Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Uptake of Fl-MPs by HK-2 at 5 – 24 h . A time-dependent increase in SI was observed (A) as well as in the percentage of Fl-MP positive cells (B). All uptake measurements were done by SSC-A mediane in flow cytometry and normalized over CTR. By confocal microscope (C), Fl-MPs were observed in the cytoplasm (red), stained with Alexa-Fluor 594-conjugated phalloidin. Nuclei were counterstained with DAPI (blue). Black arrows point MPs observed by bright field scale. Bar = 10 μm. Results represent means ± SEM obtained from three independent experiments. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. CTR; ## p < 0.001 vs. 5 h MP exposure. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Abbreviations: Fl-MPs = fluorescent microplastics; CTR=No treated cells; Norm. = normalized; SI = stain index.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques: Flow Cytometry, Microscopy, Staining

Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Effects of treatments on HK-2 viability at 24 h (MTT test). A decrease in cell viability was observed with PE-MPs and BPA + PE-MPs. For each treatment group the number of CTR cells served as baseline value 100 % and was used to express the percentage of living cells. ∗∗p < 0.01, ∗∗∗p < 0.001 vs. CTR.°p < 0.01 vs. BPA. Abbreviations: MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CTR=No treated cells; BPA = Bisphenol A; PE-MP = polyethylene microplastics.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques:

Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Effects of treatments on NOX4 (A), NRF2 (B) protein expression and MDA levels (C ). A 5-h exposure saw a rise in NOX 4, NRF2 and MDA. NOX4 and NRF2 were evaluated by Western blot. Blots were stripped and reprobed with antibody to anti β-actin. All results represent means ± SEM obtained from three independent experiments and are expressed as fold change to CTR. ∗p < 0.05; ∗∗p < 0.01 vs. CTR; ° p < 0.05 vs. BPA. Abbreviations: CTR=No treated cells; BPA = Bisphenol A; PE-MP = polyethylene microplastics; NOX4 = NADPH Oxidase 4; NRF2 = nuclear factor erythroid 2-related factor 2; MDA = malondialdehyde.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques: Expressing, Western Blot

Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Effects of treatments on inflammatory molecules . 5-hour exposure increased IL-1β (A), CCL-2 (B), CCL-5 (C) and their receptors CCR-2 (B) and CCR-5 (C). mRNAs were evaluated by RT-PCR. The results represent means ± SEM obtained from three independent experiments and are expressed as fold change to CTR. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. CTR; ° p < 0.05,°p < 0.01 vs. BPA; + p < 0.05, ++ p < 0.01, +++ p < 0.001 vs. PE-MPs. Abbreviations: IL-1β = interleukin-1β; CCL-2 = chemokine (C-C motif) ligand 2; CCL-5 = chemokine (C-C motif) ligand 5; CCR-2 = C-C chemokine receptor type 2; CCR-5 = C-C chemokine receptor type 5; RT-PCR = real-time polymerase chain reaction; CTR=No treated cells; BPA = Bisphenol A; PE-MP = polyethylene microplastics.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Treatments changed HSP90 expression . A 24-h exposition decreased HSP90 levels, as evaluated by Western blot (A) and confirmed by immunocytochemistry and image analysis (B). Blots were stripped and reprobed with antibody to β-actin. Results represent means ± SEM obtained from four independent experiments and expressed as fold change to CTR. For immunocytochemistry, staining intensity was expressed as AU and at least 200 cells for condition from three independent experiments were evaluated. ∗p < 0.05, ∗∗∗∗p < 0.0001 vs. CTR; ° p < 0.05 vs. BPA. Magnification = ×200, scale bar = 75 μm. Abbreviations: CTR=No treated cells; BPA = Bisphenol A; PE-MP = polyethylene microplastics, HSP90 = heat shock protein 90; AU = arbitrary units.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques: Expressing, Western Blot, Immunocytochemistry, Staining

Journal: Heliyon

Article Title: Coexposure to microplastic and Bisphenol A exhacerbates damage to human kidney proximal tubular cells

doi: 10.1016/j.heliyon.2024.e39426

Figure Lengend Snippet: Effects of treatments on AHR mRNA (A) and protein expression (B). A 5-h exposition increased AHR levels, but after 24 h only protein was enhanced, compared to CTR. Immunofluorescence analysis (C) at 5 h revealed AHR in nuclei, whereas after 24 h AHR was perinuclear. AHR signal was red and nuclei counterstained with DAPI (blue). mRNA was evaluated by RT-PCR. The results represent means ± SEM obtained from four independent experiments and are expressed as fold change to CTR. ∗p < 0.05 vs. CTR; ∗∗p < 0.01 vs. CTR ; ∗∗∗p < 0.001 vs. CTR; °p < 0.01 vs. BPA; °p < 0.001 vs. BPA; ++ p < 0.01, +++ p < 0.001 vs. PE-MPs. Magnification = ×400, scale bar = 75 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Abbreviations: AHR = aryl hydrocarbon receptor; HRS = hours; RT-PCR = real-time polymerase chain reaction; CTR=No treated cells; BPA = Bisphenol A; PE-MP = polyethylene microplastics; DAPI = 4′,6-diamidino-2-phenylindole; CTCF = corrected total cell fluorescence.

Article Snippet: Cells were grown to sub-confluence and exposed for 5–24 h to complete medium (CTR), 100 nmol/l BPA (Merck Group), 1.0–4.0 μm clear PE-MPs (0.2 mg/mL) (Cospheric) and BPA + PE-MPs.

Techniques: Expressing, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Fluorescence

Journal: PLoS ONE

Article Title: TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

doi: 10.1371/journal.pone.0135063

Figure Lengend Snippet: (A) 41CAFs and 311NAFs were treated with 5 ng/ml of TGF-β for 24 hours and 48 hours and analyzed for CXCL1 expression by ELISA. (B) 41CAFs were transfected with control siRNA (Con), siRNAs to Smad2 (S2), Smad3 (S3) or both (S2S3), treated with TGF-β for 24 hours, and analyzed for CXCL1 expression by ELISA. (C) 41CAFs transfected with siRNA were treated with TGF-β for 1 hour, and analyzed by western blot for expression of the indicated proteins. Expression levels of Smad2 and Smad3 were normalized to actin through densitometry analysis. Statistical analysis was performed using Two Tailed T-test (A), or One Way ANOVA followed by Bonferonni post-hoc comparisons (B). Statistical significance was determined by p<0.05; *p<0.05, **p<0.01, ***p<0.001, n.s; not significant. Values are expressed as Mean ± SEM.

Article Snippet: For each well, 24 pmol control siRNAs or siRNAs targeting Smad2, Smad3 and HGF (Santa Cruz Biotechnology were complexed to 2.4 μl lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM medium for 20 minutes at room temperature.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Control, Western Blot, Two Tailed Test

Journal: PLoS ONE

Article Title: TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

doi: 10.1371/journal.pone.0135063

Figure Lengend Snippet: (A) 41CAFs were co-transfected with Renilla luciferase and firefly luciferase reporters containing: the wildtype CXCL1 promoter (PGL3.luc.CXCL1), or mutations in SBE1 (SBEm1), SBE2 (SBEm2), or both (SBEm1/2). Cells were treated with 5 ng/ml of TGF-β for 6 hours, and analyzed for CXCL1 promoter activity by luciferase assay. Fold change was calculated relative to (-) TGF-β group. (B) 41CAFs co-expressing C/EBP-β.luc firefly and Renilla luciferase reporters were transfected with: control siRNA (Cont) or siRNAs to Smad2 (S2), Smad3 (S3). Cells were treated with 5 ng/ml of TGF-β for 24 hours and C/EBP-β activity by luciferase assay. Fold change was calculated relative to control siRNA/(-) TGF-β group. Firefly luciferase values were normalized to Renilla luciferase. Statistical analysis was performed using One Way ANOVA followed by Bonferonni post-hoc comparisons. Statistical significance was determined by p<0.05; *p<0.05, n.s; not significant. Values are expressed as Mean ± SEM.

Article Snippet: For each well, 24 pmol control siRNAs or siRNAs targeting Smad2, Smad3 and HGF (Santa Cruz Biotechnology were complexed to 2.4 μl lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM medium for 20 minutes at room temperature.

Techniques: Transfection, Luciferase, Activity Assay, Expressing, Control

Journal: PLoS ONE

Article Title: TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

doi: 10.1371/journal.pone.0135063

Figure Lengend Snippet: (A) The indicated cell lines were analyzed for HGF expression in conditioned media by ELISA. (B-C) 41CAFs expressing control siRNA (Con) or siRNAs to HGF were analyzed for expression of HGF (B) or CXCL1 (C) by ELISA. (D) 41CAFs were treated with increasing concentrations of HGF for 48 hours and analyzed for CXCL1 expression by ELISA. Statistical analysis was performed using Two Tailed T-Test (B,C), or One Way ANOVA followed by Bonferonni post-hoc comparisons (A,D). Statistical significance was determined by p<0.05; *p<0.05, **p<0.01, ***p<0.001. Values are expressed as Mean ± SEM.

Article Snippet: For each well, 24 pmol control siRNAs or siRNAs targeting Smad2, Smad3 and HGF (Santa Cruz Biotechnology were complexed to 2.4 μl lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM medium for 20 minutes at room temperature.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

doi: 10.1371/journal.pone.0135063

Figure Lengend Snippet: (A) 41CAFs were treated with 5ng/ml TGF-β for 24 or 48 hours, and analyzed for HGF expression in conditioned media by ELISA. (B) 41CAFs were transfected with control siRNA (Con), or siRNAs to Smad2 (S2) or Smad3 (S3), treated with 5 ng/ml TGF-β for 48 hours, and analyzed for HGF expression in by ELISA. (C) 41CAFs were treated with 5ng/ml TGF-β in the presence or absence of increasing concentrations of HGF for 48 hours, and analyzed for CXCL1 expression by ELISA. Statistical analysis was performed using Two Tailed T-Test (A), or One Way ANOVA followed by Bonferonni post-hoc comparisons (B, C). Statistical significance was determined by p<0.05; *p<0.05, ***p<0.001. Values are expressed as Mean ± SEM.

Article Snippet: For each well, 24 pmol control siRNAs or siRNAs targeting Smad2, Smad3 and HGF (Santa Cruz Biotechnology were complexed to 2.4 μl lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM medium for 20 minutes at room temperature.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Control, Two Tailed Test

Journal: PLoS ONE

Article Title: TGF-β Negatively Regulates CXCL1 Chemokine Expression in Mammary Fibroblasts through Enhancement of Smad2/3 and Suppression of HGF/c-Met Signaling Mechanisms

doi: 10.1371/journal.pone.0135063

Figure Lengend Snippet: (A) 41CAFs were transfected with control or HGF siRNAs, and analyzed for expression of phospho-c-Met (Tyr-1234/1235) by immunoblot. Expression of phospho-c-Met was normalized to total c-Met by densitometry analysis. (B) 41CAFs co-expressing pNF-κB.luc and Renilla luciferase reporters were transfected with control (Con) or HGF siRNAs, and analyzed for NF-κB activity by luciferase assay. Fold change was calculated relative to control siRNA group. (C) 41CAFs were treated with 36 μM SN50 or 5 μM Bay11-7085 for 24 hours, and analyzed for CXCL1 expression by ELISA. (D) 41CAFs co-expressing pNF-κB.luc and Renilla luciferase reporters were treated with 36 μM SN50 or 5 μM Bay 11–7085, and analyzed for NF-κB activity by luciferase assay. Firefly luciferase values were normalized to Renilla luciferase. Fold change was calculated relative to (-) SN50 or (-) Bay 11–7085 group. (E) 41CAFs were treated with 40 ng/ml HGF in the presence or absence of 36 μM SN50 for 24 hours, and then analyzed for CXCL1 expression by ELISA. Statistical analysis was performed using Two Tailed T-Test. Statistical significance was determined by p<0.05; *p<0.05. Values are expressed as Mean ± SEM.

Article Snippet: For each well, 24 pmol control siRNAs or siRNAs targeting Smad2, Smad3 and HGF (Santa Cruz Biotechnology were complexed to 2.4 μl lipofectamine 2000 (Invitrogen) in 100 μl Opti-MEM medium for 20 minutes at room temperature.

Techniques: Transfection, Control, Expressing, Western Blot, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test