is shown as a loading control. (F) Epitope mapping of anti-NOTCH3 mAbs using NRR3-NRR1 domain swap chimeric constructs. NRR3 domains are shown in black and NRR1 domains are shown in gray. Representative data represent mean (n = 2). (G) Binding interface of anti-N3(i) Fab on the NOTCH3-NRR domain as determined from the co-crystal structure is shown in red, LNR-A (green), LNR-B (beige), LNR-C (blue), HD1 (pink), and HD2 (magenta).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Gene Assay, Control, Quantitative RT-PCR, Gene Expression, Western Blot, Activation Assay, Construct, Binding Assay

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Cell - membrane distribution of anti-NOTCH3 mAbs depends on signaling status (A) Confocal images of anti-NOTCH3 mAbs on fixed cells. Scale bar, 10 μm. (B) Single optical sections from live-cell confocal imaging of anti-NOTCH3 mAbs simultaneously bound to cells and imaged over time with anti-N3(i)-Alexa 488 (green) and anti-N3-DyLight647 (magenta). Scale bar, 10 μm. (C) z stack of maximum intensity projections from live-cell confocal imaging of anti-NOTCH3 mAbs over time. Scale bar, left = 8 μm, right = 10 μm. (D) Cross-section of cells bound by NOTCH3-targeted mAbs from images in Figure 2 C. Scale bar, 10 μm.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Membrane, Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs have differential rates of endocytosis and lysosomal trafficking (A and B) Colocalization of mAbs with pHrodo Red dextran compartments calculated by PCC and MCC from live-cell confocal images. (A) Data represent mean ± SEM of 3 biological replicates (n = 3 fields imaged per replicate). (B) Data represent mean ± SEM of 2 biological replicates (n = 2 fields imaged per replicate), ns, non-significant. (C) Colocalization of mAbs with CT-β were calculated by PCC and MCC from live-cell confocal images. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate). (D) Single optical sections of mAbs with CT-β from live-cell confocal imaging. Scale bar, 10 μm. (E) Colocalization of mAbs with CAV-1 or clathrin were calculated by MCC in fixed cells. Data represent mean ± SEM (n = 3 fields imaged). (F) Colocalization of mAbs with pHrodo Red dextran compartments were calculated by MCC from live-cell confocal images of MβCD-treated cells. Cross hairs were set at a value of 0.2 MCC to highlight more rapid colocalization of both mAbs with pHrodo Red dextran compartments. Data represent mean ± SEM of 2 biological replicates (n = 5 fields imaged per replicate).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Imaging

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: Anti-NOTCH3 mAbs transendocytose into DLL4 ligand cells (A and B) TEC of labeled mAbs (green) that were bound to U2OS-hN3 cells and then co-cultured with HEK-DLL4 or HEK-parental cells (red) from live-cell confocal imaging. (A) Single optical sections of a U2OS-hN3 cell and a migrating HEK-DLL4 cell (asterisk) before and after contact (arrows). Scale bar, 10 μm. (B) z stack of maximum intensity projections. Arrows, anti-NOTCH3 mAbs inside HEK-DLL4 cells. (C) Confocal images of maximum intensity projections acquired from indirect immunofluorescence of mAbs bound to U2OS-hN3 cells (magenta) and then co-cultured with HEK-DLL4 (green) from. Dashed white line demarcates anti-NOTCH3 mAbs inside HEK-DLL4 cells. Scale bar, 10 μm.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Labeling, Cell Culture, Imaging, Immunofluorescence

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce cytotoxicity in both receptor and ligand cells (A) General structure of NOTCH3-targeted ADCs that were generated with mAbs, a cleavable dipeptide-based linker and the Aur0101 payload (blue). (B) NOTCH3-ADC induction of caspase-3/7 activity. Data represent mean ± SEM of 3 biological replicates (n = 3 per replicate). (C) In vitro cytotoxicity of NOTCH3-ADCs after control ( Control:siRNA ) or siRNA knockdown of NOTCH3 mRNA ( N3:siRNA ). Data represent mean ± SEM of 2 biological replicates (n = 3 per replicate). (D) In vitro cytotoxicity of NOTCH3-ADCs using parental MDA-MB-468 cells under 2D and 3D culture conditions. Data represent mean ± SEM (n = 3). (E and F) TEC of NOTCH3-ADCs induces caspase activity in HEK-DLL4 cells. (E) z stack of maximum intensity projections from live-cell confocal imaging of anti-N3(i) ADC bound to U2OS-hN3 cells and co-cultured with HEK-DLL4 cells labeled with pHrodo Red dextran. Caspase compartments (magenta line), pHrodo Red dextran compartments (blue line) and the merged image. (F) Percentage of the caspase-positive compartments that were calculated after treatment with NOTCH3-ADCs. Data represent mean ± SEM for 3 biological replicates (n = 15 fields imaged per replicate), ns, non-significant.

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Generated, Activity Assay, In Vitro, Control, Knockdown, Imaging, Cell Culture, Labeling

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet: NOTCH3-targeted ADCs induce prolonged tumor regressions (A) Tumor growth inhibition of HCC2429 CLXs treated with control and NOTCH3-targeted mAbs and ADCs. Data represent mean ± SEM (n = 7–8 per group). (B) Tumor growth inhibition of 37622 PDXs treated with NOTCH3-ADCs compared to cisplatin or control ADC. Data represent mean ± SEM (n = 9–10 per group). (C) Tumor growth inhibition of MDA-MB-468 CLXs treated with control and anti-N3 ADCs. Data represent mean ± SEM (n = 7–8 per group). (D) Tumor growth inhibition of OVCAR3 CLXs treated with control and anti-N3 ADCs compared with carboplatin alone, and carboplatin then anti-N3 ADC. Data represent mean ± SEM (n = 7–8 per group).

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Inhibition, Control

Journal: Cell Reports Medicine

Article Title: NOTCH3-targeted antibody drug conjugates regress tumors by inducing apoptosis in receptor cells and through transendocytosis into ligand cells

doi: 10.1016/j.xcrm.2021.100279

Figure Lengend Snippet:

Article Snippet: Human NOTCH3 , Origene , Cat#RC224711.

Techniques: Control, Polymer, Plasmid Preparation, Blocking Assay, Electron Microscopy, Recombinant, Adjuvant, Antibody Labeling, RNAscope, Sequencing, Software