Journal: Biochimica et biophysica acta

Article Title: The arachidonic acid-LTB4-BLT2 pathway enhances human B-CLL aggressiveness.

doi: 10.1016/j.bbadis.2014.07.016

Figure Lengend Snippet: Fig. 1. Analysis of cPLA2-alpha and iPLA2-beta expression according to disease stage. A: A representative blot from a Western blotting analysis is shown. B: Mean expression normalized to actin for n = 12 controls and n = 56 patients, as determined by densitometry analysis. Control stands for healthy B CD5 positive cells (N95% purity as ascertained by flow cytometry).

Article Snippet: Primary antibodies were purchased from Abcam for anti-actin (catalog number ab1801, rabbit polyclonal), iPLA2 (ab23706, rabbit polyclonal, does not cross-react with cPLA2 or sPLA2), pan-PLA2 (ab9014, sheep polyclonal), COX1 (ab695, mouse monoclonal), and COX2 (ab15191, rabbit polyclonal), from cell signaling technology for cPLA2 (catalog number 2832, rabbit polyclonal, does not cross-react with iPLA2 or sPLA2).

Techniques: Expressing, Western Blot, Control, Cytometry

Journal: Biochimica et biophysica acta

Article Title: The arachidonic acid-LTB4-BLT2 pathway enhances human B-CLL aggressiveness.

doi: 10.1016/j.bbadis.2014.07.016

Figure Lengend Snippet: Fig. 2. Analysis of cPLA2-alpha and iPL2-beta expression and disease progression. Analysis over a 36 months follow-up for slowly progressing (A, n = 24) or 24 months follow-up for rap- idly progressing (B, n = 19) disease. Two representative series of analyses are shown per group of patients. C and D: Mean expression normalized to actin (densitometry analysis) for patients with a slowly progressing disease (C, n = 24) or a highly progressing disease (D, n = 19).

Article Snippet: Primary antibodies were purchased from Abcam for anti-actin (catalog number ab1801, rabbit polyclonal), iPLA2 (ab23706, rabbit polyclonal, does not cross-react with cPLA2 or sPLA2), pan-PLA2 (ab9014, sheep polyclonal), COX1 (ab695, mouse monoclonal), and COX2 (ab15191, rabbit polyclonal), from cell signaling technology for cPLA2 (catalog number 2832, rabbit polyclonal, does not cross-react with iPLA2 or sPLA2).

Techniques: Expressing, Biomarker Discovery

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6–R741Q iPSCs show impaired mitochondrial respiration. (A) Schematic representation of functional domains of PLA2G6 protein with the R741Q and R747W mutation sites—seven ankyrin domains marked as Ank (between amino acids 150–382), a glycine‐rich nucleotide‐binding domain (red oval shape, centred at amino acid 485), GTSTG lipase catalytic domain with the serine active site (S519) highlighted in light red (pink oval shape, amino acids 517–521), calmodulin‐binding domain marked as CaMBMD (blue oval shape, amino acids 747–759). The locations of mutations are R741Q, present in the patient‐derived iPSCs (red) and R747W in the CRISPR‐engineered iPSCs (purple); (B) representative average trace of mitochondrial respiration of PLA2G6‐R741Q PD iPSCs (PD, red) and its familial control‐derived iPSCs (Control, black) from Seahorse mitochondrial stress assay (n=3 independent cell culture preparations, Control iPSC‐11 assay wells, PD iPSC‐12 assay wells). The time of addition of oligomycin, FCCP, rotenone, and antimycin is marked with black arrows; (C) rate of basal oxygen consumption; (D) rate of ATP production; (E) rate of proton leak; (F) rate of maximal respiration; (G) rate of non‐mitochondrial oxygen consumption; (H) spare respiratory capacity. (B‐H) represented as mean ± SEM after normalization with protein content. Each dot represents a Seahorse assay plate well, Shapiro–Wilk test was done to determine normality of the distribution, Mann‐Whitney U test * p < 0.05, ns‐non‐significant ( p ≥ 0.05). OCR‐oxygen consumption rate. Color codes: PD‐Patient‐derived iPSCs with R741Q (Red), Control‐familial control‐derived iPSCs (Black).

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Derivative Assay, CRISPR, Control, Cell Culture, MANN-WHITNEY

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: Details of primers.

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Sequencing

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6‐R741Q iPSCs show reduced mitochondrial calcium release. (A) Change in average traces of mitochondrial calcium release after treatment with 5 μM FCCP in Ca 2+ ‐free HBSS in Control and PD iPSCs measured by Rhod‐2 AM, a cell‐permeant dye specific to mitochondrial calcium, represented as mean ± SEM at each measurement, n=3 (independent cell culture preparations), Control iPSC = 306 cells, PD iPSC = 323 cells; (B) average normalized Rhod‐2 AM fluorescence of first 25 frames in the Ca 2+ ‐free HBSS represented as the basal mitochondrial calcium (mitocalcium). Each dot represents an individual cell, and the 25th and 75th percentiles are represented as the box, the mean as the bar, and SEM as the error bars. *** p < 0.001, Mann‐Whitney U test; (C) distribution of cells corresponding to different basal Rhod‐2 AM fluorescence; (D) representative images of Control and PD iPSCs stained with Rhod‐2 AM under the Airyscan microscope before and after the addition of FCCP, single Z plane, Scale bar‐10 μm, cell peripheries marked with white lines; (E) change in mitochondrial calcium levels measured by the difference from basal to minimum fluorescence after the addition of FCCP. Each dot represents an individual cell, the 25th and 75th percentiles are represented as the box, the mean as the bar, the median as the dashed bar and SEM as the error bars. ** p < 0.01, Mann‐Whitney U test; (F) mRNA expression levels of genes involved in mitochondrial calcium transport were unaltered. n=6 (except NCLX , n=5) independent cell culture preparations, each dot represents a biological replicate. Mean ± SD, Student's t‐test.

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Control, Cell Culture, Fluorescence, MANN-WHITNEY, Staining, Microscopy, Expressing

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6‐R741Q iPSCs are more sensitive to changes in mitochondrial membrane potential due to uncoupling. (A) Representative images of Control and PD iPSCs stained with TMRE‐a mitochondrial membrane potential dye under the Airyscan microscope before and after the addition of FCCP, single Z plane, scale bar‐10 μm; (B) change in average traces of mitochondrial membrane potential after uncoupling with 5 μM FCCP in HBSS in Control and PD iPSCs measured by TMRE, represented as mean ± SEM at each measurement, n=3 (independent cell culture preparations), Control iPSC = 213 cells, PD iPSC = 218 cells; and (C) change in mitochondrial membrane potential measured by the difference between the basal and the minimum fluorescence levels after the addition of FCCP. Each dot represents an individual cell, the 25th and 75th percentiles are represented as the box, the mean is the bar, the median is the dashed bar, and SEM is the error bar. ** p < 0.01, Mann‐Whitney U test ; (D) the rate of change in mitochondrial membrane potential calculated by the average of the first derivative of the change in mitochondrial membrane potential from t = 25 to 50 s. Each dot represents an individual cell, and the 25th and 75th percentiles are represented as the box, the mean as the solid bar, the median as the dashed bar, and SEM as the error bars. *** p < 0.001, Mann‐Whitney U test.

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Membrane, Control, Staining, Microscopy, Cell Culture, Fluorescence, MANN-WHITNEY

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6‐R741Q iPSCs exhibit an elevation in glycolytic activity. (A) Representative average trace of glycolytic function of PD iPSCs with R741Q‐PLA2G6 (PD, red) and its control iPSCs (Control, black) from the Seahorse glycolytic stress assay (n=3 independent cell culture preparations, Control iPSC and PD iPSC–10 assay wells each), time of addition of glucose, oligomycin, and 2‐deoxyglucose are marked with black arrows; (B) rate of glycolysis; (C) glycolytic capacity; (D) glycolytic reserve; (E) non‐glycolytic acidification. (B–E) represented as mean ± SEM after normalization with protein content. Each dot represents a Seahorse assay plate well. The Shapiro–Wilk test was done to determine normality of the distribution; (B) the Mann–Whitney U test; (C–E) the Student's t‐test with equal variance, * p < 0.05, ** p < 0.01, ns‐non‐significant ( p ≥ 0.05). ECAR‐extracellular acidification rate, Color codes: PD‐patient‐derived iPSCs with R741Q (Red), Control‐familial control‐derived iPSCs (Black).

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Activity Assay, Control, Cell Culture, MANN-WHITNEY, Derivative Assay

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6‐R741Q iPSCs have reduced store‐operated calcium entry (SOCE). (A, B) Change in average traces of cytoplasmic calcium ([Ca 2+ ] cyto ) after blocking SERCA with 1 μM TG ( t = 50 to 550 s) and adding 2 mM Ca 2+ ( t = 550 to 1050 s) in Ca 2+ free HBSS in control (n = 4 independent cell culture preparations, 348 cells) and PD iPSCs (n = 5 independent cell culture preparations, 400 cells) (A) and WT (n= 3 independent cell culture preparations, 163 cells) and mutant iPSC (n = 4 independent cell culture preparations, 139 cells) (B) measured by Fura‐2 AM, a ratiometric cell‐permeant cytoplasmic calcium dye, represented as mean ± SEM at each measurement, n ≥ 4 together includes > 300 cells; (C, D) change in [Ca 2+ ] cyto after SOCE‐Maximum change in [Ca 2+ ] cyto from the basal [Ca 2+ ] cyto after blocking SERCA with 1 μM TG and adding 2 mM Ca 2+ in Ca 2+ free HBSS; (E, F) the average rate of change in [Ca 2+ ] cyto due to SOCE calculated by the first derivative from t = 550 to 700 s in Ca 2+ free HBSS; (G, H) basal [Ca 2+ ] cyto calculated by the average of [Ca 2+ ] cyto t = 0 to 50 s in Ca 2+ free HBSS; (I, J) maximum change in [Ca 2+ ] cyto from the basal [Ca 2+ ] cyto after blocking SERCA with 1 μM TG ( t = 50 to 550 s) in Ca 2+ free HBSS. (C–J) each dot represents an individual cell, and the 25th and 75th percentiles are represented as the box, the mean as the solid bar, the median as the dashed bar and SEM as the error bars. *** p < 0.001, ns‐non‐significant ( p ≥ 0.05), Mann‐ Whitney U test. PD‐Patient‐derived iPSCs with R741Q‐PLA2G6 (Red), Control‐familial control‐derived iPSCs (Black), Mutant‐CRISPR‐engineered iPSCs with R747W‐ PLA2G6 (Purple), WT‐Wild type iPSCs (Blue).

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Blocking Assay, Control, Cell Culture, Mutagenesis, MANN-WHITNEY, Derivative Assay, CRISPR

Journal: Journal of Neurochemistry

Article Title: Altered Mitochondrial Bioenergetics and Calcium Kinetics in Young‐Onset PLA2G6 Parkinson's Disease iPSCs

doi: 10.1111/jnc.70059

Figure Lengend Snippet: PLA2G6‐R741Q iPSCs show higher oxidative stress but unaltered autophagic flux. (A) Confocal images of Control and PD iPSCs stained with DCFDA, a ROS‐specific dye at the basal level, Z‐ Stack projection, Scale bar‐ 20 μM; (B) basal ROS calculated using fluorescence from the frame with maximal Z‐projection. Each dot represents an individual cell, and the 25th and 75th percentiles are represented as the box, the mean as the solid bar, the median as the dashed bar, and SEM as the error bars. *** p < 0.001, Mann‐Whitney U test. n = 5 independent cell culture preparations, Control iPSC = 536 cells and PD iPSC = 687 cells; (C) mRNA levels of Catalase and other antioxidant enzymes. n = 6 independent cell culture preparations, each dot represents a biological replicate. Mean ± SD. Student' t –test *** p < 0.001; (D) representative images of the western blot of protein lysates of PD and iPSCs treated with DMSO (vehicle) or bafilomycin A probed for LC3 II, LC3 I, and housekeeping protein β‐ Tubulin; (E) LC3 II/LC I ratio of each group: Control iPSC (black) and PD iPSC (red) with DMSO or bafilomycin A. Each dot represents a biological sample, n = 3 independent cell culture preparations, paired t‐ test, * p < 0.05, SEM ± SD; (F) autophagic flux after treatment with bafilomycin A, calculated as the increase in LC3 II/LC3 I from the basal levels, each dot represents a biological sample, n = 3 independent cell culture preparations, paired t ‐test, ns‐ non‐significant. SEM ± SD; (G) Representative images of CTRL iPSCs and PD iPSCs treated with DMSO (vehicle) or bafilomycin A from the tandem fluorescent quenching assay (TFQA). Left to Right: Panel 1‐representative images under the red channel (RFP), Panel 2‐corresponding images under the green channel (GFP), Panel 3‐merge of both channels and Panel 4‐zoomed images of the cells (Scale bar‐10 μM). Different treatment groups are labeled;. (H) ratio of autolysosomes (AL) to autophagosomes (AP); (I) Quantification of the number of autolysosomes per cell. (H,I) Each dot represents an individual cell, the median as the dashed bar, and SEM as the error bars. * p < 0.05, ** p < 0.01, *** p < 0.001, ns‐non‐significant ( p ≥ 0.05), Shapiro‐Wilk test, Mann‐ Whitney U test. PD‐patient‐derived iPSCs with R741Q‐PLA2G6 (Red), Control‐familial control‐derived iPSCs (Black).

Article Snippet: For validation experiments, CRISPR‐edited iPSCS with R747W mutation in the PLA2G6 loci and its isogenic control were used (Synthego, CA).

Techniques: Control, Staining, Fluorescence, MANN-WHITNEY, Cell Culture, Western Blot, Labeling, Derivative Assay