Journal: Scientific Reports

Article Title: The metabolic effects of resumption of a high fat diet after weight loss are sex dependent in mice

doi: 10.1038/s41598-023-40514-w

Figure Lengend Snippet: Circulating levels of free insulin-like growth factor (IGF) 1 ( A ), total IGF1 ( B ), IGF2 ( C ), IGF binding protein (IGFBP) 2 ( D ) and leptin ( E ) in mice fed chow or a high-fat diet (HFD) for 4 months (HHHH), chow diet for 3 months and an additional month on a HFD (CCCH), or HFD for 2 months followed by chow for 1 month and then HFD for the last month (HHCH). ***p < 0.001. a: effect of the sex, b: effect of the diet. NS non-significant. n = 9.

Article Snippet: Plasma levels of free IGF1 (Ref.: AL-136, AnshLabs, Webster, TX, USA), total IGF1 (Ref.: E25; Mediagnost, Reutlingen, Germany), IGF2 (Ref.: MG200; R&D Systems, Minneapolis, Minnesota, USA), IGFBP2 (Ref.: RAB0234; Millipore, Burlington, MA, USA), insulin (Ref.: EZRMI-13K; Millipore) and leptin (Ref.: EZML-82K; Millipore) were assayed according to manufacturer’s instructions, and absorbance was read using a TECAN Infinite M200.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: The metabolic effects of resumption of a high fat diet after weight loss are sex dependent in mice

doi: 10.1038/s41598-023-40514-w

Figure Lengend Snippet: Relative mRNA levels of insulin-like growth factor (IGF) 1 ( A ), IGF2 ( B ), IGF binding protein (IGFBP)2 ( C ), neuropeptide Y (NPY; E ), Agouti-related protein (AgRP; F ), and proopiomelanocortin (POMC; G ) in the hypothalamus, as well as the correlation of hypothalamic IGF2 and IGFBP2 mRNA levels ( D ), in mice fed chow or a high-fat diet (HFD) for 4 months (HHHH), a chow diet for 3 months and an additional month on a HFD (CCCH), or HFD for 2 months followed by chow for 1 month and then HFD for the last month (HHCH). *p < 0.05, ***p < 0.001. a: effect of the sex, b: effect of the diet. n = 6.

Article Snippet: Plasma levels of free IGF1 (Ref.: AL-136, AnshLabs, Webster, TX, USA), total IGF1 (Ref.: E25; Mediagnost, Reutlingen, Germany), IGF2 (Ref.: MG200; R&D Systems, Minneapolis, Minnesota, USA), IGFBP2 (Ref.: RAB0234; Millipore, Burlington, MA, USA), insulin (Ref.: EZRMI-13K; Millipore) and leptin (Ref.: EZML-82K; Millipore) were assayed according to manufacturer’s instructions, and absorbance was read using a TECAN Infinite M200.

Techniques: Binding Assay

Journal: Scientific Reports

Article Title: The metabolic effects of resumption of a high fat diet after weight loss are sex dependent in mice

doi: 10.1038/s41598-023-40514-w

Figure Lengend Snippet: Cell number ( A ) and the relative IGF1 ( B ), IGF2 receptor (IGF2R, C ) and IGFBP2 ( D ) mRNA levels in hypothalamic astrocyte cultures from male and female rats after 24 h of IGF2 treatment at 10, 25, 100 or 200 ng/ml. **p < 0.01. a: effect of the sex; #, general effect of IGF2; δ, effect of the dose of IGF2. n = 3.

Article Snippet: Plasma levels of free IGF1 (Ref.: AL-136, AnshLabs, Webster, TX, USA), total IGF1 (Ref.: E25; Mediagnost, Reutlingen, Germany), IGF2 (Ref.: MG200; R&D Systems, Minneapolis, Minnesota, USA), IGFBP2 (Ref.: RAB0234; Millipore, Burlington, MA, USA), insulin (Ref.: EZRMI-13K; Millipore) and leptin (Ref.: EZML-82K; Millipore) were assayed according to manufacturer’s instructions, and absorbance was read using a TECAN Infinite M200.

Techniques:

Journal: Scientific Reports

Article Title: The metabolic effects of resumption of a high fat diet after weight loss are sex dependent in mice

doi: 10.1038/s41598-023-40514-w

Figure Lengend Snippet: List of TaqMan probes used for qPCR.

Article Snippet: Plasma levels of free IGF1 (Ref.: AL-136, AnshLabs, Webster, TX, USA), total IGF1 (Ref.: E25; Mediagnost, Reutlingen, Germany), IGF2 (Ref.: MG200; R&D Systems, Minneapolis, Minnesota, USA), IGFBP2 (Ref.: RAB0234; Millipore, Burlington, MA, USA), insulin (Ref.: EZRMI-13K; Millipore) and leptin (Ref.: EZML-82K; Millipore) were assayed according to manufacturer’s instructions, and absorbance was read using a TECAN Infinite M200.

Techniques:

Journal: Nature Communications

Article Title: Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression

doi: 10.1038/s41467-024-49199-9

Figure Lengend Snippet: a A phospho-receptor tyrosine kinase (RTK) array with lysates of the indicated CM-treated A549 cells for 30 min. b , f Western blot (WB) analysis of phosphorylated IGF-1R/IR (Y1135/36 for IGF-1R, Y1150/51 for IR) and IR levels in the indicated CM-treated A549 cells for 30 min. αIGF2 Ab : IGF2 neutralizing antibody (5 μg/mL) c Immunoprecipitation (IP) analysis of IGF-1R and IR phosphorylation in the indicated CM-treated A549 cells for 30 min. d Real-time PCR analysis of IGF2 , IGF1 , or INS expression in THP-1 cells ( n = 3 biologically independent replicates per group). 2DG: 5 mM 2-deoxy-D-glucose. e WB analysis of IGF2 expression in THP-1 cells. g Real-time PCR analysis of Igf2 mRNA ( n = 3/group), immunohistochemistry (IHC) analysis of pIGF-1R/IR (Y1131 for IGF-1R, Y1146 for IR) expression ( n = 25/group), and IF analysis of IGF2-expressing macrophages (IGF2 + F4/80 + ) ( n = 15/group) in LLC-Luc sc tumors. arb. units.: arbitrary units. h IGF2 ELISA using CM from CD45 - F4/80 - non-immune cells, CD45 + F4/80 - non-macrophage immune cells, and CD45 + F4/80 + macrophages isolated from LLC-Luc sc tumors ( n = 3/group). i, j Anchorage-independent colony formation and sphere formation of the indicated A549 cells treated with the CM from the indicated THP-1 cells ( n = 5 biologically independent replicates per group). k The tumor volume of primary tumors of LLC cells co-injected with the indicated BMDMs ( n = 14/group) and microscopic evaluation H&E-stained lung tissues ( n = 7/group). l The tumor volume of primary tumors ( n = 13 for LLC/sgRNA Con groups and n = 10 for LLC/sgRNA IR groups) and microscopic evaluation H&E-stained lung tissues ( n = 7 for LLC/sgRNA Con groups and n = 6 for LLC/sgRNA IR groups). The data are presented as the mean ± SD. p -values were determined by using one-way ANOVA with Tukey’s post-hoc test ( d , i , j ), a two-tailed Student’s t -test ( g , h ), or Kruskal–Wallis test with Dunn’s post-hoc test ( k , l ). The data shown in b – f , i , j are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Article Snippet: The level of IGF2 in CM was determined by an IGF2 ELISA kit (cat. no. MG200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and normalized by the protein concentration of attached cells remaining after CM collection.

Techniques: Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Isolation, Injection, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression

doi: 10.1038/s41467-024-49199-9

Figure Lengend Snippet: a Western blot (WB) analysis using the nuclear extract (NE) of CM-treated A549 cells. b Quantification of the immunofluorescence staining of nuclear phosphorylated IGF-1R/IR (pIGF-1R/IR, [Y1131 for IGF-1R, Y1146 for IR]) ( n = 5 biologically independent replicates/group). arb. units.: arbitrary units. c Streptavidin pulldown analysis on membrane fractions (MEM), cytosol extracts (CE), and NE of IGF2 (50 ng/mL)-stimulated A549 cells. d WB analysis of non-chromatin (Chr-unbound) and chromatin (Chr-bound) fractions. e Immunoprecipitation (IP) analysis of the association of IR with KPNB1 and KPNA2 in IGF2-stimulated A549 cells. f WB analysis of nuclear pIGF-1R/IR (Y1135/36 for IGF-1R, Y1150/51 for IR) and IR levels in A549 cells. WCL: whole-cell lysates. g A representative image of Coomassie blue-stained gel on resolved anti-IR immunoprecipitants and common nuclear IR-associated proteins identified by LC-MS/MS analysis. NCL: nucleolin. h IP of the association of IR with NPM1, histones, and RNA polymerase II (Pol II) in A549 cells. i Schematic diagram depicting full-length (FL) and truncation mutants of NPM1. OD: oligomerization domain. HBD: histone binding domain. NBD: nucleic acid-binding domain. j Representative WB images of IP analysis and quantitative analysis ( n = 3 biologically independent replicates/group) for the association of IR with NPM1 (FL or truncation mutants) in IGF2-stimulated H226Br cells for 3 h. k The association of IR with FL or HBD deletion mutant (ΔHBD) of NPM1 in IGF2-stimulated H226Br cells for 3 h. l Anchorage-independent colony formation ( n = 3 biologically independent replicates/group) and sphere formation ( n = 4 biologically independent replicates/group) capacities of the indicated A549 cells. m Schematic diagrams of experiments and quantitative analyzes of primary and metastatic lung tumor growth (LLC/sgRNA Con + BMDM-Veh: n = 8; LLC/sgRNA Con + BMDM-NB and LLC/sgRNA NPM1 + BMDM-Veh: n = 6; LLC/sgRNA NPM1 + BMDM-NB: n = 5). The data are presented as the mean ± SD. p -values were determined by using one-way ANOVA with Tukey’s post-hoc test ( b , j , l ) or Kruskal-Wallis test with Dunn’s post-hoc test ( m ). The data are representative of two (data shown in a – f , h , k , l ) or three (data shown in j ) independent experiments with similar results. Source data are provided as a Source Data file.

Article Snippet: The level of IGF2 in CM was determined by an IGF2 ELISA kit (cat. no. MG200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and normalized by the protein concentration of attached cells remaining after CM collection.

Techniques: Western Blot, Immunofluorescence, Staining, Membrane, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Mutagenesis

Journal: Nature Communications

Article Title: Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression

doi: 10.1038/s41467-024-49199-9

Figure Lengend Snippet: a – d Western blot (WB) analysis of the indicated protein expression in A549 and H226Br cells and their subclones. Cells were stimulated with IGF2 (50 ng/mL) for one day ( a , b ) or incubated with CM THP-Veh or CM THP-NB for one day ( c , d ). e – g Indicated LC cells were exposed to IGF2 (50 ng/mL) for 3 h (for chromatin immunoprecipitation [ChIP] assay) ( e , f ) or 24 h (for luciferase reporter assay) ( g ). e , f ChIP assay of IR ( e , f ) or NPM1 ( e ) binding to the P3 region of the CD274 promoter ( n = 3 biologically independent replicates/group). g Luciferase reporter assay of activation of the CD274 promoter ( n = 3 biologically independent replicates/group). h WB analysis of the effect of IGF2 stimulation (50 ng/mL for 24 h) on the indicated protein expression in H226Br cells. i – m B6 mice carrying LLC-Luc ortho were exposed to Veh or NB under HCD condition, either alone or together with intraperitoneal injection of anti-PD-L1 antibody (αPD-L1 Ab , 100 μg in 100 μL/mouse, twice a week). The data is representative of two independent experiments. i Schematic diagram of the experimental schedule. j Representative ex vivo bioluminescence images of analyzed organs k Quantitative analyzes of bioluminescence intensity (BLI) of analyzed organs ( n = 12/group for the Veh/HCD group; n = 11/group for the NB/HCD group; n = 13/group for the NB/HCD/αPD-L1 Ab group). l Microscopic evaluation of H&E-stained lung and liver tissues for tumor multiplicity and burden ( n = 10/group for the Veh/HCD group; n = 9/group for the NB/HCD group; n = 11/group for the NB/HCD/αPD-L1 Ab group). m Kaplan–Meier survival curve of the mice in each group ( n = 12/group). The data are presented as the mean ± SD. p -values were determined by using one-way ANOVA with Tukey’s post-hoc test ( e – g ), Kruskal–Wallis test with Dunn’s post-hoc test ( k , l ), or a log-rank test ( m ). The data shown in a – h are representative of two independent experiments with similar results. Source data are provided as a Source Data file.

Article Snippet: The level of IGF2 in CM was determined by an IGF2 ELISA kit (cat. no. MG200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and normalized by the protein concentration of attached cells remaining after CM collection.

Techniques: Western Blot, Expressing, Incubation, Chromatin Immunoprecipitation, Luciferase, Reporter Assay, Binding Assay, Activation Assay, Injection, Ex Vivo, Staining

Journal: Nature Communications

Article Title: Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression

doi: 10.1038/s41467-024-49199-9

Figure Lengend Snippet: a Representative immunofluorescence (IF) images and quantitative analyzes of the indicated markers in patient-derived lung tissues from non-smokers ( n = 3/group, 5 fields/slide [ n = 15/group]) and smokers ( n = 5/group, 6 fields/slide [ n = 30/group]). Scale bar: 100 μm. b Representative IF staining images of the indicated markers using a tissue microarray (TMA) ( n = 21 for the TNM N0 group; n = 14 for the TNM N1/2 group). Scale bars: 20 μm. c , left Correlation analysis for the Pearson correlation coefficient between IGF2 and GLUT1 levels in macrophages ( n = 35/group). c , right Correlation analysis for the Spearman rank correlation coefficient between nuclear pIGF-1R/IR (Y1131 for IGF-1R, Y1146 for IR) and PD-L1 in tumor cells ( n = 35/group). d Pie charts showing the levels of indicated markers in tumor tissues from patients with or without lymph node metastasis (N0 or N1/2, respectively; n = 21 for the TNM N0 group; n = 14 for the TNM N1/2 group). e Association of the expression level of the indicated markers with cancer stage (left, n = 16 for the stage I group; n = 14 for the stage II group; n = 5 for the stage III group) and tumor grade (right, n = 5 for the grade 1–2 group; n = 20 for the grade 2-3 group; n = 8 for the grade 3 group). f Analysis of a publicly available dataset (GSE30219) to determine the association of GLUT1 or GLUT3 levels with overall and disease-free survival (OS [ n = 136/group] and DFS [ n = 129/group], respectively) in patients with NSCLC. The data are presented as the mean ± SD. p -values were determined by using a two-tailed Student’s t -test ( a , b ), two-tailed Mann-Whitney test ( a ), one-way ANOVA with Tukey’s post-hoc test ( e ), Kruskal–Wallis test with Dunn’s post-hoc test ( e ), or a log-rank test ( f ). Source data are provided as a Source Data file.

Article Snippet: The level of IGF2 in CM was determined by an IGF2 ELISA kit (cat. no. MG200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and normalized by the protein concentration of attached cells remaining after CM collection.

Techniques: Immunofluorescence, Derivative Assay, Staining, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression

doi: 10.1038/s41467-024-49199-9

Figure Lengend Snippet: In a glucose-rich microenvironment caused by systemic NB-induced hyperglycemia, NB increases glucose utilization in macrophages through GLUT1 and GLUT3 transcription and membranous localization upregulation, thereby inducing IGF2 transcription in macrophages. IGF2 stimulates IR in tumor cells in a paracrine manner, resulting in PD-L1 expression upregulation through IR nuclear translocation and a complex formation with NPM1 and Pol II. These events lead to the acquisition of cancer stem cell-like and immune-evasive properties in tumor cells, ultimately mediating metastatic tumor formation.

Article Snippet: The level of IGF2 in CM was determined by an IGF2 ELISA kit (cat. no. MG200, R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions and normalized by the protein concentration of attached cells remaining after CM collection.

Techniques: Expressing, Translocation Assay