Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32β is the most dominant isoform in HepG2+LX-2 spheroids that increases neutral fat content, and IL32 downregulation lowers neutral fat in 2D cultured hepatocytes (A) To test the effect of IL-32 administration on intracellular fat content, immortalized human hepatic cell lines HepG2 and HepaRG were cultured in 2D and incubated with human recombinant IL-32α, IL-32β or IL-32γ isoform for 48 h. Then, intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ, showed increased intracellular neutral fat content in both (A) HepG2 (top) and HepaRG (bottom). (B) To test the effect of IL32 downregulation on intracellular fat content, 24 h after seeding, cells were transfected with scramble or IL32 siRNA and grown in regular medium without FBS (HepG2) or medium supplemented with 25 μM oleic acid (HepaRG) for an additional 48 h. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method. Intracellular neutral fat content was visualized by Oil Red O staining (ORO). ORO area quantified per DAPI stained nuclei by ImageJ showed lower intracellular neutral fat content in both HepG2 (left) and HepaRG (right). Two-sided p values were calculated by the Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. (C) HepG2+LX-2 cells were cultured as spheroids for 96 h exposing them to medium supplemented with 1% BSA or to increasing concentrations of a mixture of fatty acids (PA + OA, 1:2). IL32 gene expression measured after exposure, demonstrated higher IL32 mRNA levels with increasing intra-spheroidal triglyceride levels. The p value was calculated by test for linear trend. Data shown as mean ± SD for the reported number of experiments. (D) Percentage of IL32 gene products versus other IL-32 isoforms was measured from RNA sequencing (RNA-seq) data and IL-32β is the most dominantly expressed IL32 isoform in our spheroid model. The p value was calculated by one-way ANOVA. OA, oleic acid; PA, palmitic acid; RU, relative unit.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Recombinant, Staining, Transfection, Knockdown, MANN-WHITNEY, Gene Expression, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Incubation with human recombinant IL-32β increases while IL32 downregulation lowers intracellular triglyceride content in spheroids from immortalized and human primary hepatocytes (A) HepG2+LX-2 cells were cultured as spheroids for a total of 96 h. Initially, 48 h after seeding cells the media was supplemented with 25 nM IL-32β for 48 h. (B) HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation for total of 96 h. (C) Primary human hepatocytes (PHH) were cultured as spheroids for a total of 7 days. Initially, 48 h after seeding the media was supplemented with 25 nM IL-32β for an additional 5 days with media replacement every 48 h. (D) PHH were cultured as spheroids along with negative control (SCR) siRNA or 30 nM IL32 siRNA transfection mix for downregulation, for a total of 7 days. For both spheroid models, cellular ATP levels (marker of viability) remained stable between the experimental groups. The average of gene knockdown efficiency was ∼70%–75% as evaluated by real-time qPCR analyzed by the 2 −ΔΔCt method, relative to beta-actin. Intracellular neutral fat content measured by Oil Red O staining and AdipoRed assay showed an increase in triglycerides content after incubation with IL-32β while IL32 downregulation lowers triglyceride levels. Two-sided p values were calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD in all groups for the reported number of experiments. RFU, relative fluorescence unit; RU, relative unit (to beta-actin); SCR, scramble siRNA; untr, untreated.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Cell Culture, Generated, Negative Control, Transfection, Marker, Knockdown, Staining, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL32 downregulation lowers intracellular COL1A1, increases MMP2 levels, and lowers TIMP2 in primary di-lineage human spheroids Primary human hepatocytes and primary hepatic stellate cells, at the ratio 24:1, were seeded with negative control scramble (SCR) and IL32 siRNA, at 5,000 cells/well in ultra-low attachment 96-well U-bottom ultra-low attachment plates. Fifty percent of the total media was replenished with fresh media every 48 h. (A) After 7 days of formation, spheroids were collected and 8-μM sections were subjected to immunofluorescent staining for COL1A1. Immunofluorescence was quantified by ImageJ, normalized to number of DAPI stained nuclei. The knockdown efficiency was measured by real-time qPCR, relative to beta-actin. (B) MMP2, TIMP-1, TIMP-2, and α-SMA protein levels were measured by western blotting in the cell lysate. Calnexin was used as loading control. Representative images of protein levels are shown. For each panel, data shown as mean ± SD of the reported independent experiments. Two-sided p values were calculated by Mann-Whitney non-parametric test. COL1A1, collagen Iα1; MMP2, matrix metallopeptidase 2; TIMP1, tissue inhibitor of metalloproteinase 1; TIMP2, tissue inhibitor of metalloproteinase 2.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Negative Control, Staining, Immunofluorescence, Knockdown, Western Blot, Control, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Endogenous IL32 downregulation lowers, and incubation with recombinant IL-32 β increases, intracellular triglycerides synthesis For endogenous IL32 downregulation experiments, HepG2+LX-2 spheroids were generated by seeding cells along with negative control (SCR) siRNA or IL32 siRNA transfection mix for a total of 96 h. For IL-32β incubation experiments, initially, 48 h after seeding HepG2+LX2 cells (24:1), the media was supplemented with 25 nM IL-32β for another 48 h. In both conditions, newly synthesized triglycerides were separated by TLC and quantified by scintillation counting after incubation with 6 μCi/mL 3 H-glycerol plus 1.5 mM glycerol for 12 h. (A) Reduction in de novo triglyceride synthesis after IL-32 downregulation. (D) Increase in de novo triglyceride synthesis after 25 nM IL-32β incubation. Cells were incubated with 8.5 μCi/mL 3 H-palmitate +55 μM/L palmitic acid for 6 h, after which palmitate was precipitated with BSA and perchloric acid and quantified by scintillation counting. (B and E) Graph shows no difference in beta oxidation in both experimental groups. APOB-100 synthesis and secretion levels were measured by immunoblotting. (C) Decrease in APOB-100 in cell lysate and cell culture supernatant after IL-32 downregulation. (F) No changes in APOB-100 levels in cell lysate and culture medium, after incubation with 25 nM IL-32β. The reported number of experiments were performed independently. Representative blots are presented. For all experiments, two-sided p value was calculated by Mann-Whitney non-parametric t test. Data shown as mean ± SD. AU, arbitrary units; DPM, disintegrations per minute; TAG, triacylglycerol.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Incubation, Recombinant, Generated, Negative Control, Transfection, Synthesized, Western Blot, Cell Culture, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Differentially expressed genes reveals downregulation of key genes for lipid metabolism in HepG2+LX2 spheroids (A) Key genes of lipid metabolism differentially expressed in spheroids after IL32 downregulation as compared with scramble. (B) Top 100 differentially expressed genes after IL32 downregulation as compared with scramble. Data are presented as log2-fold change in expression and –log 10 of p values adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate (FDR). VLDL, very low-density lipoprotein.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: Co-downregulation of PLA2G2A and IL32 abolished the IL-32-mediated intracellular triglycerides lowering and IL32 downregulation reduces intracellular PI levels in human primary spheroids Primary human hepatocytes were cultured as spheroids and incubated with negative control (SCR) siRNA or 30 nM IL32 , and combination of IL32 and PLA2G2A for a total of 7 days. (A) Cellular ATP levels (marker of viability) were not different within the three groups. There was an 80%^–90% reduction in mRNA levels of IL32 and PLA2G2A , relative to beta-actin. Intracellular neutral lipid content (measured by Oil Red O staining) normalized to nuclei (stained by DAPI) were lower after IL32 downregulation while the co-downregulation of PLA2G2A and IL32 abolished this reduction. (B) IL32 downregulation results in higher PLA2G2A mRNA levels measured by real-time PCR. (C) IL32 and PLA2G2A were downregulated individually or in combination in primary hepatocyte spheroids and PLA2G2A levels were measured by human PLA2G2A ELISA in the culture medium. PLA2G2A levels were higher after IL32 downregulation and lower after co-downregulation of IL32 and PLA2G2A . (D) After 2 days from seeding, hepatocyte spheroids were incubated with 10, 25, and 50 nM human recombinant IL-32β for 5 days. PLA2G2A was measured from cell culture supernatant using human PLA2G2A ELISA and we observed a dose dependent decrease in secreted PLA2G2A levels with increasing concentration of IL-32β. The p values were calculated by test for linear trend. (E) Lipid fingerprint measured by liquid chromatography- quadrupole time-of-flight-mass spectrometry demonstrated lower total PI and triglycerides (top) levels after IL32 downregulation. There was a reduction in all PI species, except 40:5 and 40:6, with the largest effect size in 38:4 (bottom). For each part, data are shown as mean ± SD of the reported independent experiments. Two-sided p values calculated with unpaired t test for n = 3 and Mann-Whitney non-parametric t test for n > 3. Cer, ceramides; CL, cardiolipins; DAG, di-acylglycerides; GalCer, galactosyl ceramides; LPC, lysophosphatidylcholine; ORO, Oil Red O; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; RFU, relative fluorescence units; RU, relative units; PC, phosphatidylcholine; SM, sphingomyelin; TAG, triacylglycerols.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Cell Culture, Incubation, Negative Control, Marker, Staining, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Recombinant, Concentration Assay, Liquid Chromatography, Mass Spectrometry, MANN-WHITNEY, Fluorescence

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet: IL-32 rs76580947 minor allele associates with lower IL-32 expression, severe liver steatosis, and lower liver non-invasive tests (A) Regional plots of association between IL32 common genetic variants (minor allele frequency of >0.01) and ALT in the European subset of UK biobank. The x axis shows the variant positions (GRCh37); the y axis shows the –log10 p values. The gray diamond represents rs76580947, with the strongest association in the plotted region (IL-32 ± 50 Kbp), for which its pairwise LD with other variants is color coded as shown on the figure. (B) The association between rs76580947 and hepatic IL-32 mRNA levels was tested in 207 individuals from the MAFALDA cohort adjusting for age, gender, percentage of coding bases (a quality control measure from the Picard toolkit), RNA Integrity Number (RIN), and five surrogate variables detected by surrogate variable analysis. Carriers of the variant have lower IL-32 mRNA levels. Data shown as violin plots and adjusted p values are reported. (C) The association between IL32 rs76580947 stratified by genotype and IL-32 plasma protein level in 365,495 European participants from UK Biobank was tested using a linear regression analysis adjusted for age, gender, body mass index, first 10 genomic principal components, and array batch. Violin plot shows the normalized Protein eXpression (NPX) values that were rank-based inverse normal transformed prior to the analysis. (D) Forest plot of association and meta-analysis for IL32 rs76580947 with steatosis in three independent cohorts: Southern Italy (N = 425), Central Italy (N = 245), and Finnish (N = 745). The plot shows protection against severe liver steatosis (steatosis absence or mild vs. severe; fixed-effect p = 0.027). The association was tested by a binary logistic regression analysis under an additive genetic model adjusted by age, gender, body mass index, and recruitment center (only for the Finnish cohort). Pooled effect estimates were calculated using inverse-variance-weighted fixed effects meta-analysis. (E) The association between IL32 rs76580947 and clinical liver fibrosis scores and APOB levels in 365,495 European participants from UK Biobank. The analysis was performed under an additive model, using linear regression adjusting for age, gender, body mass index, the first 10 genomic principal components, and array batch. All traits were rank-based inverse normal transformed prior to the analysis. CI, confidence interval.

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Expressing, Variant Assay, Control, Clinical Proteomics, Transformation Assay

Journal: Cell Reports Medicine

Article Title: IL32 downregulation lowers triglycerides and type I collagen in di-lineage human primary liver organoids

doi: 10.1016/j.xcrm.2023.101352

Figure Lengend Snippet:

Article Snippet: After 24 h of seeding, HepG2 and HepaRG cells were exposed to human recombinant IL32α (3040-IL; R&D Systems), IL32β (6769-IL; R&D Systems) or IL32γ (4690-IL/CF; R&D Systems) for 48 h. For HepG2+LX-2 spheroids, 48 h after seeding 25 ng/mL IL32β was supplemented along with 100μL fresh medium for an additional 48 h to make a total of 200μL.

Techniques: Recombinant, Protein Extraction, Staining, Western Blot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Transfection, Gene Expression, Fluorescence, Software, Cell Counting

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: Msx2 is a downstream target of BMP2 signaling in the uterus during decidualization. (A) The primary cultures of mouse endometrial stromal cells (MESCs) were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points, as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of Msx2. The relative levels of gene expression were determined by setting the expression level of the GFP-treated sample at 24 h to 1.0 (n = 3). Rplp0, encoding a ribosomal protein, was used to normalize the level of RNA. *P < 0.05. (B) The nucleotide positions of the SBEs on the Msx2 promoter were analyzed by ChIP. (C) Mouse stromal cells were treated with E + P or E, P, and BMP2 (E + P + BMP2) for 90 min. ChIP, using the Smad4 antibody, was performed, as described in “Materials and Methods.” Chromatin enrichment was quantified by real-time PCR using primers flanking the potential SBE in the Msx2 promoter and also a negative control region in the ORF of Msx2. Enrichments were normalized to 1% of input DNA. The experiment was repeated twice, and representative data are shown.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression, Negative Control

Journal: Endocrinology

Article Title: Msx Homeobox Genes Act Downstream of BMP2 to Regulate Endometrial Decidualization in Mice and in Humans

doi: 10.1210/en.2019-00131

Figure Lengend Snippet: MSX1 and MSX2 mediate BMP2-induced HESC decidualization. The primary cultures of HESCs were established as described in “Materials and Methods.” The cells were transduced with adenovirus expressing GFP or BMP2. The cells were lysed at different time points as indicated. Total RNA was isolated, and real-time PCR was performed to analyze the levels of MSX1 and MSX2. The relative levels of gene expression were determined by setting the expression level on day 0 of the GFP-treated sample at 1.0. RPLP0, encoding a ribosomal protein, was used to normalize the level of RNA. Data were collected from three independent clinical samples, which were subjected to the same experimental conditions. *P < 0.05; **P < 0.005.

Article Snippet: The next day, the cells were either treated with E + P or E + P + BMP2 (recombinant human BMP2; R&D Systems; 355-BM-010) for 90 minutes.

Techniques: Transduction, Expressing, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT 5A in different tissue regions. (A and B) Nuclear expression of WNT5A in oral mucosa with normal appearance. (C and D) Cytoplasmic and nuclear expression of WNT5A in severe‐grade dysplasia (black arrows). (E and F) Cancer islands; black arrows indicate expression of WNT5A in the cytoplasm in the periphery of cancer islands and red arrows indicate the absence of expression of WNT5A in the central part of the cancer islands. Scale bar = 50 μ m.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Effect of recombinant WNT 5A ( rWNT 5A) on expression of β ‐catenin and E‐cadherin in SCC 9 and SCC 25. (A, E) Representative western blots of expression of active β ‐catenin, total β ‐catenin, and E‐cadherin after stimulation with rWNT 5A. (B, F) Quantification of relative E‐cadherin; (C, G) quantification of active β ‐catenin; (D, H) quantification of total β ‐catenin. All quantifications were performed on four separate experiments.

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Recombinant, Expressing, Western Blot

Journal: European Journal of Oral Sciences

Article Title: Higher expression of WNT 5A protein in oral squamous cell carcinoma compared with dysplasia and oral mucosa with a normal appearance

doi: 10.1111/eos.12352

Figure Lengend Snippet: Expression of WNT5A, β ‐catenin, and E‐cadherin in oral mucosa with a normal appearance, dysplasia, and at the invasive front of oral squamous cell carcinoma (OSCC)

Article Snippet: The cells were cultured in six‐well plates until 80% confluence was reached, rinsed with PBS, incubated with serum‐free medium overnight, rinsed again with PBS, and treated either with 0.1% BSA in PBS as the control or with 0.4 μ g/ml of recombinant WNT5A (rWNT5A; 645‐WN; R&D Systems, Minneapolis, MN, USA) in serum‐free medium.

Techniques: Expressing, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 THP-1 macrophages were treated with 15 mM lactate for 24 h, and the mRNA levels of chemokines were measured by quantitative PCR. The growth medium of control macrophages was titrated to pH6.1 using sterile HCl. (B) 3×10 5 THP-1 macrophages were incubated with different concentrations of lactate for 24 h, and CCL5 gene expression was determined with quantitative PCR. (C) 10 6 THP-1 macrophages were exposed to increasing concentrations of lactate for 48 h, and the secretion of CCL5 was measured by ELISA. (D) 10 6 human primary macrophages from breast cancer patients (n=9) were cultured with different concentrations of lactate for 48 h, and CCL5 production was detected. (E) 10 6 MDA-MB-231 cells were pre-treated with 15μM GSK 2837808A for 2 h, then the media were changed, and cells were cultured for another 24 h. The conditional media (MD-231 CM) were collected and applied to 10 6 THP-1 macrophages. CCL5 concentrations were detected with ELISA. (F) Immunohistochemical staining of CD68 and CCL5 in tumor adjacent tissues (control) and breast tumors (n=28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Real-time Polymerase Chain Reaction, Control, Sterility, Incubation, Gene Expression, Enzyme-linked Immunosorbent Assay, Cell Culture, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 48 h, and the expression of key regulators in Notch, NF-κB, STAT3 and HIF, were detected by western blot. (B) 3×10 5 THP-1 macrophages were cultured with 15 mM lactate for 24 h, and the mRNA levels of Notch ligands and receptors were measured by quantitative PCR. (C) Western blot for Notch ligands and receptors in THP-1 (10 6 ) macrophages after 48 h lactate treatment. (D) Lactate stimulated the expression of NICD in a time and dose-dependent manner. 10 6 THP-1 macrophages were treated with 15 mM lactic acid for 48 h. Data presented were representatives of at least three independent experiments. (E) 10 6 THP-1 macrophages were transfected with 50nM siNotch1, or pretreated with 50μM DAPT for 2 h, and then cultured with 15 mM lactate for 48 h. The secretion of CCL5 was measured by ELISA. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 10 6 THP-1 macrophages were treated with 15 mM lactate for 72 h, and then cells were washed twice and fresh media were added. Macrophages were cultured for another 24 h and the conditional media (lactate CM) was collected. The effect of CM on breast cancer cell migration was measured by double chamber transwell assay. 5μg/ml anti-CCL5 neutralizing antibody significantly decreased lactate CM-induced cell migration. (B) 10 6 MCF-7 cells were co-cultured with 15 mM lactate-activated macrophages in the presence of 5μg/ml anti-CCL5 antibody or not, and protein levels of EMT markers were tested by western blot. (C) 10 6 breast cancer cells were co-cultured with 10 6 lactate-activated THP-1 macrophages (or 10 6 lactate-activated primary macrophages) for different time points, and the expression of CCR5 was monitored by western blot. (D) MDA-MB-231 and MCF-7 cells were transfected with shCCR5 plasmids, or pre-treated with 5μM Maraviroc for 2 h, then cell migration induced by lactate CM was detected by double chamber transwell assay. Lactate CM was described in (A). (E) MCF-7 cells (10 6 ) were transfected with pcDNA3.1-CCR5, and then cultured with 10ng/ml CCL5 for 24 h. The expression of E-cadherin, N-cadherin and vimentin was investigated by western blot. (F) 10 6 Human primary macrophages (No. 4 and No. 9) were treated with 15 mM lactate for 72 h and CM was collected as described in (A). The migration of MDA-MB-231 cells was measured in the presence of primary macrophage CM. 5μg/ml anti-CCL5 neutralizing antibody, shRNAs designed against CCR5, or 5μM Maraviroc, significantly reduced primary macrophage CM-induced cell migration. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Migration, Transwell Assay, Western Blot, Expressing, Transfection

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) 3×10 5 MDA-MB-231 and MCF-7 cells were stimulated with 1-5ng/ml TGF-β1 for 24 h, and total RNA was isolated and tested for CCR5 mRNA by quantitative PCR. (B) Western blot for CCR5 protein in breast cancer cells (10 6 ) under TGF-β1 stimulation for 48 h. Data presented were representatives of at least three independent experiments. (C) MDA-MB-231 and MCF-7 cells (3×10 5 ) were co-transfected with pGL3-CCR5 and pRL-TK and exposed to different concentrations of TGF-β1 for 24 h, and luciferase activities were determined. (D) MDA-MB-231 and MCF-7 cells were pre-treated with 5μM SIS3 for 2 h, and cells were subjected to luciferase assay. (E) 10 6 MCF-7 cells were transfected with TGFβRI/ALK5 siRNA, and were then co-cultured with lactate-activated THP-1 macrophages (ratio 1:1) for 24 h. The protein levels of CCR5 were assayed by western blot. (F) The expression of TGF-β1, CCL5 and CCR5 in clinical samples obtained from breast cancer patients. The mRNA levels were measured by quantitative PCR, and the correlation between TGF-β1 and CCL5-CCR5 axis was shown. (G) Representative IHC staining for TGF-β1, CCL5 and CCR5 in breast cancer samples. The sample used was derived from 28 breast cancer cases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Luciferase, Cell Culture, Expressing, Immunohistochemistry, Derivative Assay

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Glucose uptake, lactic acid production and ATP levels in breast cancer cells co-cultured with lactate-activated THP-1 macrophages, with or without 5μg/ml anti-CCL5 neutralizing antibody. The co-culture system was described in Figure . (B) Western blots for glycolytic enzymes in breast cancer cells treated as in (A). (C) MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, and then subjected to cell co-culture. Glucose uptake, lactic acid production and ATP levels were measured after co-culture. The co-culture system was described in Figure . (D) The protein levels of HK2, PKM2 and LDHA in MDA-MB-231 cells cultured as in (C). (E) Recombinant human CCL5 induced aerobic glycolysis in breast cancer cells. MDA-MB-231 and MCF-7/CCR5 cells were treated with increasing concentrations of CCL5 for 12 h, and glucose uptake, lactic acid production and ATP levels were detected. (F) Western blots for glycolytic enzymes in MDA-MB-231 and MCF-7/CCR5 cells after stimulation with CCL5. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Co-Culture Assay, Western Blot, Transfection, Recombinant

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) Western blot for AMPK, c-Myc, HIF-1α and Akt in breast cancer cells co-cultured with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 72 h. Results presented were representatives of at least three independent experiments. (B) The expression of AMPK downstream signaling target ACC in breast cancer cells co-cultured as in (A). (C) MDA-MB-231 and MCF-7 cells were transfected with 50 nM AMPKα1 siRNA, or pretreated with 10μM compound C for 4 h, and then incubated with 15mM lactic acid-activated THP-1 macrophages (ratio 1:1) for 48 h. The glucose uptake, lactic acid production and ATP levels were detected. (D) The inhibition of AMPK abrogated macrophage-induced EMT in MCF-7 cells. Cells were treated as described in (C). After co-culture, the expression of EMT markers, E-cadherin and vimentin, was measured by western blot. (E) Recombinant human CCL5 induced the phosphorylation of AMPK in MDA-MB-231 and MCF-7/CCR5 cells. 10 6 cells were treated with 50ng/ml CCL5 for defferent time points as indicated, and phosphorylated AMPK and total AMPK were investigated by western blot. (F) Inhibition of CCR5 in MDA-MB-231 cells significantly attenuated macrophage-induced AMPK phosphorylation. MDA-MB-231 cells were transfected with shRNAs designed against CCR5, or pre-treated with 5μM Maraviroc for 2 h, then co-cultured with 15 mM lactate-activated macrophages as described in (A). After co-culture, the phosphorylation of AMPK was detected by western blot. (G) Expressions of CCL5, CCR5 and p-AMPK in samples obtained from breast cancer patients (n =28). Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Western Blot, Cell Culture, Expressing, Transfection, Incubation, Inhibition, Co-Culture Assay, Recombinant, Phospho-proteomics

Journal: Oncotarget

Article Title: Lactate-activated macrophages induced aerobic glycolysis and epithelial-mesenchymal transition in breast cancer by regulation of CCL5-CCR5 axis: a positive metabolic feedback loop

doi: 10.18632/oncotarget.22786

Figure Lengend Snippet: (A) MDA-MB-231 cells were co-cultured with 15 mM lactate-activated THP-1 macrophages for 7 days, in the presence of 5μg/ml anti-CCL5 neutralizing antibody or not. MDA-MB-231 cells were then collected and injected into the tail vein of nude mice. After two weeks, animals were sacrificed and metastatic nodules on lung surfaces were counted. (B) CCR5, HK2 and p-AMPK were immunostained in MDA-MB-231 metastases. Scale bars represent 50 μm. * , P<0.05; ** , P<0.01.

Article Snippet: CCL5 (DRN00B), TGF-β1 (DB100B) ELISA kits and recombinant human CCL5 (278-RN/CF) and TGF-β1 (240-B) were bought from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Injection