1 μm cccp  (Cayman Chemical)

Journal: bioRxiv

Article Title: Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma

doi: 10.1101/2024.05.27.596106

Figure Lengend Snippet: (A) Immunoblot of lysates from AML12 WT and clones c14 and c4 using an antibody against PKAc confirm heterozygous DP expression in the clones. (B) Proliferation of cellular models of FLC compared to WT AML12 cells; cells were counted on days 2, 3, and 4 after plating; n=3. (C, D) Representative traces (C) and quantification (D) of mitochondrial Ca 2+ release assays in AML12 cells; cells were treated with uncoupler CCCP, and the relative amount of Ca 2+ released was quantified using a Ca 2+ indicator dye; statistical significance was determined by one-sample t-test; n=10. (E) Representative trace of mitochondrial free Ca 2+ levels of AML12 WT and c14 cells quantified using matrix-targeted Ca 2+ reporter G-GECO (mito-G-GECO). (F, G) Baseline mito-G-GECO fluorescence normalized to minimum and maximum signals in AML12 WT and c14 (F) or c4 (G) cells; statistical significance determined by Mann Whitney test; n=20-23 (F) and n=12 (G). (H, I) Representative traces (H) and mitochondrial Ca 2+ uptake rates in AML12 cells (I); mitochondrial Ca 2+ uptake rates were calculated by monitoring Ca 2+ clearance in the presence of a Ca 2+ indicator dye; statistical significance determined by one-sample t-test; n=9. (J) Immunoblot of MCU shows comparable MCU expression in FLC clones compared to WT controls. (K) Mitochondrial Ca 2+ release after cells were treated with 5 μM PKA inhibitor BLU2864 or DMSO for 4 days was measured as in (C); fold change in released Ca 2+ is shown relative to DMSO control for each cell line; statistical significance determined by paired t-test, n=7-9. (L) Seahorse extracellular flux analysis in FLC clones compared to WT AML12 cells at baseline and after indicated treatments; n=10-16. All error bars indicate standard deviation; * indicates a p-value < 0.05, ** indicates a p-value < 0.01, *** indicates a p-value < 0.001, **** indicates a p-value < 0.0001

Article Snippet: Finally, mitochondrial membrane potential was dissipated with the addition of 1 μM CCCP (Cayman Chemical Company, 25458), and reads were taken every second for 2 min to obtain the maximum fluorescence.

Techniques: Western Blot, Clone Assay, Expressing, Fluorescence, MANN-WHITNEY, Standard Deviation

Journal: bioRxiv

Article Title: Mitochondrial Calcium Signaling Regulates Branched-Chain Amino Acid Catabolism in Fibrolamellar Carcinoma

doi: 10.1101/2024.05.27.596106

Figure Lengend Snippet: (A) Resting mitochondrial membrane potential measured by the difference in TMRM fluorescence before and after CCCP addition, normalized to WT AML12 cells. (B) Immunoblot of AML12 lysates with a PKA substrate motif antibody after 5 μM BLU2864 or DMSO treatment for 4 days. (C-G) Indicated mitochondrial parameters of AML12 cells from Seahorse extracellular flux analysis in ; statistical significance determined by the Dunnett test following Welch’s one-way ANOVA; n=10-16. All error bars indicate standard deviation; ns indicates non-significant, ** indicates a p-value < 0.01, *** indicates a p-value < 0.001, and **** indicates a p-value < 0.0001

Article Snippet: Finally, mitochondrial membrane potential was dissipated with the addition of 1 μM CCCP (Cayman Chemical Company, 25458), and reads were taken every second for 2 min to obtain the maximum fluorescence.

Techniques: Membrane, Fluorescence, Western Blot, Standard Deviation

Journal: European journal of immunology

Article Title: Nuclear retention of interleukin-1? by necrotic cells: a mechanism to dampen sterile inflammation

doi: 10.1002/eji.200939712

Figure Lengend Snippet: COS-7 cells transiently transfected with GFP or IL-1α-GFP were killed by ATP-depletion (30 minutes, 1 μM CCCP, 15mM 2-DOG) and OGD (24 hours). Control cells were kept under normoxic conditions and trypsinised. Necrotic, detached cells and live, trypsinised cells were collected and PI-stained. Cellular GFP and PI fluorescence was measured by FACS. Scatter plots (A) show PI and GFP fluorescence intensities of necrotic cells in one representative experiment. Dashed rectangle indicates PI+/GFP+ cells. Mean GFP fluorescence of GFP and IL-1α-GFP positive COS-7 cells was measured in live, trypsinised cells and in dead, PI positive cells (C). *p<0.05, **p<0.01, ***p<0.001, One-way ANOVA with post-hoc Bonferroni multiple comparison. The proportion of GFP fluorescence retained on cell death was calculated for GFP- and IL-1α-GFP-expressing cells (D). **p<0.01, Student’s t-test. Data from n = 3 independent experiments.

Article Snippet: COS-7 cells were ATP depleted by treatment with 1 μM CCCP + 15 mM 2-DOG (30 minutes) in glucose-free DMEM (OGD experiments, Invitrogen, UK) or imaging buffer [FRAP experiments, [ 19 ]].

Techniques: Transfection, Staining, Fluorescence, Expressing