Journal: Frontiers in Cell and Developmental Biology

Article Title: Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study

doi: 10.3389/fcell.2022.893677

Figure Lengend Snippet: TP53 -silenced Hke3 cells with a competent KRAS gene display significantly increased cytosolic ATP kinetics in response to extracellular substrates addition. WT, p53 KO, Hke3 and Hke3 p53 KD HCT-116 cells were separately transfected with the cytosolic ATP-sensitive (ATeam) FRET probe, loaded with 30 nM TMRM as a Δ ψ m indicator (non-quenched mode), mounted on the heated stage of an LSM 710 confocal microscope and assayed over 80 min at 37°C. (A) Experimental model schematic. C ells were allowed to equilibrate for 60 min on stage (starvation period), after which alterations in Δ ψ m and cytosolic ATP were monitored in single cells. Following a baseline of 20 min, cells were exposed to sequential additions of 2 mM lactate, 5 mM glucose and 10 µM FCCP at the indicated time points (20, 40 and 60 min) to promote mitochondrial respiration, glycolysis and, Δ ψ m depolarization and mitochondrial ATP disruption, respectively. (B) Differential interference contrast (DIC) and fluorescence measurements were recorded for TMRM, FRET, CFP, YFP by time-lapse confocal microscopy. FRET/CFP ratio and DIC representative images of Hke3 cells expressing ATeam probe are shown as a ratio of FRET/CFP normalized to baseline to monitor cytosolic ATP. Scale bar = 20 µm. (C) Kinetics of all cells monitored, expressed as means ± SEM are shown, additions are labelled with black arrows on top of the graph. (D) Analysis of the cytosolic ATP levels (FRET/CFP, normalized) before each drug treatment (20, 40 and 60 min) and at the end of the experiment (80 min). (E) Change of cytosolic ATP baseline levels (FRET/CFP, normalized) over the first 20 min, (F) quantification of the slope of cytosolic ATP (the change in FRET/CFP ratio over time in minutes), (G) the mean fold change in FRET/CFP ratio and (H) the mean duration for this change in FRET/CFP ratio after each treatment are illustrated. Means ± SEM are shown from at least three independent experiments for each cell line (WT, n = 54; p53 KO, n = 75; Hke3 n = 55; Hke3 p53 KD, n = 61). * p ≤ 0.05 compared to WT control cells; # p ≤ 0.05 between cell lines (ANOVA, post-hoc Tukey).

Article Snippet: HCT-116 cell lines, transfected with the FRET-based cytosolic or mitochondrial ATP indicators, ATeam or mitoATeam respectively, were washed twice with KB and the medium replaced with 1 ml of KB containing the membrane-permeant cationic fluorescent probe TMRM (30 nM) and 2.5 mM CaCl 2 .

Techniques: Transfection, Microscopy, Disruption, Fluorescence, Confocal Microscopy, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study

doi: 10.3389/fcell.2022.893677

Figure Lengend Snippet: TP53 -silenced Hke3 cells with a competent KRAS show significantly increased mitochondrial ATP levels during metabolic stress. WT, p53 KO, Hke3 and Hke3 p53 KD HCT-116 cells were separately transfected with the mitochondrial ATP-sensitive (mitoATeam) FRET probe, loaded with 30 nM TMRM as a Δ ψ m indicator (non-quenched mode), mounted on the heated stage of an LSM 710 confocal microscope and assayed over 80 min at 37°C. C ells were exposed as mentioned and illustrated in . (A) Differential interference contrast (DIC) and fluorescence measurements were recorded for TMRM, FRET, CFP, YFP by time-lapse confocal microscopy. FRET/CFP ratio and DIC representative images of Hke3 cells expressing mitoATeam probe are shown as a ratio of FRET/CFP normalized to baseline to monitor mitochondrial ATP. Scale bar = 20 µm. (B) Kinetics of all cells monitored, expressed as means ± SEM are shown, additions are labelled with black arrows on top of the graph. (C) Analysis of the mitochondrial ATP levels (FRET/CFP, normalized) before each drug treatment (20, 40 and 60 min) and at the end of the experiment (80 min). (D) Change of mitochondrial ATP baseline levels (FRET/CFP, normalized) over the first 20 min, (E) quantification of the slope of mitochondrial ATP (the change in FRET/CFP ratio over time in minutes), (F) the mean fold change in FRET/CFP ratio and (G) the mean duration for this change in FRET/CFP ratio after each treatment are illustrated. Means ± SEM are shown from at least three independent experiments for each cell line (WT, n = 31; p53 KO, n = 42; Hke3 n = 42; Hke3 p53 KD, n = 31). * p ≤ 0.05 compared to WT control cells; # p ≤ 0.05 between cell lines (ANOVA, post-hoc Tukey). (H) Total ATP concentration normalized to microgram of proteins for each cell line after 80 min of nutrients starvation. All data are expressed as mean ± SEM from experimental triplicates and three independent cultures. * p ≤ 0.05 compared to WT control cells; # p ≤ 0.05 between cell lines (ANOVA, post-hoc Tukey).

Article Snippet: HCT-116 cell lines, transfected with the FRET-based cytosolic or mitochondrial ATP indicators, ATeam or mitoATeam respectively, were washed twice with KB and the medium replaced with 1 ml of KB containing the membrane-permeant cationic fluorescent probe TMRM (30 nM) and 2.5 mM CaCl 2 .

Techniques: Transfection, Microscopy, Fluorescence, Confocal Microscopy, Expressing, Control, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study

doi: 10.3389/fcell.2022.893677

Figure Lengend Snippet: KRAS mutation leads to minimal mitochondrial membrane potential changes in response to metabolic substrates. WT, p53 KO, Hke3 and Hke3 p53 KD HCT-116 cells were separately loaded with 30 nM TMRM as a Δ ψ m indicator (non-quenched mode), mounted on the heated stage of an LSM 710 confocal microscope and assayed over 80 min at 37°C. Cells were exposed as mentioned and illustrated in . DIC and fluorescent measurements were recorded for TMRM by time-lapse confocal microscopy. Mitochondrial membrane potential imaging data are expressed as a fluorescence intensity normalized to baseline. (A) Mitochondrial membrane potential, before and after drugs addition, was analyzed, and TMRM fluorescence and DIC representative images of Hke3 cells are shown. (B) Kinetics of all cells monitored are shown as means ± SEM. Scale bar = 20 µm. (C) Analysis of TMRM fluorescence levels before each drug treatment (20, 40 and 60 min) and at the end of the experiment (80 min), additions are indicated with black arrows on top. (D) TMRM baseline levels over the first 20 min, (E) quantification of the slope of TMRM fluorescence (the change in TMRM fluorescence intensity over time in minutes), (F) the mean fold change in TMRM fluorescence intensity (G) and the mean duration for change in TMRM fluorescence intensity after each treatment are illustrated. Means ± SEM are shown from at least three independent experiments for each cell line (WT, n = 31; p53 KO, n = 42; Hke3 n = 42; Hke3 p53 KD, n = 31). * p ≤ 0.05 compared to WT control cells; # p ≤ 0.05 between cell lines (ANOVA, post-hoc Tukey).

Article Snippet: HCT-116 cell lines, transfected with the FRET-based cytosolic or mitochondrial ATP indicators, ATeam or mitoATeam respectively, were washed twice with KB and the medium replaced with 1 ml of KB containing the membrane-permeant cationic fluorescent probe TMRM (30 nM) and 2.5 mM CaCl 2 .

Techniques: Mutagenesis, Membrane, Microscopy, Confocal Microscopy, Imaging, Fluorescence, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: Effect of TP53 deficiency and KRAS signaling on the bioenergetics of colon cancer cells in response to different substrates: A single cell study

doi: 10.3389/fcell.2022.893677

Figure Lengend Snippet: Origin and mutations of human colorectal cancer HCT-116 cell lines.

Article Snippet: HCT-116 cell lines, transfected with the FRET-based cytosolic or mitochondrial ATP indicators, ATeam or mitoATeam respectively, were washed twice with KB and the medium replaced with 1 ml of KB containing the membrane-permeant cationic fluorescent probe TMRM (30 nM) and 2.5 mM CaCl 2 .

Techniques: Mutagenesis, Generated, shRNA