recombinant human (rh) il-1β 200-01b  (PeproTech)

Journal: bioRxiv

Article Title: Fructose utilization by GM-CSF-differentiated macrophages aggravates autoimmune inflammation via MG-derived AGE–RAGE signaling

doi: 10.64898/2026.01.26.701899

Figure Lengend Snippet: A. GM-CSF human macrophages were stimulated with LPS (50 ng/mL) for 18 h in glucose-, fructose- (10 mM), or sugar-free media. IL-1β, IL-6, and IL-23 levels were quantified by ELISA. For IL-1β detection, 4 mM ATP was added during the final 2 h (n = 6). B. IL1B mRNA expression was analyzed by RT-qPCR under identical conditions (n = 5). C. Immunoblotting of cell lysates and supernatants from (A) was performed to assess pro- and mature (m) forms of IL-1β and caspase-1 (Casp-1). Band intensities were quantified by densitometry and normalized to β-actin (n = 5). D–E. Glycolytic activity was assessed by Seahorse extracellular flux analysis of ECAR following sequential injection of glucose, fructose (10 mM), or no sugar, followed by oligomycin (2 µM) and 2-DG (50 mM). Data represents five technical replicates (n = 3). F. HIF-1α protein and mRNA levels were measured in LPS-stimulated GM-CSF macrophages under indicated sugar conditions (n = 3–4). G–J. SKG mice were injected with curdlan to induce arthritis and administered fructose in drinking water starting 3 days prior to immunization. Glucose levels in serum and synovial fluid (SF) were assessed at day 42 (n = 5–8). H–I. mRNA levels of Hif1a and HIF-1α target genes in synovial cells were analyzed by RT-qPCR (n = 5). J. Intracellular HIF-1α expression was measured by flow cytometry in CD45⁺CD11b⁺F4/80⁺ synovial cells (n = 9–10). Graphs represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 by Mann–Whitney U test (A–C, E–J) or unpaired t-test (D, F).

Article Snippet: Cells were plated in 96-well U-bottom plates and stimulated with anti-CD3/CD28-coated beads for 7 days under the following cytokine conditions: recombinant human (rh) IL-1β (30 ng/mL), rhIL-6 (30 ng/mL), rhIL-23 (10 ng/mL), and rhTGF-β (10 ng/mL) (all from R&D Systems, Minneapolis, MN) for Th17 polarization, rhIL-2 (100 IU/mL; PeproTech) and rhIL-12 (10 ng/mL; R&D Systems) for Th1 polarization, and rhIL-2 (100 IU/mL) alone for non-polarizing control.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Injection, Flow Cytometry, MANN-WHITNEY

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: Lysosomotropic beta-blocker induce IL23A release by activated MoLCs independent of beta adrenoceptors. (A) IL23A secretion was analyzed by ELISA in the supernatant of MoLCs and MoDCs stimulated with IL1B (20 ng/ml) for 24 h in the presence or absence of propranolol at different concentrations (25–150 µM). (B) Cell viability was assessed by ANXA5-FITC/PI-double-staining using flow cytometry. (C) IL23A release by MoLCs and MoDCs, quantified after stimulation with SQ 22,536 (100 µM) or forskolin (20 µM) and followed by activation with IL1B (20 ng/ml) for 24 h in the presence or absence of propranolol (75 µM). (D-G) IL23A production by MoLCs and MoDCs after stimulation with IL1B (20 ng/ml) for 24 h in the presence or absence of timolol, metoprolol, practolol or penbutolol at different concentrations (1–150 µM) was assayed by ELISA. (H) mRNA expression of IL12B and IL23A in MoLCs and MoDCs, respectively, was assessed after 24 h of stimulation with IL1B (20 ng/ml) in the presence or absence of propranolol (75 µM). Gene expression results were normalized to GAPDH and depicted relative to unstimulated MoLCs and MoDCs (set as 1.0). Propranolol does not directly polarize naïve CD4+ T cells toward Th17 development. (I) Gene expression levels of RORC and IL17A were examined in naïve CD4+ T cells, polyclonally stimulated with anti-CD3-CD28 antibodies for 5 d with or without propranolol (75 µM) or chloroquine (20 µM). Levels of mRNA were normalized to GAPDH and presented relative to untreated CD4+ T cells (set as 1.0). (J) Naïve CD4+ T cells were stimulated with anti-CD3-CD28 antibodies for 7 d in presence or absence of propranolol (75 µM) or chloroquine (20 µM), respectively. Th17 signature cytokines IL17A and IL22 in cell culture supernatants were quantified by ELISA. (A-G) *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test followed by Bonferroni posttest, (H and I) one-sample t-test. Data are representative of (A) n = 4–5, (B) n = 3–6, (C) n = 3–5, (D-J) n = 3 independent experiments and display mean values ± SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Enzyme-linked Immunosorbent Assay, Double Staining, Flow Cytometry, Activation Assay, Expressing, Cell Culture

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: Propranolol induces production of psoriasis-like inflammation associated mediators under sterile inflammatory conditions. Indicated cytokine secretion by immature and IL1B-activated (20 ng/ml) DC subsets after 24 h in the presence or absence of propranolol (75 µM) was assessed using ELISA. *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test followed by Bonferroni posttest. Data are representative of 3–4 independent experiments and display mean values + SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: Propranolol inhibits autophagic flux and impairs endosomal maturation in MoLCs and MoDCs. (A and D) Immunoblot analysis of total LC3B-I, LC3B-II, SQSTM1, and LAMP1 expression in whole-cell lysates of MoLCs and MoDCs, stimulated with IL1B (20 ng/ml) for 24 h with or without propranolol (75 µM). (B) Immunostaining of IL1B-activated MoLCs for TRAF6 and LC3A after 24 h of stimulation with propranolol (75 µM). Scale bar represents 10 µm. (C) Acidification of intracellular compartments was examined by flow cytometry of MoLCs, incubated with propranolol (75 µM), chloroquine (20 µM) or bafilomycin A1 (1 µM) for 0.5 h or 4 h, respectively. Cells were pre-incubated for 0.5 h in medium supplemented with acidotropic LysoTracker Red DND-99 (100 nM). pH indicator-specific detection of mean fluorescence intensity (MFI) was quantified and depicted relative to untreated controls (assigned as 1.0). (E) Co-localization of LC3A and EGFR with LAMP1 was analyzed by immunofluorescence microscopy in MoLCs and MoDCs after 24 h of stimulation with propranolol (75 µM). Scale bar represents 25 µm. (F) Immunoblot analysis of whole-cell lysates from MoLCs probed with anti-Ub for total ubiquitin-conjugated constituents, stimulated with IL1B (20 ng/ml) for 24 h with or without propranolol (75 µM) or MG132 (10 µM). (G) IL23A release was analyzed by ELISA in the supernatant of IL1B-stimulated MoLCs (20 ng/ml) for 24 h in the presence or absence of propranolol (75 µM) or MG132 (10 µM). (A, D and F) Protein expression was quantified by densitometric analysis with ACTB/β-actin serving as control. (A, F and G) *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test followed by Bonferroni posttest, (C) unpaired two-tailed t-test. Data are representative of n = 3–4 independent experiments and display mean values ± SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Western Blot, Expressing, Immunostaining, Flow Cytometry, Incubation, Fluorescence, Immunofluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: NLRP3 inflammasome activity is dispensable for propranolol-mediated induction of IL23. (A) Immunoblot analysis of cell pellets from MoLCs probed with anti-NLRP3, after 24 h activation with IL1B (20 ng/ml) in the presence or absence of propranolol (75 µM) or MG132 (10 µM). (B) Quantification of IL1B mRNA in immature and IL1B-activated (20 ng/ml) DC subsets stimulated for 3 h with or without propranolol (75 µM). Gene transcripts were normalized to GAPDH and presented relative to unstimulated controls (set as 1.0). (C and D) ELISA of IL23A released by MoLCs and MoDCs activated with IL1B (20 ng/ml) or curdlan (20 µg/ml), respectively, and stimulated with or without propranolol (75 µM) and NLRP3 inflammasome inhibitor MCC950 (5 µM) for 24 h. (E) Immunoblot analysis of IL1B in whole-cell lysates from MoLCs obtained after 24 h of stimulation with IL1B (20 ng/ml) with or without propranolol (75 µM) or MG132 (10 µM). (A and E) Protein expression was normalized to ACTB and quantified by densitometry. (A and E) *P < 0.05, **P < 0.01, one-way ANOVA test followed by Bonferroni posttest, (B) one-sample t-test. Data are representative of (A) n = 4, (B-E) n = 3 independent experiments and display mean values + SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Activity Assay, Western Blot, Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: Propranolol-induced inhibition of autophagic flux is accompanied by oxidative stress and abundance of ROS-producing mitochondria in MoLCs. (A and B) qRT-PCR quantification of ATF3 and ATF6 copy numbers in immature and IL1B-activated (20 ng/ml) DC subsets stimulated for 3 h with or without propranolol (75 µM). Levels of mRNA were normalized to GAPDH and presented relative to untreated DCs (set as 1.0). (C) Flow cytometry analysis of total intracellular ROS formation in IL1B-activated DCs, pre-incubated with ROS assay stain for 1 h and subsequently stimulated with or without propranolol (75 µM) and N-acetyl-L-cysteine (20 mM) for 1 h and 3 h, respectively. H2O2 (200 µM) was used as a positive control. ROS quantification is presented relative to unstimulated controls (set as 1.0). (D) MoLCs stimulated with IL1B (20 ng/ml) were stimulated with or without propranolol (75 µM) and NAC (20 mM). Levels of IL23A were detected by ELISA. (E) Flow cytometry analysis of total intracellular ROS formation in naïve CD4+ T cells, pre-incubated with ROS assay stain for 1 h and followed by stimulation with or without propranolol (75 µM) and N-acetyl-L-cysteine (20 mM) for 1 h and 3 h, respectively. H2O2 (200 µM) served as positive control. ROS levels were displayed relative to untreated controls (set as 1.0). (F) Detection of mitochondrial-generated ROS was examined by flow cytometry and mean fluorescence intensity (MFI) was quantified in immature and IL1B-stimulated MoLCs, pre-loaded with MitoSOX (5 µM) for 10 min and subsequently cultivated for 24 h in the presence or absence of propranolol (75 µM) or bafilomycin A1 (1 µM). Detected ROS were depicted relative to unstimulated MoLCs (set as 1.0). (G) Copy numbers of mitochondrial DNA (hmito3) in unstimulated and IL1B-stimulated (20 ng/ml) with or without propranolol (75 µM), chloroquine (20 µM) or bafilomycin A1 (1 µM) were assayed after 48 h. mtDNA was normalized to ALDOA, used as a loading control for genomic DNA and displayed relative to IL1B-activated cells (set as 1.0). (H) Immunoblot analysis of PINK1 and PRKN in whole-cell lysates from MoLCs obtained after 24 h of stimulation with IL1B (20 ng/ml) with or without propranolol (75 µM) or MitoTEMPO (20 µM). Protein expression was evaluated by densitometric analysis with ACTB/β-actin assisting as control. (I) ELISA of IL23A levels collected by MoLCs activated with IL1B (20 ng/ml) and stimulated with or without propranolol (75 µM) and MitoTEMPO (20 µM) for 24 h. (A-C, F and G) *P < 0.05, one-sample t-test. (D, H and I) *P < 0.05, **P < 0.01, ***P < 0.001, one-way ANOVA test followed by Bonferroni posttest. Data are representative of (A, D and I) n = 3, (B, C, E, F and H) n = 4, (G) n = 3–6 independent experiments and display mean values + SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Inhibition, Quantitative RT-PCR, Flow Cytometry, Incubation, ROS Assay, Staining, Positive Control, Enzyme-linked Immunosorbent Assay, Generated, Fluorescence, Western Blot, Expressing

Journal: Autophagy

Article Title: Lysosomotropic beta blockers induce oxidative stress and IL23A production in Langerhans cells

doi: 10.1080/15548627.2019.1686728

Figure Lengend Snippet: MAPK14 activity crucially regulates IL23A release. (A) IL23A secretion was analyzed by ELISA of IL1B-stimulated (20 ng/ml) MoLCs, stimulated for 24 h with or without propranolol (75 µM) in the presence or absence of a selective MAPK1-MAPK3 inhibitor (U0126, 10 µM), inhibitor of MAPK14 (SB 202,190, 10 µM) and MAPK8 inhibitor (SP 600,125, 10 µM). (B) Analysis of IL12B and IL23A mRNA expression in IL1B-activated (20 ng/ml) and propranolol-stimulated (75 µM) MoLCs with or without U0126 (10 µM), SB 202,190 (10 µM) or SP 600,125 (10 µM), respectively after 24 h. Gene transcripts were normalized to GAPDH and depicted relative to IL1B-activated MoLCs concomitantly treated with propranolol (set as 1.0). (C) IL23A release by immature and IL1B-activated (20 ng/ml) DC subsets after 24 h in presence or absence of propranolol (75 µM) and NFKB inhibitor Bay 11–7082 (10 µM) was determined by ELISA. (D) Levels of IL12B and IL23A mRNA expression in MoLCs and MoDCs, stimulated with IL1B (20 ng/ml) for 24 h with or without propranolol (75 µM) and Bay 11–7082 (10 µM). Gene copy numbers were normalized to GAPDH and presented relative to respective IL1B-stimulated DCs (set as 1.0). (E and F) Analysis of NFKB1 and NFKB2 as well as RELA, RELB and REL mRNA expression in MoLCs and MoDCs, activated with IL1B (20 ng/ml) alone or with propranolol (75 µM) for 3 h. Levels of mRNA were normalized to GAPDH and displayed relative to untreated controls (set as 1.0). (G) Binding of RELA, RELB and REL to promoters of IL12B, IL23A and TNF in MoLCs stimulated for 4 h with IL1B (20 ng/ml) in presence or absence of propranolol (75 µM) and inhibitor of MAPK14 (SB 202,190, 10 µM), respectively, assayed by ChIP and subsequently quantified by qRT-PCR. Data are presented relative to total input DNA (GAPDH). (H) Immunoblot analysis of total NFKB2 p100, NFKB2 p-NFKB2 p100, and NFKB2 p52 expression in whole-cell lysates of MoLCs, stimulated with IL1B (20 ng/ml) for 24 h with or without propranolol (75 µM) and MitoTEMPO (20 µM). (H) Protein expression was assessed by densitometric analysis with ACTB as control. (A-D, G and H) *P < 0.05, **P < 0.01, one-way ANOVA test followed by Bonferroni posttest. Data are representative of (A) n = 5, (B and H) n = 4, (C) n = 3, (D-F) n = 3–4, (G) n = 5–7 independent experiments and display mean values + SEM.

Article Snippet: For subsequent stimulation, the following agonists were applied to the cell culture medium: rh-IL1B/IL-1β (20 ng/ml; eBioscience, 14–8018), rh-IL36G (100 ng/ml; PeproTech, 200-36G), ultrapure LPS from Escherichia coli serotype 0111:B4 (1 µg/ml; InvivoGen, tlrl-3pelps), curdlan, a beta-1,3-glucan extracted from Alcaligenes faecalis (20 µg/ml; InvivoGen, tlrl-curd).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Binding Assay, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of mesenchymal stem cells (MSCs) by tumor necrosis factor α (TNFα) + interleukin 1β (IL-1β), the resulting cells acquire elongated cancer-associated fibroblast (CAF)-like morphology. Human MSCs were exposed to TNFα (50 ng/mL) + IL-1β (0.5 ng/mL) or to vehicles. Cytokine concentrations were selected based on the considerations described in the Materials and Methods section. ( A ) At different time points, the cells were photographed by light microscopy. Images from a representative experiment out of n > 3 are presented. Bar, 50 μm. ( B ) Determination of cell characteristics using IN Cell technology in cells that were treated using the cytokines/vehicles for 18 days and were then subjected to IN Cell analysis. ( B1 ) Cell morphology was detected by calcein (green) and Hoechst (blue) staining. Images of cell morphology from a representative experiment out of n = 3 are presented. Bar, 100 µm. ( B2 ) Quantification of cell characteristics by the IN Cell technology. *** p < 0.001. The results of a representative experiment of n = 3 are presented.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Light Microscopy, Cell Analysis, Staining

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Persistent stimulation of MSCs by TNFα + IL-1β leads to prominent alterations in gene expression and in fibroblast-relevant transcriptional programs in the resulting CAF-like cells. Human MSCs were exposed to “persistent” stimulation by TNFα and/or IL-1β for 14 days or to “short” stimulation of 48 h (concentrations as in ) in three independent biological repeats, followed by RNAseq analyses. Control cells were treated using the vehicles of the cytokines. ( A ) Heat maps. Deseq-normalized counts, values were centered. ( B ) Venn diagrams. Upregulated genes: FC > 2, padj < 0.05; downregulated genes: FC < 0.5, padj < 0.05. ( C ) Fibroblast-relevant transcriptional programs that were significantly modified by persistent TNFα + IL-1β stimulation, at FC > 2 or FC < 0.5 and padj < 5 × 10 −10 , are demonstrated.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Gene Expression, Control, Modification

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, the resulting CAF-like cells express typical CAF markers. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. The expressions of vimentin ( A ) and fibroblast activation protein (FAP) ( B ) were determined by confocal analyses. ( A1 , B1 ) Two representative images are demonstrated for each treatment, derived from a representative experiment out of n = 3. Bar, 25 μm. ( A2 , B2 ) Quantitative analyses of the fluorescence intensity performed by the ImageJ program on images of the representative experiment ( n ≥ 5 images for each treatment). ** p < 0.01.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Activation Assay, Derivative Assay, Fluorescence

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, the resulting CAF-like cells express typical CAF functions. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. The figure demonstrates the results of a collagen contraction assay, in which the change in collagen gel size was determined along the process. ( A ) Kinetics analyses of changes in collagen gel diameter compared to original gel size, measured by ImageJ. The average ± SEM of 5 independent experiments is demonstrated. *** p < 0.001. ( B ) An image from a representative experiment out of n = 5 is presented. Three wells are demonstrated for each treatment. The photo was taken at day 10 of the collagen contraction assay. Bar, 15.6 mm.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Contraction Assay

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Persistent stimulation with TNFα + IL-1β leads to the conversion of MSCs to inflammatory CAFs. ( A , B ) Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. ( A ) αSMA expression was determined by confocal analyses. ( A1 ) Two images of each treatment, derived from a representative experiment out of n > 3, are presented. Bar, 25 μm. ( A2 ) Quantitative analyses of the fluorescence intensity performed by the ImageJ program on images of the representative experiment ( n ≥ 5 images for each treatment). ** p < 0.01. ( B ) Cell proliferation was determined by cell counts at the end of the stimulation process. Average ± SD of n > 3 is presented. * p < 0.05. Time-dependent analysis of MSC cell numbers along the stimulation process are demonstrated in . ( C ) Expression of pro-inflammatory genes and secreted proteins, determined at the end of the stimulation process, is demonstrated. ( C1 ) mRNA expression was determined by transcriptome analyses performed at day 14, with three independent biological repeats. padj < 10 × 10 −10 –padj < 5 × 10 −271 , depending on the gene. ( C2 ) Expression of secreted proteins, determined by secretome analyses performed at days 18–19 with three independent biological repeats. p < 10 −3 – p < 7.5 × 10 −7 , depending on the protein. Data are presented as the fold induction of values obtained in cytokine-stimulated cells compared to vehicle-treated cells for all genes and proteins. demonstrate the 60 top upregulated or downregulated genes and proteins, obtained following persistent stimulation of the MSCs by TNFα + IL-1β.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Expressing, Derivative Assay, Fluorescence

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, cancer-relevant programs are promoted in the resulting inflammatory CAFs. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles for 14 days in transcriptome analyses and for 18–19 days in secretome analyses. ( A ) Heat maps of protein expression determined by secretome analysis of three independent biological repeats, comparing cytokine-stimulated cells and control cells. ( B ) Disorder analysis of cancer-related genes and secreted proteins for which expression was modified in cytokine-stimulated cells compared to vehicle-treated cells. ( B1 ) Gene expression data were obtained by RNAseq analysis, in which genes were filtered using a cutoff of FC > 2 or FC < 0.5, padj < 5 × 10 −10 . ( B2 ) Data of secreted proteins were obtained by secretome analysis, in which proteins were filtered using a cutoff of FC ≥ 2 or FC ≤ 0.5, p < 0.05. The INGENUITY program was used to identify affected disorders. Indicated disorders obeyed the cutoff of p < 10 −17 in transcriptome analyses and p < 5 × 10 −8 in secretome analyses.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Expressing, Control, Modification, Gene Expression

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, the resulting inflammatory CAFs release factors that induce morphological changes in luminal-A breast cancers (BC) cells. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. At the end of the stimulation period, cytokines were replaced by fresh cytokine-devoid media, and 48 h later, conditioned media (CM) were collected and administered to human luminal-A BC cells. ( A ) Tumor cell characteristics were quantified 4 days or 3 days after the addition of CM to MCF-7 cells ( A1 ) and to T47D cells, repectively ( A2 ) by IN Cell technology. The results of a representative experiment out of n = 3 are presented. *** p < 0.001. ( B ) Tumor cell morphology was photographed by confocal microscopy 2–3 days after the addition of CM, using phalloidin staining of actin filaments (green) and Hoechst staining of nuclei (blue). ( B1 ) MCF-7 cells. ( B2 ) T47D cells. Images from a representative experiment out of n = 3 are presented. Bar, 75 μm.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Confocal Microscopy, Staining

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, the resulting inflammatory CAFs release factors that promote scattering of luminal-A BC cells. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. Cytokine-devoid CM were collected as described in and were administered to mCherry-expressing MCF-7 ( A ) and T47D ( B ) human luminal-A BC cells, followed by determination of tumor cell scattering out of spheroids (MCF-7 cells) or the ability to form spheroids (T47D cells). ( A1 , B1 ) Representative images of spheroid formation at days 8 or 4 for MCF-7 cells and T47D cells are demonstrated, respectively. Bar, 200 µm. The results of a representative experiment out of n = 3 are presented. ( A2 , B2 ) Quantitative analyses of spheroid sizes performed by the ImageJ program on images of the representative experiment ( n ≥ 7 images for each treatment). *** p < 0.001.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Expressing

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Following persistent stimulation of MSCs with TNFα + IL-1β, the resulting inflammatory CAFs release factors that promote migration of luminal-A BC cells. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. Cytokine-devoid CM were collected as described in and were administered to mCherry-expressing MCF-7 and T47D human luminal-A BC cells. ( A ) Tumor cell migration was determined in fibronectin-coated transwells in response to serum-containing medium. ( A1 ) MCF-7 cells. ( A2 ) T47D cells. Images and quantitative analyses of a representative experiment out of n = 3, for each cell type, are presented. *** p < 0.001. ( B ) MCF-7 wound closure assay, performed in IncuCyte ® . ( B1 ) Representative images taken at 24 h. Bar, 150 µm. ( B2 ) The kinetics graphs of tumor cell migration demonstrate the proportion (%) of the original wound area that became covered by migrating cells at each time point. The results presented are mean ± SEM of 5 replicates for each treatment and are of a representative experiment out of n > 3. *** p < 0.001.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Migration, Expressing, Wound Closure Assay

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Tumor cell migration, elevated by inflammatory CAF-derived factors, is mediated through cooperativity between Ras-activating receptors and G protein-coupled receptors (GPCR) that signal via Gαi. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. Cytokine-devoid CM were collected as described in and were administered to MCF-7 cells in the presence of ( A ) FTS, an inhibitor of Ras; ( B ) PTx, an inhibitor of Gαi; or ( C ) both inhibitors together. Control cells were treated by the vehicles of inhibitors. Inhibitor concentrations were selected based on the considerations described in the Materials and Methods section. Cell numbers and viability were not affected by the inhibitors. Then, kinetics analyses of wound closure assays were performed in IncuCyte ® , as described in B. The results are presented as mean ± SEM of 8–9 replicates for each treatment. *** p < 0.001, for differences between tumor cells treated by CM of inflammation-derived CAFs and control CAFs. ### p < 0.001, for differences between tumor cells treated by the inhibitors and tumor cells that were not treated by the inhibitors. The results of a representative experiment out of n = 3 are presented.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Migration, Derivative Assay, Control

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Tumor cell migration, elevated by inflammatory CAF-derived factors, is mediated through cooperativity between the chemokine receptors CCR2, CCR5, and CXCR1/2. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. Cytokine-devoid CM were collected as described in and were administered to MCF-7 cells in the presence of inhibitors. ( A ) Chemokine receptor inhibitors. ( A1 ) CCR2i = the CCR2 inhibitor CAS 445479–97-0; ( A2 ) CCR5i = the CCR5 inhibitor Maraviroc; and ( A3 ) CXCR1/2i = the CXCR1/2 inhibitor Reparixin. ( B ) Combined inhibitory measures directed to chemokine receptors compared to inhibition of Gαi by PTx performed in the same experiment. ( B1 ) All three chemokine receptor inhibitors together. ( B2 ) Inhibition by PTx. In all treatments, cell numbers and viability were not affected by the inhibitors. Kinetics analyses of wound closure assays were performed in IncuCyte ® , as described in . The results are presented as mean ± SEM of 6–9 replicates for each treatment. *** p < 0.001, for differences between tumor cells treated by the CM of inflammation-derived CAFs and control CAFs. ### p < 0.001, for differences between tumor cells treated by the inhibitors and tumor cells that were not treated by the inhibitors. The results of a representative experiment out of n = 3 are presented (with the exception of n = 2/3 in (A1)).

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Migration, Derivative Assay, Inhibition, Control

Journal: Cancers

Article Title: Persistent Inflammatory Stimulation Drives the Conversion of MSCs to Inflammatory CAFs That Promote Pro-Metastatic Characteristics in Breast Cancer Cells

doi: 10.3390/cancers13061472

Figure Lengend Snippet: Tumor cell migration, elevated by inflammatory CAF-derived factors, is mediated through cooperativity between Ras-activating receptors and chemokine receptors. Human MSCs were exposed to persistent TNFα + IL-1β stimulation (concentrations as in ) or to vehicles, generally for 14–18 days. Cytokine-devoid CM were collected as described in and were administered to MCF-7 cells in the presence of inhibitors. In the same experiment, different inhibitory conditions were used: ( A ) combined inhibition of CCR2, CCR5, and CXCR1/2 together, as in . ( B ) Inhibition of Ras-mediated signaling by FTS. ( C ) Combined inhibitory measures directed to chemokine receptors and Ras-mediated signaling. In all treatments, cell numbers and viability were not affected by the inhibitors (except for the inhibitory treatment included in part C, where 15–25% decrease in cell numbers was noted). Kinetics analyses of wound closure assays were performed in IncuCyte ® , as described in . The results are presented as mean ± SEM of 8–9 replicates for each treatment. *** p < 0.001, for differences between tumor cells treated by the CM of inflammation-derived CAFs and control CAFs. ### p < 0.001, for differences between tumor cells treated by the inhibitors and tumor cells that were not treated by the inhibitors. The results of a representative experiment out of n = 3 are presented.

Article Snippet: Based on preliminary titration studies performed in our lab and on other in vitro reports using TNFα [ , , ] and IL-1β [ , ], MSCs were stimulated by recombinant human (rh) TNFα (50 ng/mL; #300-01A, PeproTech, NJ, USA) and/or rhIL-1β (0.5 ng/mL; #200-01B, PeproTech).

Techniques: Migration, Derivative Assay, Inhibition, Control