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    Thermo Fisher gene exp pkd1 mm00465436 g1
    Recombinant PC1 localizes to mitochondria in a subset of cells. ( a . The CTF can be further processed to release a cytoplasmic tail (CTT). Constructs used in the study include full-length human PC1 (hFL) with N-terminal (eGFP or mCherry) and C-terminal (HA or eGFP) tags. ( b ) Lysates from MDCK cells with either stable, inducible expression of vector control (pcDNA5) or <t>eGFP-PKD1-HA</t> constructs probed with GFP or HA antibodies show that the majority of cellular PC1 is free NTF, with only a small amount of uncleaved FL and CTF. ( c – f ) NIH3T3 cells transfected with mCherry-PKD1-eGFP show three main patterns: (1) NTF in the ER with undetectable CTF ( c ); (2) FL and/or NTF/CTF either mostly co-localized in the ER ( d ) or with partial co-localization in the ER and with CTF/CTT in distinct subcellular regions ( e , panel insert); (3) NTF localized to the ER and CTF or CTT in mitochondria ( f ). The endoplasmic reticulum (ER) is identified by transient expression of pEF.myc.ER-E2-Crimson or stained with ER-Tracker™ Blue-White DPX ( c , d ) and mitochondria (Mito) are identified by transient expression of mito-BFP or stained with MitoTracker Deep Red.
    Gene Exp Pkd1 Mm00465436 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Recombinant PC1 localizes to mitochondria in a subset of cells. ( a . The CTF can be further processed to release a cytoplasmic tail (CTT). Constructs used in the study include full-length human PC1 (hFL) with N-terminal (eGFP or mCherry) and C-terminal (HA or eGFP) tags. ( b ) Lysates from MDCK cells with either stable, inducible expression of vector control (pcDNA5) or eGFP-PKD1-HA constructs probed with GFP or HA antibodies show that the majority of cellular PC1 is free NTF, with only a small amount of uncleaved FL and CTF. ( c – f ) NIH3T3 cells transfected with mCherry-PKD1-eGFP show three main patterns: (1) NTF in the ER with undetectable CTF ( c ); (2) FL and/or NTF/CTF either mostly co-localized in the ER ( d ) or with partial co-localization in the ER and with CTF/CTT in distinct subcellular regions ( e , panel insert); (3) NTF localized to the ER and CTF or CTT in mitochondria ( f ). The endoplasmic reticulum (ER) is identified by transient expression of pEF.myc.ER-E2-Crimson or stained with ER-Tracker™ Blue-White DPX ( c , d ) and mitochondria (Mito) are identified by transient expression of mito-BFP or stained with MitoTracker Deep Red.

    Journal: Scientific Reports

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed

    doi: 10.1038/s41598-018-20856-6

    Figure Lengend Snippet: Recombinant PC1 localizes to mitochondria in a subset of cells. ( a . The CTF can be further processed to release a cytoplasmic tail (CTT). Constructs used in the study include full-length human PC1 (hFL) with N-terminal (eGFP or mCherry) and C-terminal (HA or eGFP) tags. ( b ) Lysates from MDCK cells with either stable, inducible expression of vector control (pcDNA5) or eGFP-PKD1-HA constructs probed with GFP or HA antibodies show that the majority of cellular PC1 is free NTF, with only a small amount of uncleaved FL and CTF. ( c – f ) NIH3T3 cells transfected with mCherry-PKD1-eGFP show three main patterns: (1) NTF in the ER with undetectable CTF ( c ); (2) FL and/or NTF/CTF either mostly co-localized in the ER ( d ) or with partial co-localization in the ER and with CTF/CTT in distinct subcellular regions ( e , panel insert); (3) NTF localized to the ER and CTF or CTT in mitochondria ( f ). The endoplasmic reticulum (ER) is identified by transient expression of pEF.myc.ER-E2-Crimson or stained with ER-Tracker™ Blue-White DPX ( c , d ) and mitochondria (Mito) are identified by transient expression of mito-BFP or stained with MitoTracker Deep Red.

    Article Snippet: Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Techniques: Recombinant, Construct, Expressing, Plasmid Preparation, Transfection, Staining

    Pkd1 ko/ko cells have differences in mitochondrial function and morphology. ( a ) Representative distribution of mitochondrial membrane potential in 96784-LTL cells measured by flow cytometry and showing increased frequency of mutant cells with higher TMRM fluorescence. ( b ) Curves of the difference between mutant and wild type TMRM fluorescence for each centile of the TMRM distribution, showing that mutant cells tend to have higher TMRM intensity, particularly in the upper centiles. Each dotted line corresponds to one experiment, colored by cell line. The blue solid line is the best fit of the data. ( c ) P-values comparing TMRM intensity at each centile and showing that mutant cells have significantly higher TMRM for cells in the upper two thirds of the TMRM distribution. Red line: p = 0.05, n = 10. ( d ) Representative image showing 96784-LTL control and Pkd1 ko/ko cells stained with mitochondrial marker (gray: MitoTracker Deep Red; blue: Hoechest 33342 nuclear stain). The panels on the right show higher magnification of the areas inside the red squares. Mitochondria form a more elongated and interconnected network in controls. ( e ) Representative cumulative distribution in 96784-LTL control (blue line) and Pkd1 ko/ko cells showing that at most centiles (y-axis) the solidity is higher (i.e. mitochondrial network is more fragmented) in mutants (red line). ( f ) Measure of mitochondrial fragmentation in independent experiments comparing three matched mutant and control cell lines. The y-axis shows the 25 th percentile solidity (higher values correspond to more fragmented mitochondrial network). Lines connect matched control (left) and mutant (right) for each independent experiment (n = 13 experiments; average number of analyzed mitochondria/dot: 799; p-value

    Journal: Scientific Reports

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed

    doi: 10.1038/s41598-018-20856-6

    Figure Lengend Snippet: Pkd1 ko/ko cells have differences in mitochondrial function and morphology. ( a ) Representative distribution of mitochondrial membrane potential in 96784-LTL cells measured by flow cytometry and showing increased frequency of mutant cells with higher TMRM fluorescence. ( b ) Curves of the difference between mutant and wild type TMRM fluorescence for each centile of the TMRM distribution, showing that mutant cells tend to have higher TMRM intensity, particularly in the upper centiles. Each dotted line corresponds to one experiment, colored by cell line. The blue solid line is the best fit of the data. ( c ) P-values comparing TMRM intensity at each centile and showing that mutant cells have significantly higher TMRM for cells in the upper two thirds of the TMRM distribution. Red line: p = 0.05, n = 10. ( d ) Representative image showing 96784-LTL control and Pkd1 ko/ko cells stained with mitochondrial marker (gray: MitoTracker Deep Red; blue: Hoechest 33342 nuclear stain). The panels on the right show higher magnification of the areas inside the red squares. Mitochondria form a more elongated and interconnected network in controls. ( e ) Representative cumulative distribution in 96784-LTL control (blue line) and Pkd1 ko/ko cells showing that at most centiles (y-axis) the solidity is higher (i.e. mitochondrial network is more fragmented) in mutants (red line). ( f ) Measure of mitochondrial fragmentation in independent experiments comparing three matched mutant and control cell lines. The y-axis shows the 25 th percentile solidity (higher values correspond to more fragmented mitochondrial network). Lines connect matched control (left) and mutant (right) for each independent experiment (n = 13 experiments; average number of analyzed mitochondria/dot: 799; p-value

    Article Snippet: Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Techniques: Flow Cytometry, Cytometry, Mutagenesis, Fluorescence, Staining, Marker

    . Alternatively, PC1-CTT may directly change mitochondrial fusion/fission rates, changing the mitochondrial network and function. How these changes would result in cystic change is not clear, but as discussed in the text, a similar cascade was observed in lymphangiogenesis and planar cell polarity, processes previously linked to Pkd1 . This model does not include PC2 or regulation of NTF/CTF dissociation, aspects likely relevant, but not investigated in this study.

    Journal: Scientific Reports

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed

    doi: 10.1038/s41598-018-20856-6

    Figure Lengend Snippet: . Alternatively, PC1-CTT may directly change mitochondrial fusion/fission rates, changing the mitochondrial network and function. How these changes would result in cystic change is not clear, but as discussed in the text, a similar cascade was observed in lymphangiogenesis and planar cell polarity, processes previously linked to Pkd1 . This model does not include PC2 or regulation of NTF/CTF dissociation, aspects likely relevant, but not investigated in this study.

    Article Snippet: Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Techniques:

    Pkd1 ko/ko cells have metabolic differences. ( a ) Fluxomics. Principal components bi-plot showing clustering of three replicates of a mutant and control immortalized kidney epithelial cell line (94414-LTL) according to flux of 13 C from labeled glucose through different metabolites. Circles are samples, and their location in the plot is determined by a linear combination of specific factors (metabolites). The direction and weight each metabolite contributes to the location of the sample is represented by the direction and size of the corresponding arrow. Mutant (red circles) and control (blue circles) samples cluster in opposite corners of the figure, and labeled arrows show the metabolites that have the highest influence in separating groups. ( b ) Fatty acid uptake assay showing that mutant cells have increased number and size of lipid droplets (green: mitochondria stained with MitoTracker Green; Magenta: BODIPY 558/568 C 12 ). The panels on the right show higher magnification of the areas inside the white squares. ( c ) Quantile plot showing distribution of lipid droplet size quantified in ten random fields in two proximal tubule kidney cell lines (each line is one experiment for one cell line). The insert on the left shows only up to the 80 th quantile, to highlight differences within the lower range of area values (n = 4 experiments, p = 0.044; line: percentile values of the lipid droplet area for one experiment, colored by genotype).

    Journal: Scientific Reports

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed

    doi: 10.1038/s41598-018-20856-6

    Figure Lengend Snippet: Pkd1 ko/ko cells have metabolic differences. ( a ) Fluxomics. Principal components bi-plot showing clustering of three replicates of a mutant and control immortalized kidney epithelial cell line (94414-LTL) according to flux of 13 C from labeled glucose through different metabolites. Circles are samples, and their location in the plot is determined by a linear combination of specific factors (metabolites). The direction and weight each metabolite contributes to the location of the sample is represented by the direction and size of the corresponding arrow. Mutant (red circles) and control (blue circles) samples cluster in opposite corners of the figure, and labeled arrows show the metabolites that have the highest influence in separating groups. ( b ) Fatty acid uptake assay showing that mutant cells have increased number and size of lipid droplets (green: mitochondria stained with MitoTracker Green; Magenta: BODIPY 558/568 C 12 ). The panels on the right show higher magnification of the areas inside the white squares. ( c ) Quantile plot showing distribution of lipid droplet size quantified in ten random fields in two proximal tubule kidney cell lines (each line is one experiment for one cell line). The insert on the left shows only up to the 80 th quantile, to highlight differences within the lower range of area values (n = 4 experiments, p = 0.044; line: percentile values of the lipid droplet area for one experiment, colored by genotype).

    Article Snippet: Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Techniques: Mutagenesis, Labeling, Staining

    PC1-CTT levels are not altered by cellular stress or inhibition of degradation pathways. ( a – c ) Immunoblot of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5. PC1-FL and PC1-CTT amounts are unchanged after ( a ) treatment with 1 μM CCCP for 15 min or 18 hours; ( b ) 2 h hypoxia (0.01% O 2 , 5% CO 2 ); or ( c ). ( d , e ) 24 h treatment with protease inhibitors ( d ) or γ-secretase inhibitor ( e ) had no effect on PC1-CTT detection.

    Journal: Scientific Reports

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed

    doi: 10.1038/s41598-018-20856-6

    Figure Lengend Snippet: PC1-CTT levels are not altered by cellular stress or inhibition of degradation pathways. ( a – c ) Immunoblot of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5. PC1-FL and PC1-CTT amounts are unchanged after ( a ) treatment with 1 μM CCCP for 15 min or 18 hours; ( b ) 2 h hypoxia (0.01% O 2 , 5% CO 2 ); or ( c ). ( d , e ) 24 h treatment with protease inhibitors ( d ) or γ-secretase inhibitor ( e ) had no effect on PC1-CTT detection.

    Article Snippet: Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Techniques: Inhibition, Expressing