Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: High NRP1 expression predicts shorter time to relapse- and distant metastasis-free survival in ER-negative breast cancer patient cohorts. a Association of NRP1 expression quartiles (Q1–Q4) with overall survival in the BrCa TCGA cohort . Data were obtained from UCSC Xena . KM Plotter analysis of relapse-free survival (left panel; RFS) and distant metastasis-free survival (right panel; DMFS) in an b unselected patient cohort (RFS; n = 4929, DMFS; n = 2765; months survival displayed as median survival), c ER-positive only (RFS; n = 3768, DMFS; n = 2016; months survival displayed as median survival) and d ER-negative only (RFS; n = 1161, DMFS; n = 749; months survival displayed as upper quartile survival) tumor subcohorts [ , ]

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 is over-expressed in the claudin-low molecular subtype of breast cancer. a Heatmap showing NRP1 expression association with PAM50, claudin-low, core claudin-low (CoreCL), ER and HER2 tumor status, as well as core claudin-low signature genes. b NRP1 mRNA expression (log2 signal) in intrinsic breast cancer subtypes and claudin-low tumors (CLDN low ) in the METABRIC patient dataset ( n = 1904), obtained through cBioPortal . c NRP1 mRNA expression across intrinsic subtypes subdivided into claudin-low (CL) and non-claudin-low tumors. Correlation of claudin-low di up-gene (CLDN low UP GES) and dii down-gene (CLDN low DN GES) GSVA-derived signature scores with NRP1 expression. Claudin-low gene signature scores were obtained via GSVA. Sample subtype is represented according to color scheme used in A-C. e NRP1 mRNA expression in METABRIC core claudin-low (CoreCL), non-core claudin-low (OtherCL) and non-claudin-low tumors . Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, Derivative Assay

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 expression is associated with in vivo tumor progression, cancer stemness and spheroid-initiating potential. a Western blot analysis of NRP1 expression in non-targeting control (shNT) and NRP1 shRNA-silenced (shNRP1 ) SUM159 cells inoculated into mice. b Post-inoculation tumor volumes, c tumor volume at week 8 post-inoculation and d Kaplan–Meier analysis of overall survival in SUM159 shNT and NRP1 knockdown groups. e Representative images of NRP1 immunohistochemistry in shNT and shNRP1 tumors and f quantification of NRP1 IHC staining across shNT and NRP1 knockdown groups. g NRP1 expression across CL1, CL2 and CL3 claudin-low subtypes as well PAM50 classifiers in the METABRIC dataset obtained via cBioportal [ , ]. h qPCR (left and center panel; n = 3) and Western blot (right panel) analysis of ZEB1 expression in HS578T cells after 72 h NRP1 knockdown versus NT control. i qPCR analysis of ITGA6 mRNA expression in HS578T cells (leftmost panel; NRP1 expression shown in h ) and SUM159 cells (center and right panel) after 72 h NRP1 knockdown versus NT control ( n = 3). j Western blot showing ITGA6 expression in HS578T, SUM159 and MDA-MB-231 cells after 72 h NRP1 knockdown versus NT control. k Western blot showing expression of ZEB1 and NRP1 in FACS sorted CD44 + /CD24 lo and CD44 + /CD24 hi populations of SUM159 cells. l Number of spheroids (> 50 µM) formed by day 6 following seeding of single cell SUM159 and Hs578T cell cultures containing 1,200 cells in the presence of 50 µg/ml Vesencumab (red lines) or IgG control (black lines). n = 5, along with m representative images of ( mi ) SUM159 and ( mii ) Hs578T spheroid cultures at days 4 and 6 post-seeding. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001, **** p ≤ 0.0001, error bars represent SEM

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Expressing, In Vivo, Western Blot, Control, shRNA, Knockdown, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses in vivo growth of claudin-low SUM159 orthotopic xenografts. a Luciferase-derived luminescence signal from SUM159 luc primary tumors as imaged by the IVIS Spectrum In Vivo Imaging System at 7 weeks post-tumor inoculation. Endpoint (7 weeks) mean b tumor luciferase intensity, c tumor weight and d tumor volume. e Tumor growth curves in IgG control and Vesencumab groups. f Endpoint (7 weeks) tumors from IgG control and Vesencumab groups; two additional tumors from the Vesencumab-treated group were too small to collect. g Representative images of H&E staining and Ki67 and CD31 immunohistochemistry of Vesencumab and IgG control treated tumors, with quantification of hi Ki67 and hii CD31 staining across all tumors. ‘Ves’; Vesencumab. Error bars represent SEM; ** p < 0.001; **** p < 0.0001. For a – e , n = 12–14. For g – h , n = 12

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, In Vivo, Luciferase, Derivative Assay, In Vivo Imaging, Control, Staining, Immunohistochemistry

Journal: Breast Cancer Research : BCR

Article Title: Neuropilin-1 is over-expressed in claudin-low breast cancer and promotes tumor progression through acquisition of stem cell characteristics and RAS/MAPK pathway activation

doi: 10.1186/s13058-022-01501-7

Figure Lengend Snippet: NRP1 inhibition suppresses EGFR and PDGFRα signaling in claudin-low cells. ai Receptor tyrosine kinase array showing EGFR and PDGFRα expression in SUM159 cells after 60 min treatment with 50 μg/mL IgG or Vesencumab (top panel), or 72 h after transfection with NRP1 targeting siRNA (siNRP1 or siNRP1 ) versus non-targeting (NT) control (bottom panel), with aii corresponding densitometry. Ref1 and Ref2 represent positive controls. b Western blot showing NRP1, total EGFR, phospho-EGFR (pEGFR Y1068), total PDGFRα and phospho-PDGFRα (Y1018) expression in SUM159, MDA-MB-231 and HS578T cells after 72 h NRP1 knockdown versus NT control. c Correlation analysis between NRP1 and EGFR (right panel; Pearson: 0.24, p = 2.7e−26) and PDGFRα (left panel; Pearson: 0.52, p = 3.47e−130) mRNA expression in the METABRIC dataset ( n = 1904) . Data was obtained from cBioportal. d Immunohistochemical analysis of phosphorylated (T202/Y204) p42/44 levels in endpoint (7 weeks post-tumor inoculation) Vesencumab or IgG control treated tumors, with e representative images (right panel); scale bars = 50 µm, n = 5–6. Error bars represent SEM, * p ≤ 0.05; ** p ≤ 0.01 versus control

Article Snippet: Anti-human APC-conjugated NRP1 antibody (R&D System, FAB3870A) or IgG-APC control (R&D System, IC003A) was added and cells incubated on ice for 45 min.

Techniques: Inhibition, Expressing, Transfection, Control, Western Blot, Knockdown, Immunohistochemical staining

Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: FTO Promotes Leukemic Cell Proliferation by Facilitating Cell Cycle and Suppressing Cell Apoptosis. (A) CCK-8 analysis of cell growth in the FTO-silenced OCI-AML3. (B) EdU analysis of cell proliferation in the FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (C) Flow cytometric analysis of cell cycle distribution in FTO-silenced OCI-AML3 cells. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (D) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. (E) Western blot analysis of Cyclin A2, t-Caspase 9, and c-Caspase 9 protein level in the FTO-silenced OCI-AML3. β-actin was used as the internal control. The bar graph showed the relative level of protein. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: CCK-8 Assay, Staining, Western Blot, Control

Journal: Frontiers in Oncology

Article Title: Mutant NPM1-Regulated FTO-Mediated m 6 A Demethylation Promotes Leukemic Cell Survival via PDGFRB/ERK Signaling Axis

doi: 10.3389/fonc.2022.817584

Figure Lengend Snippet: The Oncogenic Function of FTO is Dependent on m 6 A RNA Demethylase Activity. (A) CCK-8 analysis of cell proliferation in OCI-AML3 cells following transfection of the HA-FTO-WT and HA-FTO-MUT plasmids. (B) m 6 A dot blot analysis of the m 6 A level of global RNAs in OCI-AML3 cells with MA or DMSO treatment for 24 h. Methylene blue staining served as a loading control. (C) Dose-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 0, 10, 25, 50, and 100 μM MA for 24 h. (D) Time-dependent effect of MA on cell viability. CCK-8 analysis of cell proliferation in OCI-AML3 cells exposed to 100 μM MA for 0, 24, and 48 h. (E) EdU analysis of cell proliferation in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of EdU positive cells. Scale bar: 50 μm. (F) Flow cytometric analysis of the cell cycle distribution in OCI-AML3 cells upon 50 μM and 100 μM MA treatments of 24 h. The bar graph showed the percentage of G0/G1, S, and G2/M phase cells. (G) Cell apoptosis was measured by flow cytometric analysis of the Annexin V/DAPI stained cells. The bar graph showed the cell apoptosis rate. Data were presented as the mean ± SD of three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The cell apoptosis was determined by an Annexin V-APC/DAPI Apoptosis Detection Kit (Elabscience, Wuhan, China, #E-CK-A258).

Techniques: Activity Assay, CCK-8 Assay, Transfection, Dot Blot, Staining, Control