Journal: Nature communications

Article Title: Differential trafficking of ligands trogocytosed via CD28 versus CTLA4 promotes collective cellular control of co-stimulation.

doi: 10.1038/s41467-022-34156-1

Figure Lengend Snippet: Fig. 4 | Dissecting mechanisms underlying trogocytosis. a Cartoons depicting putative mechanisms of trogocytosis via intraluminal vesicle (ILV) generation or membrane fusion between donor and recipient cells. b CD80 experiences different pH-levels upon trogocytosis via CD28 versus CTLA4. CD28- or CTLA4-expressing 58αβ A2 T cells were co-cultured with CD80-TagRFP-pHluorin expressing CHO cells. Quantification of pHluorin/TagRFP ratio and extrapolation of pH-levels experienced by trogocytosed CD80 based on standard curve shown in Figure S3. c, d Analysis of membrane fusion by bi-molecular fluorescence complementation (BIFC) assay. c Cartoon depicting putative outcomes of BIFC trogocytosis assay. CD8+ T cells (d) or CD28- or CTLA4-expressing 58αβ A2 T cells (e) expressing cytosolic GFP1–10 were co-cultured with CD80-mScarlet-GFP11-expressing CHO cells. Positive control: GFP1–10 transgenic T cells transduced with CD80-mScarlet- GFP11 (d). Negative control: GFP1–10 transgenic T cells cultured with CD80- mScarlet-transgenic CHO cells. Plot shows increase of GFP gMFI over negative

Article Snippet: Alternatively, 3 × 104 58αβ T cells were added to a monolayer of 4 × 104 CHO cells plated the day before into 96-well flat-bottom plates and co-cultured for up to 6 h. For DC-T cell co-culture 4 × 104 peptide-pulsed DCs were co-cultured with 105 P14+ CD8+ T cells for 24 h. In some experiments 100nM Bafilomycin A1 (Selleckchem)was added.

Techniques: Membrane, Expressing, Cell Culture, Bimolecular Fluorescence Complementation Assay, Trogocytosis Assay, Positive Control, Transgenic Assay, Transduction, Negative Control

Journal: Cells

Article Title: Decanoic Acid Stimulates Autophagy in D. discoideum

doi: 10.3390/cells10112946

Figure Lengend Snippet: Decanoic acid induces autophagosome formation in Dictyostelium . ( A ) Exogenous GFP-Atg8 is integrated into the membranes of the phagophore and autophagosome to allow visualization of autophagy in cells. ( B ) Representative images of Dictyostelium cells expressing GFP-Atg8, untreated (control) or starved (1 h), and following analysis for ( C ) autophagosome number and ( D ) autophagosome size, shown as mean ± SEM ( n = 6, Mann–Whitney t -test). ( E ) Representative images of Dictyostelium cells expressing GFP-Atg8 untreated (control, DMSO) or treated with decanoic acid (DA, 60 µM) or octanoic acid (OA, 60 µM) for 4 or 24 h, autophagy inducer (AR-12, 2.5 µM), or protease inhibitor (2.5×). Images were analyzed for ( F ) autophagosome number and ( G ) autophagosome size ( n ≥ 6), shown as mean ± SEM (Kruskal–Wallis with Dunn’s multiple comparisons test). Significance against control is indicated by ns p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001, or between decanoic acid and octanoic acid, ## p ≤ 0.01. Scale bars represent 20 µm. Each data point is derived from at least 30 individual cells or autophagosomes, with box and whisker plots showing 10–90th percentile range.

Article Snippet: Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control.

Techniques: Expressing, Control, MANN-WHITNEY, Protease Inhibitor, Derivative Assay, Whisker Assay

Journal: Cells

Article Title: Decanoic Acid Stimulates Autophagy in D. discoideum

doi: 10.3390/cells10112946

Figure Lengend Snippet: Decanoic acid enhances autophagic flux in Dictyostelium . Autophagic flux can be measured in Dictyostelium cells expressing GFP-Atg8 by western blot using a GFP-specific antibody to visualise total GFP-Atg8, cleavage to free GFP, and proteolytic degradation of free GFP, with MCCC1 as a loading control, and additionally visualized with total protein. Autophagic flux was analyzed in Dictyostelium cells, under control conditions (solvent-treated growing cells), or cells treated with decanoic acid (DA) or octanoic acid (OA), both at 60 µM, for 4 or 24 h, or with an autophagy inducer or protease inhibitor, showing ( A ) a representative western blot of GFP-Atg8, Free GFP, and loading control (MCCC1), and total protein levels. Quantification of ( B ) GFP-Atg8 and ( C ) free GFP levels, enabled the assessment of autophagic flux provided by ( D ) the ratio of free GFP:GFP-Atg8. Data, analyzed by a Kruskal–Wallis with Dunn’s multiple comparisons test, from n = 8, provides significance indicated by ns p > 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Article Snippet: Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control.

Techniques: Expressing, Western Blot, Control, Solvent, Protease Inhibitor

Journal: Cells

Article Title: Decanoic Acid Stimulates Autophagy in D. discoideum

doi: 10.3390/cells10112946

Figure Lengend Snippet: Medium-chain fatty acids do not reduce transient PIP 3 production in Dictyostelium at concentrations that trigger autophagy. To visualize PIP 3 production, cells expressing PHcrac-GFP in early development were treated with decanoic acid or octanoic acid (both at 60 µM, 4 h), PI3K inhibitor (100 µM LY294002, 15 min), or solvent control (DMSO) and stimulated with 1 µM cAMP to trigger production of PIP 3 visualized by transient movement of PHcrac-GFP to the membrane. ( A ) Cells show transient movement of PHcrac-GFP to cell membranes, reflecting induction of PIP 3 production, and ( B ) membrane fluorescence was quantified normalized to whole cell fluorescence, and ( C ) maximum membrane fluorescence (PIP 3 production) and ( D ) total area under the curve (total PIP 3 production) were compared between treatments. Data represent mean and SEM from six independent experiments (three individual cells were analyzed per experiment). Scale bar represents 10 µm. Significance is indicated by ns > 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (Kruskal–Wallis with Dunn’s multiple comparisons test).

Article Snippet: Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control.

Techniques: Expressing, Solvent, Control, Membrane, Fluorescence

Journal: Cells

Article Title: Decanoic Acid Stimulates Autophagy in D. discoideum

doi: 10.3390/cells10112946

Figure Lengend Snippet: Medium-chain fatty acids do not reduce protein kinase B (PKB) phosphorylation in Dictyostelium at concentrations that trigger autophagy. To visualize the activation of PKB, cells in early development were treated with decanoic acid or octanoic acid (both at 60 µM, 4 h), PI3K inhibitor (100 µM LY294002, 15 min), or solvent control (DMSO) and stimulated with 1 µM cAMP to trigger transient phosphorylation of PKBA, and PKBR1 over 120 s was visualized by western analysis. ( A ) Under these conditions, cells indicated transient phosphorylation of both PKBA and PKBR1 compared to loading control (MCCC1). Quantification of ( B ) PKBA phosphorylation showed a small but significant decrease 20 s after induction in the presence of decanoic acid with no effect on total activation (area under the curve) and a large decrease in the presence of a PI3K inhibitor. Quantification of ( C ) PKBR1 phosphorylation showed a small but significant decrease in phosphorylation in the presence of a PI3K inhibitor, 20 s after induction, and a large decrease in total activation (area under curve). Data were derived from n ≥ 9 independent experiments. Significance is indicated by ns > 0.05, * p ≤ 0.05, *** p ≤ 0.001 (Kruskal–Wallis with Dunn’s multiple comparisons test).

Article Snippet: Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control.

Techniques: Phospho-proteomics, Activation Assay, Solvent, Control, Western Blot, Derivative Assay

Journal: Cells

Article Title: Decanoic Acid Stimulates Autophagy in D. discoideum

doi: 10.3390/cells10112946

Figure Lengend Snippet: Decanoic acid but not octanoic acid increases autophagy gene expression after 24 h. Cells treated with decanoic acid (60 µM) (or control) were analyzed by RT-qPCR measuring the relative abundance of ( A ) atg1 and ( B ) atg8a . Cells treated with octanoic acid (120 µM) (or control) were also analyzed by RT-qPCR measuring the relative abundance of ( C ) atg1 and ( D ) atg8a . mRNA was normalized to the housekeeping gene gapdh in all conditions with the mRNA levels normalized so that untreated cells have a relative expression level of 1 ( n = 3). Data represented are mean ± SEM (Kruskal–Wallis with Dunn’s multiple comparisons test). Significance is indicated by * p ≤ 0.05.

Article Snippet: Cells were treated with decanoic acid (60 μM), octanoic acid (60 μM), or DMSO control for 22 or 2 h to provide final treatment durations of 24 and 4 h. Autophagy inducer AR-12 (Selleckchem, Houston, TX, USA, OSU-03012), or DMSO control, was added, and shaking was continued for 1 h before addition of protease inhibitors (Roche, Basel, Switzerland, 11873580001) at a final concentration of 2.5-fold or HL5 control.

Techniques: Gene Expression, Control, Quantitative RT-PCR, Expressing