Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 1 Protein expression of TRPA1 in the LSC group and control group. (A) Western blot results for TRPA1 expression in skin tissues

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control, Western Blot

Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 2 Expression of TRPA1 in the LSC group and control group by Immunohistochemical analysis. (A) The expression of TRPA1 in skin tissue of

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control, Immunohistochemical staining

Journal: Annals of palliative medicine

Article Title: Down-regulated expression of transient receptor potential ankyrin 1 in lichen simplex chronicus.

doi: 10.21037/apm-20-1712

Figure Lengend Snippet: Figure 3 TRPA1 and inflammatory mediators in the skin tissue specimens mRNA expression in the LSC group and control group. (A)

Article Snippet: The proteins in a sample were separated using sodium dodecyl sulfate-polyacrylamide gel (Beyotime Biotech, Shanghai, China) electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Beyotime Biotech, Shanghai, China) at a constant voltage of 80 V. After being blocked with a rapid blocking buffer for 10–15 minutes, and the membrane was incubated at 4 °C in an ice room overnight with primary antibodies against TRPA1 (1:1,000; ACC-037, Alomeno Labs, Israel) and β-actin (1:500; Wuhan Boster Biological Technology, China).

Techniques: Expressing, Control

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Primers used for RT-qPCR analysis.

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Amplification

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Expression of the lipid metabolism regulator PPARβ and downstream effectors (CPT1b, FAS, and ACC2). mRNA levels in obese rats under hypoxic conditions, with hypoxic training, and with regulated miR-122 expression were determined using qRT-PCR. OE, obese rats with miR-122 overexpression and hypoxic training; IE, obese rats with miR-122 depletion and hypoxic training; CE, obese rats with hypoxic training only; H, obese sedentary rats without regulation of miR-122 expression. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 vs rats in group H; # p < 0.05, ## p < 0.01 vs rats in group CE; & p < 0.05, && p < 0.01 vs rats in group OE (1-way analysis of variance).

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Expressing, Quantitative RT-PCR, Over Expression

Journal: Heliyon

Article Title: miR-122/PPARβ axis is involved in hypoxic exercise and modulates fatty acid metabolism in skeletal muscle of obese rats

doi: 10.1016/j.heliyon.2024.e26572

Figure Lengend Snippet: Expression of the lipid metabolism regulator PPARβ and downstream effectors (CPT1b, FAS, and ACC2). Protein levels in obese rats under hypoxic conditions, with hypoxic training, and with regulated miR-122 expression were determined using Western blot analysis. OE, obese rats with miR-122 overexpression and hypoxic training; IE, obese rats with miR-122 depletion and hypoxic training; CE, obese rats with hypoxic training only; H, obese sedentary rats without regulation of miR-122 expression. Data are presented as mean ± SD. *p < 0.05, **p < 0.01 vs rats in group H; # p < 0.05, ## p < 0.01 vs rats in group CE; & p < 0.05, && p < 0.01 vs rats in group OE (1-way analysis of variance).

Article Snippet: The protein samples were then transferred to a PVDF membrane (0.45-μm pore size) at a constant pressure of 100 V for 10 min, followed by blocked overnight in Tris-buffered saline with 0.1% Tween-20 (TBST), which contained 5% bovine serum albumin, and then probed overnight at 4 °C with primary antibodies against PPARβ (1:2000; GTX113250) and FAS (1:1000; GTX13550) (both from GeneTex, Irvine, CA, USA) and ACC2 (1:500; A03668-2) and CPT1b (1:500; PB9491) (both from Boster Bio, Pleasanton, CA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: (a,b) Microarray-based gene expression analysis of Wnt6 +/+ and Wnt6 −/− BMDMs infected for 24 hours with Mtb H37Rv (MOI 3:1). Fold expression of statistically significantly regulated genes associated with fatty acid uptake and degradation (a) or lipid synthesis and storage (b) are depicted; n=3. (c) qRT-PCR based gene expression analysis of Wnt6 +/+ and Wnt6 −/− BMDMs infected for 24 h with various doses (MOIs) of Mtb H37Rv; n=3. (d) qRT-PCR based gene expression analysis of WNT6-overexpressing (WNT6) or control (ctrl (LacZ)) NIH3T3 cells; n=3. (e) qRT-PCR based gene expression analysis of hMDMs treated with WNT6 conditioned medium (WNT6 CM) or control conditioned medium (ctrl) CM for 24 hours. Fold change relative to control (ctrl CM) is shown. For statistical comparison, raw data were used. Data from 3 independent experiments using cells from different donors are shown; n=3. (f) qRT-PCR based gene expression analysis of ACACB (ACC2) mRNA expression in Mtb-infected hMDMs at day 7 p.i‥ Cells were infected with Mtb H37Rv (MOI) 1:1), washed (4 h p.i.) and incubated for 7 days; n=3. (g) CFU analysis of Mtb-infected (MOI 1:1) Wnt6 +/+ or Wnt6 −/− BMDMs at day 0 (4 h), 3 and 7 p.i. (h) CFU analysis of Mtb-infected (MOI 0.1:1) Wnt6 +/+ or Wnt6 −/− BMDMs at day 7 p.i. after incubation of cells various concentrations of oleic acid (oleate-BSA). Bacterial growth was related to the number of macrophages (normalized CFU) at the individual timepoint/condition (given as CFU per 100.000 cells). Shown is the mean +/− SEM of a total of 3 (g) or 4 (h) independent experiments. Statistical analyses were carried out using One-Way ANOVA with a suitable post-hoc test for multiple comparison except microarray-based gene expression analysis (c,d), which was conducted as described in Material and Methods . *p≤0.05, **p≤0.01, ***p≤0.001. All data are depicted as mean +/− SEM.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Microarray, Expressing, Infection, Quantitative RT-PCR, Incubation

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: (a) CFU analysis of Mtb-infected (MOI 0.5:1) wild-type (WT), ACC1 KO and ACC2 KO human macrophage-like cells (BLaER1 macrophages) at day 3 p.i‥ n=3. (b,d,e) CFU analysis of Mtb-infected (MOI 1:1) hMDMs treated with pharmacological ACC2 inhibitors at day 7 p.i‥ Uptake was determined 4h p.i‥ After washing, cells were incubated in the absence (solvent ctrl, DMSO) or presence of different ACC2 inhibitors with the concentrations indicated; n=3. (c) In the same set of experiments shown in (b), additionally to the treatment with ACC2 inhibitor 1 alone (300nM), cells were also treated with isoniazid (INH (0.03 μg/ml)) or as a combination of ACC2 inhibitor plus INH. Shown is the relative reduction of CFU (%). (f) CFU analysis of Mtb-infected (MOI 0.5:1) hMDMs treated with ACC2 inhibitors in the presence of exogenous fatty acids at day 7 p.i‥ Uptake was determined 4h p.i‥ After washing, cells were incubated with oleate-BSA or palmitate-BSA (both 400μM) in the absence or presence of ACC2 inhibitors 2 (blue triangles, 300 nM) or ACC2 inhibitor 3 (green triangles, 400 nM); n=4. (g,h) hMDMs were pulsed with isotopically labelled 13 C-Oleate-BSA prior to infection with Mtb (MOI 1:1) and subsequently incubated in the absence (solvent, ctrl) and presence of ACC2 inhibitor 3 (400 nM) for 7 days. Mass spectrometry-based analyses show (g) ratios of TAG (left panel) and CE (right panel) normalized to PC and (h, left panel) the change (%) in isotope labeling ( 13 C18) in the Mtb-specific membrane lipid PI 16:0_19:0 tuberculostearic acid (TSA) from the same sample. In parallel, cells were subjected to CFU analysis, revealing the % of CFU reduction (h, right panel) from each individual experiment (donor); n=4. (i) Flow cytometry-based quantification of - Rhodamine 123 signals (relative to MitoTracker Deep Red signals (both aMFI) x100) in Mtb-infected (MOI 0.1:1) and ACC2 inhibitor 3 (400nM) treated hMDMs at day 3 p.i.; n=4. (j) Quantification of Lactate Dehydrogenase Release (LDH) from hMDM cultures at day 7 p.i.; n=5. Cells were equally infected as described for (b). UI, uninfected; TAG, Triacylglycerols; PC, Phosphatidylcholines; CE, Cholesterolester. Statistical analyses were carried out using One-Way ANOVA with a suitable post-hoc test for multiple comparison; *p≤0.05, **p≤0.01, ***p≤0.001. All data are depicted as mean +/− SEM.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Infection, Incubation, Mass Spectrometry, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: (a) Analysis of human macrophage viability in the presence of ACC2 inhibitor 1 as determined by real-time impedance measurements (expressed as cell index). hMDMs were incubated in the presence of solvent (DMSO, ctrl), ACC2 inhibitor 1 or Staurosporine (1 μg/ml) for the indicated time on a xCELLigence System; Depicted is representative data from 2 independent experiments with similar results. (b) Mtb growth in the absence (solvent) and presence of various ACC2 inhibitors or the TB drug rifampicin as determined by measuring fluorescence of GFP-expressing Mtb in liquid culture. Bacteria were cultured in 7H9 medium supplemented with 10% OADC and growth was measured as relative light units at 528 nm after excitation at 485 nm in a fluorescence microplate reader at the indicated time point; n=2 (left panel), n=3 (right panel). (c) TNFα release of hMDMs infected for 24 hours with Mtb H37Rv and simultaneously incubated with solvent (DMSO, ctrl) or the indicated concentrations of ACC2 inhibitor 1. Mean +/− SEM from 3 independent experiments/donors is shown. (d) Effect of addition of fatty acids on Mtb CFU in primary human macrophages. After infection with Mtb (MOI 0.5:1), cells were washed and incubated in the absence (BSA) or presence of different concentrations of oleate- and palmitate-BSA. Data are derived from the same set of experiments shown in ; n=4. (e) Flow cytometry-based quantification of Rhodamine 123 signals (relative to MitoTracker Deep Red signals (both aMFI) x100) in Mtb-infected wild-type (WT) and ACC2 KO human macrophage-like cells (BLaER1 macrophages) (MOI 0,1:1) normalized to uninfected WT cells at day 3 p.i‥ (f) Enhanced viability of hMDMs during infection with Mtb H37Rv when treated with ACC2 inhibitor (lower panel) in comparison to solvent control (DMSO, upper panel). Depicted is a representative observation of 2 independent experiments with similar results. Statistical analyses were carried out using One-Way ANOVA with a suitable post-hoc test for multiple comparison (d); * p≤0.05, ***p≤0.001. All data are depicted as mean +/− SEM.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Incubation, Fluorescence, Expressing, Cell Culture, Infection, Derivative Assay, Flow Cytometry

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: (a,b) Immunohistochemical analyses of formalin-fixed and paraffin-embedded lung tissue derived from a tuberculosis patient. Consecutive sections (1μm) were incubated with antibodies specific for ACC2 (a) or the macrophage/monocyte marker CD68 (b). Antigens were visualized with a Horseradish peroxidase (HRP)-based detection system using AEC as chromogen (red). Scale bar, 100 μm (c,d) Immunohistochemical analyses of formalin-fixed and paraffin-embedded lung tissue of Mtb-infected C57Bl/6 (~1000 CFU, d42 p.i.) or 129/Sv mice (~200 CFU, d28 p.i.). Sections (2 μm) were incubated with antibodies specific for ACC 1/2 and antigens visualized with a Horseradish peroxidase (HRP)-based detection system using AEC as chromogen (red). (e-h) In vivo efficacy of ACC2 inhibitor treatment when combined with the first-line anti-TB drug INH. After 28 days of infection with Mtb H37Rv (~200CFU), 129/Sv mice were either left untreated (pretreatment, d28 p.i., n=4, white bars) or were treated for 14 days with INH alone (10 mg/ per kg bodyweight (BW), n=8, grey bars) or with ACC2 inhibitor 3 (ND-646, 25 mg/kg BW) plus INH (n=10, red bars). Lung weights (e), lung cytokine and chemokine levels (f), TAG and CE abundance in the lung (g), as well as mycobacterial loads in lung, liver and spleen were determined. Statistical analyses were carried out using an one-tailed, unpaired Student’s t-test; *p≤0.05, **p≤0.01, ***p≤0.001; n.s.= not significant. Data are depicted as Min-Max bar with line at mean.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Immunohistochemical staining, Derivative Assay, Incubation, Marker, Infection, In Vivo, One-tailed Test

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: (a) Immunohistochemical analyses of formalin-fixed and paraffin-embedded lung tissue derived from a tuberculosis patient (Patient 1). The upper panel shows consecutive sections (1 μm) incubated with primary antibodies directed against ACC2 (left panel), ACC1/2 (middle panel) and the macrophage/monocyte marker CD68 (right panel). The lower panels show the respective consecutive section, which was incubated without primary antibody (sec. AB ctrl). Antigens were visualized with a Horseradish peroxidase (HRP)-based detection system using AEC as chromogen (red). (b) Effect of a low-dose and short-term ACC2 inhibitor treatment on mycobacterial loads in Mtb-infected mice. After 28 days of infection with a low dose of Mtb H37Rv (~200CFU), 129/Sv mice were either left untreated (white bars), were treated with vehicle solution (grey bars) or with ACC2 inhibitor (ND-646, 25 mg/kg BW) for a period of 7 days. At day 35 p.i., Mtb bacterial burden was determined in lung, liver and spleen (n=6-10 animals per group). Statistical analyses were carried out using an one-tailed, unpaired Student’s t-test; n.s.= not significant. Data are depicted as Min-Max bar with line at mean.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Immunohistochemical staining, Derivative Assay, Incubation, Marker, Infection, One-tailed Test

Journal: bioRxiv

Article Title: WNT6-ACC2-induced accumulation of triacylglycerol rich lipid droplets is exploited by M. tuberculosis

doi: 10.1101/2020.06.26.174110

Figure Lengend Snippet: Homeostatic-or activation (TLR2/4)-dependent WNT6-signaling via Frizzled receptors induces the expression of various key lipid metabolic genes including acyl-CoA:diacylglycerol acyltransferase (DGAT2) and Acetyl-CoA Carboxylase-2 (ACC2). ACC2 is known to generate Malonyl-CoA, which inhibits carnitine palmitoyltransferase 1 (CPT1)-dependent import of fatty acids into mitochondria thereby reducing cellular fatty acid oxidation. Intracellular fatty acids are converted by different enzymes including DGAT2 into triacylglycerols(TAG), which are sequestered into lipid droplets. M. tuberculosis gains access to host derived fatty acids, e.g. via the interaction of bacteria containing phagosomes with TAG-rich lipid droplets. Intracellular accumulation of fatty acids also induces necrotic cell death (lipotoxicity) thereby promoting Mtb dissemination and release of lipid droplets from the dying host cell.

Article Snippet: Slides were incubated in Antibody Diluent (Zytomed Systems, Berlin, Germany) in the presence of a primary antibody specific for WNT6 (purchased from Abcam (ab50030, 5 μg/ml) or Bio-Techne (AF4109, 6.6 μg/ml)), CD68 (clone PG-M1, 1:100, purchased from Agilent Technologies), PLIN2 (Abcam (# ab78920, 1:100), ACC2 (LS-C11360; LSBio, Seattle, USA) and ACC1/2 (mAb, C83B10; Cell signalling, Frankfurt, Germany).

Techniques: Activation Assay, Expressing, Derivative Assay