lif neutralizing antibody  (R&D Systems)

Journal: JCI Insight

Article Title: EGFR -mutant transformed small cell lung cancer harbors intratumoral heterogeneity targetable with MEK inhibitor combination therapy

doi: 10.1172/jci.insight.197008

Figure Lengend Snippet: ( A ) Heatmap of cell viability after 5 days’ treatment with 1 μmol/L of each single agent (rows) per various lung cancer cell lines (columns) relative to DMSO control. Commercially available agents used are in . ( B ) Bar graph of IC 50 values of various indicated lung cancer cell lines in response to trametinib. IC 50 of NE and non-NE tSCLC cell lines is shown in red and blue, respectively. ( C ) Dose-response curve of trametinib in tSCLC cell lines (blue: NE, red: non-NE). Cell viability was assessed by CTG after 120 hours ( n = 6, technical replicate, mean ± SD). ( D ) NE tSCLC cell viability was assayed by CTG following treatment with indicated concentration of LIF and IL-6 for 5 days. ( E ) qPCR analysis of LIF mRNA expression levels in non-NE tSCLC cell lines treated with 10 nmol/L trametinib 2 and 6 hours. Gene expression was normalized to GUSB and shown as relative to that of DMSO-treated control ( n = 3, technical replicate, mean ± SD). ****= P ≤ 0.001 by 2-way ANOVA with Šídák’s multiple comparisons test. ( F ) Various cell lines were analyzed for LIF expression by ELISA. Data are presented as mean ± SD ( n = 3. Technical replicates). ( G ) GFP-expressing DFCI112F and DFCI190F cells were incubated with CM from their corresponding adherent cell line pairs pretreated with either LIF neutralizing antibody (500 ng/mL) or control IgG and monitored in real time via IncuCyte imager for green fluorescence intensity. ( H and I ) GFP expressing DFCI112F ( H ) and DFCI190 ( I ) cells were incubated with CM from their adherent cell line pairs pretreated with siRNA against LIF or unrelated sequence and monitored via IncuCyte imager. LIF knockdown efficiency was tested using ELISA ( n = 3, technical replicate, mean ± SD). ( J ) The schema illustrating the interaction between NE and non-NE tSCLC cells through LIF.

Article Snippet: LIF neutralizing antibody (AF-250-NA), IL-6 neutralizing antibody (AF-206-NA), and control antibody (AB-108-C) were purchased from R&D Systems.

Techniques: Control, Concentration Assay, Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Incubation, Fluorescence, Sequencing, Knockdown

Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Ectopic LIF expression (left panel) or treatment with rhLIF (right) enhanced lactate production of different breast cancer cell lines analyzed by measuring lactate levels in the medium using a lactate assay kit. B LIF neutralization antibody (LIF neu-ab) blocked the promoting effect of LIF on lactate production in MCF7, MDA-MB 231 and T47D cells. C Knockdown of endogenous LIF decreased lactate production in MDA-MB 231 cells. D , E Ectopic LIF expression enhanced glycolysis rates ( D ), whereas knockdown of endogenous LIF decreased glycolysis rates ( E ) in different breast cancer cell lines as calculated by ECAR measured by using a Seahorse analyzer. F The diagram of glycolysis. G – I The fold change of inter-metabolites in glycolysis resulted from ectopic LIF expression in MCF7 and MDA-MB 231 cells ( G ), rhLIF treatment in MCF7 cells ( H ) or LIF knockdown in MDA-MB 231 cells ( I ) as determined by LC/MS metabolomics analysis. J The fold change of inter-metabolites in glycolysis induced by ectopic LIF expression in MCF7 xenograft tumors determined by LC/MS metabolomics analysis. Data are presented as mean ± SD ( n = 3/group). * p < 0.05; ** p < 0.01; *** p < 0.001; unpaired Student’s t -test.

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Expressing, Lactate Assay, Neutralization, Knockdown, Liquid Chromatography with Mass Spectroscopy

Journal: Cell Death & Disease

Article Title: Leukemia inhibitory factor drives glucose metabolic reprogramming to promote breast tumorigenesis

doi: 10.1038/s41419-022-04820-x

Figure Lengend Snippet: A Glut1 mRNA levels were detected by quantitative real-time PCR (qPCR) in MCF7, MDA-MB 231, and T47D cells with or without ectopic LIF expression. B , C Ectopic LIF expression ( B ) or rhLIF treatment (100 ng/ml for 12 h) ( C ) promoted endogenous Glut1 PM translocation in MCF7, MDA-MB 231, and T47D cells as determined by Western-blot assays. D Knockdown of LIF decreased endogenous Glut1 PM translocation in MDA-MB 231 cells. E Ectopic LIF expression increased the PM translocation of ectopically expressed Myc-Glut1 in MCF7, MDA-MB 231 and T47D cells as determined by Western-blot assays. F LIF neutralization antibody (LIF neu-ab) largely abolished exogenous Glut1 PM translocation promoted by LIF. G The rhLIF treatment promoted exogenous Glut1 PM translocation in cells. H Knockdown of LIF decreased Myc-Glut1 PM translocation in MDA-MB 231 cells. I Ectopic LIF expression promoted Myc-Glut1 PM translocation (left panels) while knockdown of LIF decreased Myc-Glut1 PM translocation (right panels) in MDA-MB 231 cells as determined by IF staining assays. Scale bar, 10 μm. J Ectopic LIF expression promoted the PM translocation of Myc-Glut1 in MCF7, MDA-MB 231, and T47D cells as determined by flow cytometry assays. Left panels: representative images of flow cytometry analysis. Right panels: quantifications of relative fluorescence intensity of Myc-Glut1 on the cell membrane normalized with total Myc-Glut1 fluorescence intensity in cells. In A , J data are presented as mean ± SD. n = 3/group. * p < 0.05; ** p < 0.01; NS: non-significant; unpaired Student’s t -test. Uncropped Wes t ern-blot images are shown in Supplementary Fig .

Article Snippet: LIF neutralization antibody (AF-250-NA) was purchased from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Translocation Assay, Western Blot, Knockdown, Neutralization, Staining, Flow Cytometry, Fluorescence, Membrane