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

Journal: bioRxiv

Article Title: A pathway to produce non-coding piRNAs from endogenous protein-coding regions supports Drosophila spermatogenesis

doi: 10.1101/2022.10.02.510544

Figure Lengend Snippet: (A) Mapping of 23∼29-nt RNAs accumulating in D. melanogaster testes to endogenous protein-coding genes. Abundance (TPM ; transcripts per kilobase million) of 23∼29-nt RNAs mapping to exons (5’UTR + CDS [coding sequence] + 3’UTR) was obtained for individual genes. Mean of 6 data was shown in X-axis. Mean mRNA abundance (TPM) was obtained from 2 data, and enrichment of 23∼29-nt RNAs relative to mRNAs (TPM/TPM) was shown in Y-axis. (B) Size distribution of 18∼29-nt exon-mapping RNAs. (C) Nucleotide probability of 23∼29-nt exon-mapping RNAs. (D, E) Binding of 23∼29-nt RNAs to Piwi or Aub. Abundance (TPM) of 23∼29-nt RNAs in Piwi- or Aub-bound fraction was given as mean of Piwi-IP and GFP-Piwi-IP data, or as mean of Aub-IP and GFP-Aub-IP data. Enrichment (Piwi-bound/total or Aub-bound/total) was shown in X-axis. Genes enriched with 23∼29-nt RNAs relative to mRNAs (TPM/TPM>10, n=2449) were analyzed. (F) Genes producing piRNAs most abundantly for Aub. Abundance (mean RPM [Read per million] of Aub-IP and GFP-Aub-IP) was shown in Y-axis. Seventeen or 324 genes (RPM >100 or >10) were grouped and analyzed later. (G) Loss of aub, ago3 , or rhi effect on exon-mapping piRNAs. Seventeen genes (Aub-IP RPM >100) were analyzed. (H) Origins of piRNAs within gene exons. Bedgraph shows 23∼29-nt RNAs in testis total RNAs (blue), Aub-bound piRNAs (red), Piwi-bound piRNAs (green), and mRNA transcriptome (gray). Gene model shows CDS (bold line) and UTRs (thin line). (I) Density of mapping piRNAs compared between 5’UTR, CDS, and 3’UTR. pira, vas, CG9010 accumulating rhi - and ago3 -dependent piRNAs were excluded from 17 genes. Remaining 14 genes were analyzed in box plot. p (two-tailed paired t -test). (J) Strand orientation of Aub-bound piRNAs mapping to pcf11 and phlpp . Arrowhead highlights exon-exon junction reads.

Article Snippet: The membrane was blocked in 4%(w/v) skim milk (Nacalai) in ×1 phosphate-buffered saline (PBS) supplemented with 0.1%(v/v) Tween-20, and further incubated with the following antibodies; anti-Aub antibody (guinea pig, 1:500) , anti-GFP antibody (Clontech, rabbit, 1:2000), anti-Ago1 antibody (Abcam, Ab5070, rabbit, 1:1000), anti-Ago2 antibody (guinea pig, 1:100) , anti-Piwi (mouse, P4D2, 1:10), anti-FLAG M2-peroxidase (HRP) (SIGMA, #A8592, 1:5000), anti-H3K18Ac (active motif, #39756, 1:2000), anti-H3K27Ac (active motif, #39136, 1:2000), anti-H4K8Ac (active motif, #61104, 1:2000), anti-H4K12Ac (active motif, #39928, 1:2000), anti-guinea pig immunoglobulins-HRP (DAKO, 1:1000), anti-rabbit IgG-HRP (BioRad, 1:3000), anti-mouse IgG-HRP (BioRad, 1:3000).

Techniques: Sequencing, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: A pathway to produce non-coding piRNAs from endogenous protein-coding regions supports Drosophila spermatogenesis

doi: 10.1101/2022.10.02.510544

Figure Lengend Snippet: (A) Principles of germline BioID/TurboID developed in this study. Using free biotins, an engineered biotinylation enzyme called mini(m)Turbo generates diffuse pattern of biotin-AMP, which adds biotins to lysine residues of proteins close to the selected bait. Not only stable (Sta) but also transient (Tra) interactors such as chaperone clients can be biotin-labeled. mTurbo was fused to bait proteins including Cyp40, non-functional Cyp40 derivatives (RKAA or Δ TPR), or GFP. RKAA and Δ TPR lack affinity to Hsp90. (B) Silver staining of proteins purified with streptavidin. Cyp40-dependent signals were enriched in >∼80kDa area from which peptides were extracted and analyzed by mass spectrometry. (C) Volcano plot summarizing mass-spectrometry data. Spectrum counts of proteins purified from testes expressing mTurbo-FLAG-Cyp40 (2 data; in the presence or absence of endogenous cyp40 ) were compared with those in control conditions (4 data; single replicate of mTurbo-FLAG-Cyp40 RKAA and mTurbo-FLAG-Cyp40 Δ TPR , duplicate of mTurbo-GFP). X-axis; enrichment of spectrum counts (Cyp40/control), Y-axis; p (two-tailed unpaired t -test). Names of significantly ( p <0.01) enriched proteins were indicated. (D) Immunoblotting of Ago proteins and mTurbo-FLAG-Cyp40 present in testes (Input) and purified with streptavidin (Pulldown). For Ago2, both 3FLAG-HA-tagged (3FH-)Ago2 expressed under native promoter activity and endogenous Ago2 were analyzed. Of note, mTurbo-FLAG-Cyp40 proteins were barely detectable in input (indicated by asterisk), but strongly enriched by streptavidin pulldown, indicating efficient self biotinylation. Steady state level of mTurbo-FLAG-Cyp40 RKAA was much lower and only visible in pulldown fraction (Arrow).

Article Snippet: The membrane was blocked in 4%(w/v) skim milk (Nacalai) in ×1 phosphate-buffered saline (PBS) supplemented with 0.1%(v/v) Tween-20, and further incubated with the following antibodies; anti-Aub antibody (guinea pig, 1:500) , anti-GFP antibody (Clontech, rabbit, 1:2000), anti-Ago1 antibody (Abcam, Ab5070, rabbit, 1:1000), anti-Ago2 antibody (guinea pig, 1:100) , anti-Piwi (mouse, P4D2, 1:10), anti-FLAG M2-peroxidase (HRP) (SIGMA, #A8592, 1:5000), anti-H3K18Ac (active motif, #39756, 1:2000), anti-H3K27Ac (active motif, #39136, 1:2000), anti-H4K8Ac (active motif, #61104, 1:2000), anti-H4K12Ac (active motif, #39928, 1:2000), anti-guinea pig immunoglobulins-HRP (DAKO, 1:1000), anti-rabbit IgG-HRP (BioRad, 1:3000), anti-mouse IgG-HRP (BioRad, 1:3000).

Techniques: Labeling, Functional Assay, Silver Staining, Purification, Mass Spectrometry, Expressing, Two Tailed Test, Western Blot, Activity Assay