Journal: Stem Cell Reports

Article Title: Impairment of Mitochondrial Calcium Buffering Links Mutations in C9ORF72 and TARDBP in iPS-Derived Motor Neurons from Patients with ALS/FTD

doi: 10.1016/j.stemcr.2020.03.023

Figure Lengend Snippet: Impairment of Mitochondrial Ca 2+ Buffering Due to Reduced MCU and MICU2 Levels in C9ORF72 MNs (A and B) (A) Representative image of Rhod 2AM-loaded neurons and (B) representative Ca 2+ traces. (C) Ca 2+ uptake in the mitochondria is lower in C9ORF72 MNs compared with the C9-Ed MNs and healthy control MNs on glutamate stimulation; n = 15–62 neurons from two to three independent differentiations. (D) Mitochondrial Ca 2+ uptake in response to caffeine is significantly reduced in C9ORF72 MNs ( ∗∗∗ p < 0.001); n = 8–23 neurons from two to three independent differentiations. (E) Representative Ca 2+ traces recorded in response to glutamate stimulation and CCCP. (F) Amplitude of Ca 2+ released from mitochondria is significantly lower in C9ORF72 MNs ( ∗∗ p < 0.01, ∗∗∗ p < 0.001); n = 27–39 neurons from two independent differentiations. (G and H) Immunoblotting of MCU (G) shows reduced levels in C9ORF72 MNs (H) ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (I–K) (I) RNA-seq showing MICU2 levels are downregulated in C9ORF72 iPS-derived MNs compared with C9-Ed and (J) immunoblotting confirming reduced expression of MICU2 in C9-02 and (K) qPCR confirming reduced MICU2 expression ( ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001). Data are shown as mean ± SEM with one-way ANOVA and Sidak's post hoc tests. See also Figure S5 .

Article Snippet: The stimuli used for calcium release were 50 mM KCl (Sigma), 1 mM L-glutamic acid (Sigma), 4 μM CCCP (Tocris Bioscience), 20 mM caffeine (Tocris Bioscience), 100 μM CNQX (Tocris Bioscience), and 10 μM memantine (Tocris Bioscience).

Techniques: Control, Western Blot, RNA Sequencing, Derivative Assay, Expressing

Journal: PLOS Pathogens

Article Title: Microsporidian Encephalitozoon hellem inhibits host mitophagy by inducing ERAD to degrade BNIP3L

doi: 10.1371/journal.ppat.1014078

Figure Lengend Snippet: (A) HEK293 cells were treated with CCCP (20 μM) for 6 hours. Western blot analysis was performed to detect the protein levels of tubulin (control), LC3I and LC3II, the latter of which indicates the activation of mitophagy. (B) HEK293 cells were transfected with plasmids expressing EhPTP4 and EGFP (control) for 48 hours, respectively. Subsequently, the cells were treated with CCCP (20 μM) for 6 hours. Western blot analysis was used to detect the protein levels of tubulin, LC3I and LC3II. (C) BNIP3L expression of HEK293 cells was knocked down by transfecting siBNIP3L for 48 hours. RT-qPCR was employed to detect the mRNA levels of BNIP3L, and Western blot was conducted to detect the protein levels of BNIP3L and tubulin (control). (D) HEK293 cells were transfected with siBNIP3L for 48 hours and then treated with CCCP (20 μM) for 6 hours. Western blot analysis was carried out to detect the protein levels of LC3I, LC3II and tubulin (control) using specific antibodies, respectively. The data are presented as the mean ± SEM of three independent experiments. Statistical analysis was performed using the Student’s t-test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: Following transfection, the culture medium was replaced with fresh medium containing the mitochondrial uncoupler CCCP (Selleck Chemicals, #S6494) at a final concentration of 20 μM.

Techniques: Western Blot, Control, Activation Assay, Transfection, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: MIC of compounds targeting mycobacterial bioenergetics.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques:

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: 2B8 collapses both components of the mycobacterial PMF: Δψ and ΔpH. The fluorescent dyes DiSC 3 (5) and ACMA were used to evaluate 2B8’s effect on mycobacterial Δψ ( a , b ) and ΔpH ( c , d ), respectively. Uptake by live M . smegmatis slowly quenched DiSC 3 (5) fluorescence ( a , b ). Likewise, ACMA fluorescence was quenched upon energizing M . smegmatis IMVs with NADH (arrow), to create a ΔpH ( c , d ). Thereafter, bacteria or IMVs were treated with different drugs and monitored for fluorescence reversal. As indicated by fluorescence reversal, treatment with 31.25–125 µM 2B8 abruptly depolarized M . smegmatis membrane potential ( a ) and collapsed the ΔpH generated by M . smegmatis IMVs ( c ). Both parameters were also affected by 15 µM CCCP and 80 µM TRZ ( b , d ). The ΔpH ( d ) but not Δψ ( b ), was collapsed with 18 µM BDQ. A similar pattern was observed for 125 µM RA13 ( a , c ). 5 µM valinomycin and 10 µM nigericin were used as positive controls for Δψ and ΔpH. Experiments were repeated three separate times and representative data are shown. ***p < 0.001 by ANOVA.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques: Fluorescence, Bacteria, Membrane, Generated

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: 2B8 increases M . tuberculosis respiration at low concentrations. Oxygen consumption of M . tuberculosis mc 2 6206 strain was monitored in real-time using high-resolution respirometry. Upon bacterial inoculation, the oxygen consumption rate (OCR) was measured at steady state (white columns, without compounds). Compounds were added in stepwise increments (depicted under each drug) and the change in OCR reported as fold-increase compared to the steady state without treatment. The lower right panel is a composite of the actual OCR levels achieved after stepwise increments in compound concentration, as depicted for respective compounds (for clarity, statistical significance and compound concentrations were not included for the lower right panel). Treatment with 2B8 and CCCP resulted in a bell-shaped effect: increased OCR was observed at lower concentrations, whereas higher concentrations resulted in OCR levels similar to no treatment. RA13 induced minimal changes in OCR, with only a small increase at 125 µM. BDQ increased OCR until 9 µM and this was maintained (albeit with a decreasing trend), upon treatment with additional BDQ. The OCR increase induced by TRZ was sustained at all the tested concentrations. Experiments were repeated three different times and all biological replicates were analyzed together. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: 2B8 reduces intracellular ATP levels in M . tuberculosis . Intracellular ATP levels in M . tuberculosis were quantified by RLUs at 2 and 24 h after treatment. Number of CFUs was also determined in parallel and data normalized to RLUs/CFUs. After 2 h treatment, only BDQ and TRZ significantly reduced M . tuberculosis intracellular ATP levels. After 24 h, in addition to BDQ and TRZ, intracellular ATP was also reduced with 62.5 and 125 µM 2B8, and CCCP. In contrast, RA13 had no effect. Experiments were repeated three separate times and all replicates were pooled together for analysis. **p < 0.01, ***p < 0.001 by ANOVA.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques:

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: 2B8 increases M . tuberculosis NADH/NAD + ratio. After 2 or 24 h treatment, intracellular NADH and NAD + concentrations were determined and the NADH/NAD + ratio was calculated. Treatment with 62.5 and 125 µM 2B8 for 2 h resulted in a significant increase of the NADH/NAD + ratio. Treatment with BDQ and TRZ also increased the ratio significantly. In contrast, 125 µM RA13 did not. After 24 h treatment, 62.5 and 125 µM 2B8, BDQ, and TRZ resulted in a significantly elevated NADH/NAD + ratio. However, RA13 and CCCP were ineffective at changing the NADH/NAD + ratio. Experiments were repeated three separate times and all replicates were pooled for analysis. *p < 0.05, **p < 0.01, ***p < 0.001 by ANOVA.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques:

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: Inhibition of NADH oxidation by 2B8 is partially restored by CFZ. ( a ) NADH oxidation by M . smegmatis IMVs was evaluated by monitoring the rate of fluorescence decay at 340 ex /460 em . Treatment with 2B8 at 78.125–125 µM, 5 mM KCN and 80 µM TRZ potently inhibited NADH oxidation. In contrast, NADH oxidation was minimally inhibited by a lower concentration of 2B8 (62.5 µM) or 125 µM RA13. BDQ at 18 µM had an intermediate inhibitory activity, whereas NADH oxidation was accelerated with 15 µM CCCP. ( b , c ). Restoration of NADH oxidation by clofazimine (CFZ) was measured in M . smegmatis IMVs treated with 5 mM KCN ( b ), 80 µM TRZ ( b ), or 125 µM 2B8 ( c ). Addition of 42 µM CFZ (dotted lines) restored NADH oxidation in KCN-treated IMVs ( b ). As expected, CFZ did not restore NADH oxidation in TRZ-treated IMVs ( b ). In contrast, CFZ also restored NADH oxidation in 2B8-treated IMVs ( c ), albeit partially in comparison to KCN-treated samples. Experiments were done three separate times and representative data are shown. ***p < 0.001 by ANOVA.

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques: Inhibition, Fluorescence, Concentration Assay, Activity Assay, Comparison

Journal: Scientific Reports

Article Title: 2-aminoimidazoles collapse mycobacterial proton motive force and block the electron transport chain

doi: 10.1038/s41598-018-38064-7

Figure Lengend Snippet: Fold β-lactam potentiation with Bioenergetics-Targeting Compounds a .

Article Snippet: Cultures were treated with DMSO control, 7.825–250 μM 2B8 and RA13 (provided by Dr. Christian Melander), 15 μM carbonyl cyanide m-chlorophenyl hydrazine (CCCP), 80 μM TRZ or 18 μM BDQ (BOC Sciences, Shirley, NY, USA).

Techniques:

Journal: PLoS ONE

Article Title: Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

doi: 10.1371/journal.pone.0101840

Figure Lengend Snippet: Effect of efflux inhibitor, CCCP and phytochemicals in accumulation of ethidium bromide in P. aeruginosa strain.

Article Snippet: Cyanide-m-chlorophenylhydrazone (CCCP), ethidium bromide, carbenicillin and levofloxacin were purchased from Hi-Media, India.

Techniques:

Journal: PLoS ONE

Article Title: Identification of Natural Compound Inhibitors for Multidrug Efflux Pumps of Escherichia coli and Pseudomonas aeruginosa Using In Silico High-Throughput Virtual Screening and In Vitro Validation

doi: 10.1371/journal.pone.0101840

Figure Lengend Snippet: Effect of efflux inhibitor, CCCP and phytochemicals in accumulation of ethidium bromide in E. coli strain.

Article Snippet: Cyanide-m-chlorophenylhydrazone (CCCP), ethidium bromide, carbenicillin and levofloxacin were purchased from Hi-Media, India.

Techniques:

Journal: PLoS ONE

Article Title: Transcriptional Analysis of MexAB-OprM Efflux Pumps System of Pseudomonas aeruginosa and Its Role in Carbapenem Resistance in a Tertiary Referral Hospital in India

doi: 10.1371/journal.pone.0133842

Figure Lengend Snippet: MIC reduction assay using meropenem.

Article Snippet: Efflux pump activity of the strains were phenotypically detected by using meropenem (10 mg, Himedia, Mumbai) with and without CCCP (carbonyl cyanide m-chlorophenylhydrazone, 100mM, Himedia, Mumbai)[ ].

Techniques:

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