Journal: Oncotarget

Article Title: A hypoxic signature marks tumors formed by disseminated tumor cells in the BALB-neuT mammary cancer model

doi: 10.18632/oncotarget.8859

Figure Lengend Snippet: A. Heat map of differentially regulated genes; the green color depicts genes that are downregulated and the red denotes genes that are up-regulated in DTC tumors (DTC 1-4) and mammary tumors (T 1-4). B. Ingenuity pathway analysis (IPA) illustrating HIF-1α as a possible master regulator of differentially regulated genes in DTC tumors compared to mammary tumors. Up-regulated and down-regulated genes are shown in red and in green, respectively. C-D-E. VEGF C. and BNIP3 D. mRNA expression and miR-210 quantification E. in mammary and DTC tumors. The histograms depict relative expression compared to mammary tumor expressed as fold change. Data are expressed as mean values (± SD) of 4 different samples/group. F. Histopathologic evaluation of necrotic areas in mammary and DTC tumors. Representative haematoxylin and eosin-stained sections (H&E) of each tumor type were photographed on the left at 10x (scale bar 200 μm). The right histogram illustrates the quantification of necrotic areas over total tumor area. Data are expressed as mean values (± SD) of 6 different samples/group. G. Immunohistochemical analysis of HIF-1α expression in frozen sections of mammary and DTC tumors. A representative image of each tumor stained for HIF-1α is shown on the left at 20x; the quadrant indicates the area photographed at 40x (scale bar 100 μm). The right histogram shows the quantification of HIF-1α positive nuclei; data are expressed as mean values (± SD) of 5 different samples/group.* P < 0.05, ** P < 0.01.

Article Snippet: The membranes were hybridized overnight with rabbit anti-mouse actin (1:5,000; Sigma-Aldrich) and rabbit anti-mouse HIF-1α (1:1,000; Novus Biologicals), followed by incubation with anti-rabbit HRP-conjugated antibody (1:5,000 dilution, Amersham-Pharmacia, Little Chalfont, U.K.).

Techniques: Expressing, Staining, Immunohistochemical staining

Journal: Oncotarget

Article Title: A hypoxic signature marks tumors formed by disseminated tumor cells in the BALB-neuT mammary cancer model

doi: 10.18632/oncotarget.8859

Figure Lengend Snippet: A. Western blot analysis of HIF-1α protein levels in mammary and DTC tumor cell lines cultured under normoxic (N) or hypoxic (H) conditions. The molecular weight of HIF-1α and Actin, used as a loading control is indicated on the right. B. Quantification of HIF-1α protein expression by densitometry. The histograms represent quantification of HIF-1α normalized to individual actin levels, and relative to HIF-1α protein levels in normoxia of mammary tumor cell line. C. Immunofluorescence analysis of HIF-1α localization in mammary and DTC tumor cell lines in normoxic (top row) and hypoxic conditions (bottom row). In green HIF-1α staining, in blue nuclei. Images acquired at 40x and 63x magnification, scale bar 10 μm. D-E-F. qPCR analysis of VEGF D. and BNIP3 E. mRNA levels and miR-210 expression F. in cells derived from mammary and DTC tumor cell lines. G-H. qPCR analysis of VEGF , BNIP3 and miR-210 levels following incubation under hypoxia in mammary G. and DTC tumor cell lines H. Data are presented as relative to level under normoxic conditions. All data represent mean values ± SD. * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were hybridized overnight with rabbit anti-mouse actin (1:5,000; Sigma-Aldrich) and rabbit anti-mouse HIF-1α (1:1,000; Novus Biologicals), followed by incubation with anti-rabbit HRP-conjugated antibody (1:5,000 dilution, Amersham-Pharmacia, Little Chalfont, U.K.).

Techniques: Western Blot, Cell Culture, Molecular Weight, Control, Expressing, Immunofluorescence, Staining, Derivative Assay, Incubation

Journal: Oncotarget

Article Title: A hypoxic signature marks tumors formed by disseminated tumor cells in the BALB-neuT mammary cancer model

doi: 10.18632/oncotarget.8859

Figure Lengend Snippet: A. HIF-1α , VEGF and BNIP3 mRNA levels were measured by qPCR in DTC transduced with a lentiviral vector silencing HIF-1α (shHIF-1α) compared to control (shRNA). B. Tumor growth curves of control (shRNA, black dots) and HIF-1α (shHIF-1α, white dots) transduced DTC cells. Mean tumor volume (n=6 samples/group) ± SD was plotted as a function of time (days). C. Representative pictures of tumors at sacrifice of the mice (day 21). D. Representative haematoxylin and eosin-stained sections (H&E) of tumors. Pictures were acquired at 1.25x magnification, scale bar 10 mm. E. Analysis of HIF-1α , VEGF and BNIP3 mRNA levels in shRNA and shHIF-1α DTC tumors by qPCR. F. HIF-1α expression in early and late DTC by immunofluorescence analysis. On the left, representative confocal images of early (upper panels) and late (lower panels) DTC taken at 63x magnification, scale bar 20 μm. Nuclei were stained in blue, CK8-18 in red and HIF-1α in green. On the right, histogram reporting mean numbers of CK8-18 + /HIF-1α + DTC per 2 × 10 6 BM cells. * P < 0.05.

Article Snippet: The membranes were hybridized overnight with rabbit anti-mouse actin (1:5,000; Sigma-Aldrich) and rabbit anti-mouse HIF-1α (1:1,000; Novus Biologicals), followed by incubation with anti-rabbit HRP-conjugated antibody (1:5,000 dilution, Amersham-Pharmacia, Little Chalfont, U.K.).

Techniques: Transduction, Plasmid Preparation, Control, shRNA, Staining, Expressing, Immunofluorescence

Journal: Oncotarget

Article Title: A hypoxic signature marks tumors formed by disseminated tumor cells in the BALB-neuT mammary cancer model

doi: 10.18632/oncotarget.8859

Figure Lengend Snippet: A. Schematic representation of mammary, DTC and metastasis tumor generation; the right panel shows the histopathological analysis of mammary, DTC or metastasis tumors. A representative of each tumor frozen section was stained with H&E and HER2. Light microscopy images were taken at a magnification 1.25x (scale bar 10 mm), 2.5x (scale bar 1 mm) and 20x (scale bar 100 μm). B. Immunohistochemical analysis of HIF-1α expression in metastasis, mammary and DTC tumors. A representative tumor frozen section is shown, photographed at 20x and at 40x magnification. C-D. qPCR analysis of mRNA levels of BNIP3 C. and VEGF D. in mammary, DTC and metastasis tumors. Data represent mean relative expression of mRNA compared to mammary tumor (± SD), expressed as fold change. E. qPCR analysis of miR-210 expression in mammary, DTC and metastasis tumors. Data are expressed as mean values (± SD), relative to the level of miR-210 in mammary tumors. F. Flow cytometric analysis of HER2 expression in mammary, DTC and metastasis tumor cell lines. A representative plot of the percentages of HER2 + cells in the population of mammary, DTC and metastasis tumor cell lines is shown. G. Immunofluorescence analysis of HIF-1α localization in mammary, DTC and metastasis tumor cell lines in normoxic and hypoxic conditions. In green HIF-1α staining, in blue nuclei; scale bar 20 μm. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The membranes were hybridized overnight with rabbit anti-mouse actin (1:5,000; Sigma-Aldrich) and rabbit anti-mouse HIF-1α (1:1,000; Novus Biologicals), followed by incubation with anti-rabbit HRP-conjugated antibody (1:5,000 dilution, Amersham-Pharmacia, Little Chalfont, U.K.).

Techniques: Staining, Light Microscopy, Immunohistochemical staining, Expressing, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Extracellular in vivo levels of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) were significantly increased in human breast cancer. A total of 17 breast cancer patients underwent microdialysis before surgery. One catheter was inserted into the breast cancer, and another catheter was inserted into adjacent normal breast tissue as control. Proteins were analyzed by Luminex in cohort 1 (expressed as pg/ml), n = 6 and by proximity extension assay (PEA) technology in cohort 2 (expressed as linear NPX), n = 11 as described in Section “ .” The bars to the right represent data from both cohorts normalized to the control in each cohort (relative protein expression), n = 17. (A) IL-8 in vivo measurements in microdialysis samples from breast cancer patients. (B) VEGF in vivo measurements in microdialysis samples from breast cancer patients. Results are presented as mean ± SEM and were analyzed pairwise by Wilcoxon signed-rank test, * p < 0.05, *** p < 0.001. IL-8 and VEGF exhibited a significant positive correlation by Spearman’s correlation test, r = 0.51 and p < 0.05.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: In Vivo, Luminex, Expressing

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Addition of breast adipocytes (BAd) significantly increased interleukin-8 (IL-8) secretion but decreased vascular endothelial growth factor (VEGF) secretion compared to breast cancer cells (BCC) cultured alone and anti-IL-8 and anti-VEGF significantly decreased BAd-induced angiogenesis in primary tumors in zebrafish. BCC were cultured in 3D spheres alone or in combination with BAd. Secreted IL-8 and VEGF were analyzed as described in Section “ .” Prior injections, breast pre-adipocytes were differentiated for 12 days and estrogen receptor positive (ER+) BCC were cultured ± β-estradiol (E2) 1 nM for 48 h. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye. Cells were injected ± anti-IL-8, anti-VEGF, or isotype control at 0.1 mg/ml ± E2 1 nM into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) BAd mammospheres alone and low metastatic ER+ MCF-7 ± 90% BAd mammospheres were cultured ± E2 1 nM during 7 days, n = 4–5 in each group. (B) BAd mammospheres alone an ER+ T47D with intrinsically higher metastatic capacity ± 90% BAd mammospheres were cultured ± E2 1 nM during 7 days, n = 5–6 in each group. (C) Estrogen receptor negative (ER−) metastatic MDA-MB-23 ± 90% BAd and BAd mammospheres were cultured during 7 days, n = 4–5 in each group. (D) MCF-7 cells were injected alone or in combination with 50% BAd ± E2 1 nM, tumor angiogenesis was analyzed 3 days post-injections, n = 12–18 in each group. (E) T47D cells were injected alone or in combination with 50% Bad ± E2 1 nM, tumor angiogenesis was analyzed 3 days post-injections, n = 10 in each group. (F) MDA-MB-231 cells were injected alone or in combination with 50% BAd, tumor angiogenesis was analyzed 3 days post-injections, n = 7–10 in each group. Representative confocal images are shown for each cell line. BV = blood vessels. Results are presented as mean ± SEM and analyzed by Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (IL-8) treatment significantly decreased breast cancer cells (BCC) dissemination induced by breast adipocytes (BAd). Prior injections, breast pre-adipocytes were differentiated for 12 days and estrogen receptor positive (ER+) BCC were cultured ± β-estradiol (E2) 1 nM for 48 h. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye. Cells were injected ± anti-IL-8, anti-VEGF, or isotype control at 0.1 mg/ml ± E2 1 nM into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) MCF-7 cells were injected alone or in combination with 50% BAd ± E2 1 nM. BCC dissemination was evaluated 3 days post-injections, n = 18–27 in each group. (B) T47D cells were injected alone or in combination with 50% BAd ± E2 1 nM. BCC dissemination was evaluated 3 days post-injections, n = 16–28 in each group. (C) MDA-MB-231 cells were injected alone or in combination with 50% BAd. BCC dissemination was evaluated 3 days post-injections, n = 14–18 in each group. (D) MCF-7, T47D, and MDA-MB-231 cells were cultured alone or in combination with 50% breast pre-adipocytes in vitro during 24 h, and migration of cells was determined as described in Section “ ,” n = 6 in each group. BV = blood vessels. Arrows indicate disseminated BCC. Results are presented as mean ± SEM and analyzed by Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection, In Vitro, Migration

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (αIL-8) decreased vascular endothelial growth factor (VEGF) and CCL5 secretion, which affected estrogen receptor positive (ER+) breast cancer cells (BCC) dissemination. For monolayer co-cultures, breast pre-adipocytes were differentiated for 5 days before ER+ BCC were added at 4 × 10 3 cells/well. For zebrafish experiments, breast pre-adipocytes were differentiated for 12 days, MCF-7 cells were cultured + β-estradiol (E2) 1 nM for 48 h and labeled with 4 µg/ml Fast DiI™ oil red dye before injected into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) MCF-7 cells were co-cultured with 50% breast adipocytes (BAd) in the presence or absence of αIL-8, anti-VEGF (αVEGF), or control isotype (Iso) antibodies at 1 µg/ml during 3 days in the presence of E2 1 nM, and secreted cytokines were quantified as described in Section “ ,” n = 5–4 in each group. (B) T47D cells were co-cultured with 50% BAd in the presence or absence of αIL-8, αVEGF, or control Iso antibodies at 1 µg/ml during 3 days in the presence of E2 1 nM, and secreted cytokines were quantified as described in Section “ ,” n = 6–5 in each group. (C) MCF-7 cells were injected in zebrafish embryos alone or in combination with 50% BAd ± anti-CCL5 (αCCL5) or Iso control antibody at 0.1 mg/ml and E2 1 nM, as described in Section “ .” MCF-7 dissemination was analyzed 3 days post-injections, n = 13–27 in each group. Representative images of zebrafish embryos are shown. Arrows show disseminated BCC cells. BV = blood vessels. Results are presented as mean ± SEM, Student’s t -test, * p < 0.05, *** p < 0.001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Cell Culture, Labeling, Injection

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (αIL-8) decreased CCL5 and anti-vascular endothelial growth factor (αVEGF) increased interleukin-8 (IL-8) and CCL2, which affected the dissemination of estrogen receptor negative (ER−) breast cancer cells (BCC). For monolayer co-cultures, breast pre-adipocytes were differentiated for 5 days before MDA-MB-231 cells were added at 3 × 10 3 cells/well. For zebrafish experiments, breast pre-adipocytes were differentiated for 12 days and MDA-MB-231 cells were labeled with 4 µg/ml Fast DiI™ oil red dye before injected into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) MDA-MB-231 cells were co-cultured with 50% breast adipocytes (BAd) in the presence or absence of αIL-8, αVEGF, or control isotype (Iso) antibodies at 1 µg/ml during 3 days, and secreted cytokines were quantified as described in Section “ ,” n = 4 in each group. (B) MDA-MB-231 cells were injected in zebrafish embryos alone or in combination with 50% BAd ± anti-CCL2 (αCCL2), anti-CCL5 (αCCL5), or control Iso antibodies at 0.1 mg/ml, as described in Section “ .” BCC dissemination was analyzed 3 days post-injections, n = 20–24 in each group. Representative images of zebrafish embryos are shown. Arrows show disseminated BCC. BV = blood vessels. Results are presented as mean ± SEM, Student’s t -test, * p < 0.05, ** p < 0.01. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Labeling, Injection, Cell Culture

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Anti-interleukin-8 (αIL-8) reduced lymphocyte function-associated antigen 1 (LFA-1) expression in neutrophils and the neutrophil-mediated dissemination of breast cancer cells (BCC) in the presence of breast adipocytes (BAd). For immunocytochemistry, neutrophils were cultured at 1 × 10 6 cells/ml in BAd-conditioned or control medium and incubated 45 min at 37°C. Prior zebrafish injections, breast pre-adipocytes were differentiated for 12 days, BCC were labeled with 4 µg/ml Fast DiI™ oil red dye, and neutrophils were labeled with 6 µg/ml DiB. All BCC were injected ± αIL-8, anti-LFA-1 (αLFA-1), or isotype (Iso) control antibodies at 0.1 mg/ml into the perivitelline space of 2 days old zebrafish embryos, which expressed enhanced green fluorescent protein in endothelial cells. (A) Neutrophils were cultured ± conditioned medium (CM) from BAd ± αIL-8 or Iso control at 1 µg/ml, and whole cells were stained with anti-human LFA-1 as described in Section “ ” ( n = 15 random fields per group). Insets show magnification of the cells. (B) Breast pre-adipocytes were differentiated during 12 days and cultured in DMEM supplemented medium during 24 h, and secreted interleukin-8 (IL-8) was measured in control and CM as described in Section “ ,” n = 4 in the CM group. (C) MCF-7 cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “ .” BCC dissemination was analyzed at 1 day post-injections, n = 11–25 in each group. (D) T47D cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “ .” BCC dissemination was analyzed at 1 day post-injections, n = 15–26 in each group. (E) MDA-MB-231 cells were injected in zebrafish embryos together with 33% BAd ± 33% neutrophils, as described in Section “ .” BCC dissemination was analyzed at 1 day post-injections, n = 12–21 in each group. (F) Number of co-disseminated MCF-7/neutrophil cells was quantified as described in Section “ ,” n = 17–21 in each group. (G) Number of co-disseminated T47D/neutrophil cells was quantified as described in Section “ ,” n = 21–26 in each group. (H) Number of co-disseminated MDA-MB-231/neutrophil cells was quantified as described in Section “ ,” n = 12–21 in each group. Results are presented as mean ± SEM, Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Expressing, Immunocytochemistry, Cell Culture, Incubation, Labeling, Injection, Staining

Journal: Frontiers in Immunology

Article Title: Adipocytes Promote Early Steps of Breast Cancer Cell Dissemination via Interleukin-8

doi: 10.3389/fimmu.2018.01767

Figure Lengend Snippet: Interleukin-8 (IL-8) increased dissemination and MUC-1 expression in estrogen receptor positive (ER+) breast cancer cells (BCC) and IL-8 gene silencing affected the dissemination and expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and mucin-1 (MUC-1) integrins in estrogen receptor negative (ER−) BCC. Prior injections, MCF-7 and T47D cells were cultured 48 h in β-estradiol (E2) 1 nM. All BCC were labeled with 4 µg/ml Fast DiI™ oil red dye and then injected into the perivitelline space of 2 days old zebrafish embryos, which express enhanced green fluorescent protein in endothelial cells. (A) MCF-7 cells were injected into zebrafish embryos ± recombinant human IL-8 (rhIL-8) at 1 µg/ml. BCC dissemination was analyzed after 3 days post-injections as described in Section “ ,” n = 18–20 in each group. (B) Western blot analysis of MCF-7 cells treated ± rhIL-8 at 10 ng/ml during 5 days to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH load control was reused for illustrative purposes. (C) T47D cells were injected into zebrafish embryos ± rhIL-8 at 1 µg/ml. BCC dissemination was analyzed after 3 days post-injections as described in Section “ ,” n = 17–23 in each group. (D) Western blot analysis of T47D cells treated ± rhIL-8 at 10 ng/ml during 3 days to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH is shown as load control. (E) MDA-MB-231 cells transfected with or without an IL-8 gene silencer RNA were injected into the perivitelline space of zebrafish embryos, and BCC dissemination was analyzed at 3 days post-injections, n = 19–22 in each group. (F) Western blot analysis of MDA-MB-231 cells transfected with/without an IL-8 silencer RNA (siIL-8) to evaluate the expression of MUC-1, VCAM-1, and ICAM-1. GAPDH load control was reused for illustrative purposes. (G) ELISA quantification of extracellular in vitro IL-8 in cell culture supernatants of MDA-MB-231 cells transfected with negative control silencer RNA (siRNA Control) or IL-8 silencer RNA (siRNA IL-8) during 2 days showed the successful knockdown of IL-8, n = 6 in each group. Representative images of zebrafish embryos are shown. Arrows show disseminated BCC. BV = blood vessels. Results are presented as mean ± SEM, Student’s t -test, * p < 0.05, **** p < 0.0001. Western blots shown in this figure were prepared by cropping and pasting from original membranes shown in full in Supplementary Figure 1. Data are representative of at least two independent experiments.

Article Snippet: After 24 h co-culture, treatment ± E2 1 nM, rabbit anti-human IL-8, rabbit anti-human VEGF (Acris Antibodies GmbH Cat # PP1073P1, RRID:AB_1008432), or rabbit isotype antibodies at 1 µg/ml was performed for 3 days.

Techniques: Expressing, Cell Culture, Labeling, Injection, Recombinant, Western Blot, Transfection, Enzyme-linked Immunosorbent Assay, In Vitro, Negative Control