leptin neutralizing antibody nab experiment  (R&D Systems)

Journal: bioRxiv

Article Title: Obesity promotes breast epithelium DNA damage in BRCA mutation carriers

doi: 10.1101/2022.07.29.502090

Figure Lengend Snippet: ( A ) Experimental schematic showing the collection of breast adipose tissue conditioned media (CM) from lean and overweight/obese women. ( B ) MCF-10A cells were treated with CM for 24 hours. DNA damage assessed by IF (#γH2AX foci/100 cells) is shown correlated with BMI in BRCA1 +/- (n=36 CM cases) and ( C ) BRCA2 +/- (n=13 CM cases) MCF-10A cells. Spearman’s rank correlation coefficient (ρ) and associated P value are shown along with 95% confidence intervals. ( D ) DNA damage in BRCA1 +/- and BRCA2 +/- MCF-10A cells and in ( E ) primary BRCA1 +/- breast epithelial cells treated with leptin (400ng/µl) for 24 hours. ( F ) DNA damage in BRCA1 +/- MCF-10A cells after 24-hour treatment with lean CM, obese (ob) CM, or ob CM in the presence of a leptin neutralizing antibody (Lep Ab). ( G ) DNA damage in BRCA1 +/- and BRCA2 +/- MCF-10A cells and in ( H ) primary BRCA2 +/- breast epithelial cells treated with insulin (100nM) for 24 hours. ( I ) DNA damage in BRCA1 +/- MCF-10A cells after 24-hour treatment with lean CM, ob CM, or ob CM in the presence of PI3K inhibitor BKM120 (1µM). Student’s t-test was used to determine significant differences in ( D-I) . All experiments in MCF-10A cells were conducted a minimum of two times with representative results from one experiment shown. Data in primary cells were generated from cells treated in triplicate. Data is presented as mean +/- SD. * P <0.05, ** P <0.01, *** P <0.001, ns= not significant.

Article Snippet: In leptin neutralization studies, obese CM was pre-incubated with a leptin neutralizing antibody (Lep ab, 13.3μg/mL, Fisher Scientific #AF398) for 1 hour at 4°C and then cells were treated with lean or obese CM alone or obese CM + Lep ab.

Techniques: Generated

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: Leptin was involved in obese adipocyte-induced 5-FU resistance in CRC cells. (A and B) mRNA and protein levels of leptin in non-obese and obese adipocytes were analyzed by quantitative RT-PCR (A) and Western blot analysis (B), respectively. (C) Amounts of leptin in M-CM and P-CM were measured by ELISA. (D and E) H3347 and HCT116 cells were pre-incubated with M-CM or P-CM with/without 2 μg/ml leptin neutralizing antibodies for 48 hours followed by 5-FU treatment for 48 hours. Cell viability was evaluated by MTT analysis (D). The protein expressions of apoptosis-related molecules, cleaved caspase3, Bax and Bcl-2, were analyzed by Western blot analysis (E). (F and G) H3347 and HCT116 cells pre-treated with 100 ng/ml leptin recombinant protein for 48 hours were subjected to 5-FU treatment for another 48 hours. MTT assay and Western blot analysis were used to detect cell viability (F) and the expressions of apoptosis-related molecules (G), respectively. M-CM, non-obese adipocyte-derived conditioned media. P-CM, obese adipocyte-derived conditioned media. GAPDH served as the loading control. Data are expressed as the mean ± SEM. SEM, error bars. *P<0.05 by Student’s t test or one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, MTT Assay, Derivative Assay

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: AXL was involved in leptin-induced 5-FU resistance in CRC cells. (A) Protein levels of AXL, phospho-PLCγ (Tyr783) and PLCγ were determined by Western blot analysis in H3347 and HCT116 cells incubated with M-CM or P-CM with/without 2 μg/ml leptin neutralizing antibodies for 48 hours. (B) The expressions of AXL, phospho-PLCγ (Tyr783) and PLCγ in cells treated with 100 ng/ml leptin recombinant protein for 48 hours were analyzed by Western blot analysis. (C and D) mRNA and protein levels of AXL in H3347 and HCT116 cells transfected with non-targeting siRNA or AXL siRNA were examined by quantitative RT-PCR (C) and Western blot analysis (D). (E) CRC cells transfected with non-targeting siRNA or AXL siRNA were treated with 100 ng/ml leptin recombinant protein for 48 hours. The expressions of AXL, phospho-PLCγ (Tyr783) and PLCγ were detected by Western blot analysis. (F and G) H3347 and HCT116 cells transfected with non-targeting siRNA or AXL siRNA were treated with 100 ng/ml leptin recombinant protein for 48 hours followed by 5-FU treatment for another 48 hours. Cell viability was determined by MTT assay (F). The expressions of apoptosis-related molecules, including cleaved caspase3, Bax and Bcl-2, were analyzed by Western blot analysis (G). M-CM, non-obese adipocyte-derived conditioned media. P-CM, obese adipocyte-derived conditioned media. SC siRNA, non-targeting siRNA. GAPDH was used as the loading control. Data are expressed as the mean ± SEM. SEM, error bars. *P<0.05 by Student’s t test or one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Western Blot, Incubation, Recombinant, Transfection, Quantitative RT-PCR, MTT Assay, Derivative Assay

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: AMPK was the downstream regulator of leptin-induced activation of the YAP-TEAD complex in CRC cells. H3347 and HCT116 cells were treated with 100 ng/ml leptin recombinant protein with/without 0.5 mM of the AMPK activator, AICAR, for 48 hours. A. Protein levels of phospho-AMPK (Thr172), AMPK, phospho-LATS1 (Ser909), LATS1, phospho-YAP (Ser127), YAP and AXL were analyzed by Western blot analysis. B. Subcellular distributions of YAP and TEAD were investigated by Western blot analysis of the cytoplasmic and nuclear fractions of CRC cells after the indicated treatments. Lamin B and GAPDH served as nuclear and cytoplasmic controls, respectively. C. The interaction between YAP and TEAD was evaluated using co-immunoprecipitation. Protein extracts of CRC cells were immunoprecipitated with anti-YAP or anti-TEAD antibodies. The expressions of YAP and TEAD in the precipitated proteins were detected by Western blot analysis. D. The association between TEAD and the AXL promoter was examined by ChIP assay. The ChIP assay was carried out on the AXL promoter using anti-TEAD antibodies followed by PCR to amplify the DNA region containing TEAD binding sites. E. pAXL-cypridina luc plasmid and internal control pTK-red firefly luc plasmid were co-transfected into H3347 and HCT116 cells followed by treatment with 100 ng/ml leptin recombinant protein with/without 0.5 mM AICAR for 48 hours. The relative promoter activity of AXL was subsequently detected using a dual luciferase assay. GAPDH served as the loading control. Anti-rabbit IgG was used as the isotype control. Data are expressed as the mean ± SEM. SEM, error bars. *P<0.05 by one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Activation Assay, Recombinant, Western Blot, Immunoprecipitation, Binding Assay, Plasmid Preparation, Transfection, Activity Assay, Luciferase

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: The YAP-TEAD complex was involved in the leptin-induced AXL expression in CRC cells. (A and B) The mRNA and protein expressions of YAP in H3347 and HCT116 cells transfected with either non-targeting siRNA or YAP siRNA were examined by quantitative RT-PCR (A) and Western blot analysis (B). (C) H3347 and HCT116 cells transfected with non-targeting siRNA or YAP siRNA were treated with 100 ng/ml leptin recombinant protein for 48 hours. The expressions of phospho-YAP (Ser127), YAP and AXL were analyzed by Western blot analysis. SC siRNA, non-targeting siRNA. GAPDH served as the internal control. Data are expressed as the mean ± SEM. SEM, error bars. *P<0.05 by Student’s t test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Recombinant

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: Leptin induced p-glycoprotein expression and activity via the AXL/PLCγ axis in CRC cells. H3347 and HCT116 cells transfected with non-targeting siRNA or AXL siRNA were treated with 100 ng/ml leptin recombinant protein for 48 hours. (A and B) mRNA and protein levels of p-glycoprotein (P-gp) were analyzed by quantitative RT-PCR (A) and Western blot analysis (B). (C) The activity of P-gp was determined using a multidrug resistance assay. SC siRNA, non-targeting siRNA. GAPDH served as the loading control. Data are expressed as the mean ± SEM. SEM, error bars. *P<0.05 by one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Expressing, Activity Assay, Transfection, Recombinant, Quantitative RT-PCR, Western Blot

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: Leptin inhibition sensitized CRC cells to 5-FU in obese mice. C57BL/6 mice were fed on a HFD or LFD from 5 weeks of age until the end of the experiments. MC-38 cells (1×106) were subcutaneously implanted 10 weeks after initiating the diet, and treatment was started when the tumor burden reach about 100 mm3. 5-FU (25 mg/kg) and neutralizing antibodies against leptin (50 μg per mouse) were injected intraperitoneally into tumor-bearing mice for 2 weeks. Goat IgG antibody was served as the control. The tumor volume was calculated using the formula: V = (width2 × length)/2. A. Timeline of the animal experiments and treatment schedule for the tumor-bearing mice. B and C. Food intake and body weight changes of the mice fed on a HFD or LFD. D. Triglyceride (TG) levels in mice serum were detected using a TG colorimetric assay. E. ELISA was used to measure the serum level of leptin in the mice. F and G. Average tumor growth curves and tumor weights of CRC tumors in the mice with the indicated treatments. Tumor growth was monitored every 3-4 days. H. Representative immunohistochemistry images for phospho-YAP (Ser127), AXL and P-gp staining in CRC tumor sections (Scale bar, 50 μm). Data are expressed as the mean ± SEM of at least three mice in each group. SEM, error bars. n.s., not significant. *P<0.05 by Student’s t test or one-way ANOVA followed by Bonferroni’s post hoc test.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Inhibition, Injection, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: American Journal of Cancer Research

Article Title: Obesity-associated leptin promotes chemoresistance in colorectal cancer through YAP-dependent AXL upregulation

doi:

Figure Lengend Snippet: Obese adipocyte-associated leptin increased 5-FU resistance through YAP-dependent AXL upregulation. Leptin secreted by obese adipocytes resulted in 5-FU resistance in CRC cells by increasing AXL expression and PLCγ activation. Mechanistically, leptin induced AXL expression by suppressing AMPK and LATS1 followed by stimulation of YAP activation and nuclear translocation. Nuclear YAP further interacted with TEAD, thereby promoting the association between TEAD and the AXL promoter and eventually increasing AXL promoter activity. In addition, the AXL/PLCγ axis led to 5-FU resistance by inducing P-gp expression in CRC cells after leptin treatment.

Article Snippet: 5-FU (25 mg/kg) was injected intraperitoneally daily, and leptin neutralizing antibodies (50 μg per mouse) (R&D Systems, Minneapolis, MN, USA) were injected intraperitoneally every 3 days.

Techniques: Expressing, Activation Assay, Translocation Assay, Activity Assay