cav2 3  (Alomone Labs)

Journal: Journal of Cellular and Molecular Medicine

Article Title: Overexpression of sortilin is associated with 5‐FU resistance and poor prognosis in colorectal cancer

doi: 10.1111/jcmm.15752

Figure Lengend Snippet: Sortilin protein expression increases upon 5‐FU treatment in tumours from Nude mice xenografted with CRC cell lines. A, Tumour volume of WiDr and SW620 xenografted Nude mice untreated (CTL) or exposed to 5‐FU (5‐FU) was determined every 3 days from the graft and during the treatment protocol, as described in material and methods ( V = [L × W(L + W)]ᴨ/12). T. I. refers to Treatment Initiation. B, Sortilin protein expression analysis by Western blotting on whole lysates obtained from tumours of untreated xenografted Nude mice and 5‐FU‐treated ones ( n = 5 per conditions). Actin was used as loading control. Histograms represent means of 5 different samples (significant P ‐values are indicated in the graphs * P < .05, ** P < .01, *** P < .001). C, Representative illustration of sortilin protein expression observed by immunohistochemistry (magnification x100) on paraffin‐embedded xenografted Nude mice tumours ( n = 5 per conditions). Histograms represent the immunochemistry intensity score, as described in material and methods

Article Snippet: For immunohistochemistry analysis, primary antibody used was mouse anti‐sortilin (ANT‐016, Alomone Labs).

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Journal of Cellular and Molecular Medicine

Article Title: Overexpression of sortilin is associated with 5‐FU resistance and poor prognosis in colorectal cancer

doi: 10.1111/jcmm.15752

Figure Lengend Snippet: High SORT1 expression is associated with poorer survival, DFS and RFS, and sortilin expression is associated with higher CRC tumour grades. SORT1 expression levels by risk group according to survival ( n = 77; A), DFS ( n = 545; B) and RFS ( n = 37; C) of patients suffering from CRC, extracted from ‘SurvExpress’ database. Significant P‐values are indicated in the graphs * P < .05, ** P < .01, *** P < .001. D, Sortilin protein expression, observed by immunohistochemistry (magnification, 50×) on tissue microarrays including seven samples of benign tissue and 126 samples from CRC tumours of different grades ( n = 8, 98, and 20 for grades I, II and III, respectively). Quantification was performed by calculating the immunochemistry intensity score described in materials and methods. E, Sortilin expression in whole‐cell lysates of WiDr and SW620. Actin was used as a loading control

Article Snippet: For immunohistochemistry analysis, primary antibody used was mouse anti‐sortilin (ANT‐016, Alomone Labs).

Techniques: Expressing, Immunohistochemistry

Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: NLC drive expression of NTSR2-TrkB and pro-survival signaling in B-CLL cells. ( a ) Brightfield microscopy of co-cultured NLC (red arrows) and B-CLL cells (blue arrows). ( b ) Confocal microscopy analysis of CD14 (green) and CD68 (red) (left panel), and CD163 (green) and CD68 (red) (right panel), expression by NLC (n = 3). Nuclei are stained with DAPI. ( c ) Western blot analysis of CD68, CD163, and CD206 expression by NLC (n = 3). ( d ) Representative western blots showing expression of NTSR2, TrkB, p-Src, Src, and Bcl-2 by B-CLL cells isolated from two independent patients and cultured alone or with autologous NLC for 48 h. ( e ) Quantification of NTSR2 (n = 15), TrkB (n = 6), p-Src/Src (n = 17) and Bcl2 (n = 16) expression in independent patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05 and ** p < 0.01). Blots are cropped for clarity; full-length blots are shown in the Supplementary Fig. .

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Expressing, Microscopy, Cell Culture, Confocal Microscopy, Staining, Western Blot, Isolation

Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: The effects of BDNF on B-CLL pro-survival signaling are similar to those of BAFF, APRIL, and CXCL12 combined. ( a ) Representative western blot showing expression of NTSR2, p-Src, Src, and Bcl-2 by B-CLL cells isolated from patients. Cells were either co-cultured for 48 h with autologous NLC or cultured alone in complete medium supplemented (as indicated) with exogenous human (h) CXCL12 (100 ng/mL), BAFF (2 ng/mL), APRIL (25 ng/mL), or BDNF (100 ng/mL). (b,c,d), full-length blots are shown in the Supplementary Fig. . Quantification of NTSR2 ( b ), p-Src ( c ), and Bcl-2 ( d ) expression in four different patient samples. Data are presented as the mean ± SEM from at least three independent experiments, (* p < 0.05, ** p < 0.01). Blots are cropped for clarity; full blots are shown in the Supplementary Fig. .

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: Inhibiting BDNF in addition to BAFF, APRIL, and CXCR4 reverses NLC-mediated protection of B-CLL cells from apoptosis. ( a ) Representative western blot showing expression of p-Src and Bcl-2 by B-CLL cells isolated from patients. Cells were cultured (for 72 h) alone, with autologous NLC, or with autologous NLC plus single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). ( b – d ), full-length blots are shown in the Supplementary Fig. .Quantification of NTSR2 ( b ), p-Src ( c ) and Bcl-2 ( d ) expression in six independent experiments using six different patient samples. ( e ) Flow cytometry analysis of cell death, assessed by annexin V-fluorescein isothiocyanate/propidium iodide dual staining of B-CLL cells cultured (for 72 h) either alone, with autologous NLC, or with autologous NLC and single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 inhibition (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). Cell death was assessed by exclusion of annexin V/propidium iodide-negative cells. Experiments were performed using n = 9 different patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). ns not significant. ( f ) Schematic representation of the obtained results. NLC produce and secrete BDNF, which promotes survival of B-CLL cells by activating the Src signaling pathway and upregulating expression of Bcl-2. This newly described member of the NLC secretome appears to exert both complementary (alongside BAFF, APRIL and CXCL12) and independent effects. Here, we propose a model in which BDNF or pro-survival cytokines secreted by NLC within survival centers balance each other out to facilitate survival of B-CLL cells, and argue that simultaneous inhibition of BDNF signaling through the NTSR2-TrkB conditional oncogenic platform, along with inhibition of BAFF, APRIL and CXCR4/CXCL12, could cancel out NLC-mediated protection of B-CLL cells from apoptosis and restore normal cell death.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-NTSR2 (1:400, #ANT-016, Alomone Labs), mouse monoclonal anti-BDNF (1:1,000, #MAB648, R&D Systems), rabbit polyclonal anti-TrkB (1:500, #orb214339, Biorbyt), mouse monoclonal anti-CD68 (1:500, #333801, BioLegend), mouse monoclonal anti-CD163 (1:500, #326502, BioLegend), mouse monoclonal anti-CD206 (1:500, #32502, BioLegend), rabbit polyclonal anti-phospho-Src Tyr-416 (1:1,000, #2,101, Cell Signaling Technology, Ozyme, France), rabbit polyclonal anti-Src (1:1,000, #2,108, Cell Signaling Technology, Ozyme), rabbit monoclonal anti-Bcl-2 (1:1,000, #2,870, Cell Signaling Technology, Ozyme), anti-BAFF (1:1,000, #orb76960, Biorbyt), anti-APRIL (0.2 µg/mL; #AF884, R&D Systems) and mouse monoclonal anti-β-actin (1:10,000, #A5441, Sigma-Aldrich).

Techniques: Western Blot, Expressing, Isolation, Cell Culture, Inhibition, Flow Cytometry, Staining