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    Structured Review

    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.
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    Images

    1) Product Images from "A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-20856-6

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used:

    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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Inhibition, Expressing

    2) Product Images from "A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed"

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-20856-6

    Recombinant PC1 localizes to mitochondria in a subset of cells. ( a ) Schematics showing PC1 domains and predicted topology. Full-length PC1 (FL) is a 4,302-aa protein with 11 predicted transmembrane (TM) domains 3 . N-terminal to the first TM, a G-protein-coupled receptor cleavage site (GPS) undergoes autocatalytic cleavage to produce 3,048-aa N-terminal (NFT; ~325 kDa) and 1,254-aa C-terminal fragments (CTF; ~150 kDa) that remain non-covalently associated 4 . 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.
    Figure Legend Snippet: Recombinant PC1 localizes to mitochondria in a subset of cells. ( a ) Schematics showing PC1 domains and predicted topology. Full-length PC1 (FL) is a 4,302-aa protein with 11 predicted transmembrane (TM) domains 3 . N-terminal to the first TM, a G-protein-coupled receptor cleavage site (GPS) undergoes autocatalytic cleavage to produce 3,048-aa N-terminal (NFT; ~325 kDa) and 1,254-aa C-terminal fragments (CTF; ~150 kDa) that remain non-covalently associated 4 . 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.

    Techniques Used: 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
    Figure Legend 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

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

    Model of how PC1 signaling could alter nephron architecture. PC1 could respond to ligand binding or mechanical stimuli (from cilia, cell-cell/matrix) by modulating the amount of PC1-CTT that is released and traffics to mitochondria. In mitochondria, PC1-CTT could impact fatty acid oxidation (FAO) either by regulating fatty acid uptake or mitochondrial function. Altered FAO could change the pool of acetyl-CoA (and possibly ROS and NAD+/NADH), with effects in tubulin acetylation (affecting trafficking and cytoskeleton) and histone acetylation (possibly with global gene expression reprogramming), both previously linked to PKD 77 , 78 . 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 function 45 , 79 , 80 . This model does not include PC2 or regulation of NTF/CTF dissociation, aspects likely relevant, but not investigated in this study.
    Figure Legend Snippet: Model of how PC1 signaling could alter nephron architecture. PC1 could respond to ligand binding or mechanical stimuli (from cilia, cell-cell/matrix) by modulating the amount of PC1-CTT that is released and traffics to mitochondria. In mitochondria, PC1-CTT could impact fatty acid oxidation (FAO) either by regulating fatty acid uptake or mitochondrial function. Altered FAO could change the pool of acetyl-CoA (and possibly ROS and NAD+/NADH), with effects in tubulin acetylation (affecting trafficking and cytoskeleton) and histone acetylation (possibly with global gene expression reprogramming), both previously linked to PKD 77 , 78 . 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 function 45 , 79 , 80 . This model does not include PC2 or regulation of NTF/CTF dissociation, aspects likely relevant, but not investigated in this study.

    Techniques Used: Ligand Binding Assay, Expressing

    PC1 CTT is enriched in mitochondria. ( a ) Immunoblot showing total cell lysates (“mito−”) and mitochondrial enriched fractions (“mito+”) of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5 and of MEFs obtained from wild type or transgenic Pkd1 F/H BAC mice expressing PKD1-HA. While Full-length (FL) and CTF (CTF) are detected in both total lysates and in the mitochondrial fractions, only the PC1 C-terminal fragment (CTT, molecular weight between 15~20 KDa) is consistently enriched in the mitochondrial fraction. The ratio of the lower two bands varied from experiment to experiment (see also Fig. 6a and e , and Supplementary Fig. S3 ). The cropped blots at the bottom of each panel show subsequent staining of the same membrane with nuclear (HP1β) and mitochondrial (Tim23) markers. ( b ) Constructs of mouse PC1 CTT used in panel “c” to map the putative MTS, a sequence which it is highly conserved. A previously reported nuclear localization sequence (NLS) 7 is shown in red. ( c ) Fluorescence imaging in live cells expressing eGFP or mPC1-CTT truncation constructs described in panel “b” (Mito: MitoTracker Deep Red). ( d ) NIH3T3 cells transiently transfected with various constructs. Previously published constructs 8 that express hPC1-CTT either fused to a membrane anchor (CD16.7-h-PC1) or as a smaller truncated form, both including the MTS and NLS sequences. The top panel shows the predominant membrane-anchored pattern of CD16.7-hPC1; the middle panel shows that in some cells this construct is presumably cleaved, releasing a fragment that localizes to mitochondria; in the lower panel the truncated hPC1-CTT is seen predominantly in mitochondria, with diffuse expression elsewhere in the cell but without nuclear enrichment. ( e ) Live cell imaging of human MTS sequence variants fused to eGFP (in green) co-localized with mitotracker (magenta). L4137P and L4139P are predicted to be pathogenic while V4146I is predicted to be a normal variant.
    Figure Legend Snippet: PC1 CTT is enriched in mitochondria. ( a ) Immunoblot showing total cell lysates (“mito−”) and mitochondrial enriched fractions (“mito+”) of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5 and of MEFs obtained from wild type or transgenic Pkd1 F/H BAC mice expressing PKD1-HA. While Full-length (FL) and CTF (CTF) are detected in both total lysates and in the mitochondrial fractions, only the PC1 C-terminal fragment (CTT, molecular weight between 15~20 KDa) is consistently enriched in the mitochondrial fraction. The ratio of the lower two bands varied from experiment to experiment (see also Fig. 6a and e , and Supplementary Fig. S3 ). The cropped blots at the bottom of each panel show subsequent staining of the same membrane with nuclear (HP1β) and mitochondrial (Tim23) markers. ( b ) Constructs of mouse PC1 CTT used in panel “c” to map the putative MTS, a sequence which it is highly conserved. A previously reported nuclear localization sequence (NLS) 7 is shown in red. ( c ) Fluorescence imaging in live cells expressing eGFP or mPC1-CTT truncation constructs described in panel “b” (Mito: MitoTracker Deep Red). ( d ) NIH3T3 cells transiently transfected with various constructs. Previously published constructs 8 that express hPC1-CTT either fused to a membrane anchor (CD16.7-h-PC1) or as a smaller truncated form, both including the MTS and NLS sequences. The top panel shows the predominant membrane-anchored pattern of CD16.7-hPC1; the middle panel shows that in some cells this construct is presumably cleaved, releasing a fragment that localizes to mitochondria; in the lower panel the truncated hPC1-CTT is seen predominantly in mitochondria, with diffuse expression elsewhere in the cell but without nuclear enrichment. ( e ) Live cell imaging of human MTS sequence variants fused to eGFP (in green) co-localized with mitotracker (magenta). L4137P and L4139P are predicted to be pathogenic while V4146I is predicted to be a normal variant.

    Techniques Used: Expressing, Transgenic Assay, BAC Assay, Mouse Assay, Molecular Weight, Staining, Construct, Sequencing, Fluorescence, Imaging, Transfection, Live Cell Imaging, Variant Assay

    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).
    Figure Legend 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).

    Techniques Used: 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 ) 48 h serum starvation. The bottom panel in “b” confirms Hif1α induction in the total lysate of the corresponding samples. In panel “b”, the same blot is shown at low signal intensity in the high molecular weight range, and at high signal intensity in the low molecular weight range, to optimize visualization of the relevant bands (original images are presented in Supplementary Fig. S6A ). The samples in panels “c” and “e” were run on the same gel for each experiment, with the middle, irrelevant lanes removed for clarity (see original images, Supplementary Fig. S6B and C ). ( d , e ) 24 h treatment with protease inhibitors ( d ) or γ-secretase inhibitor ( e ) had no effect on PC1-CTT detection.
    Figure Legend 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 ) 48 h serum starvation. The bottom panel in “b” confirms Hif1α induction in the total lysate of the corresponding samples. In panel “b”, the same blot is shown at low signal intensity in the high molecular weight range, and at high signal intensity in the low molecular weight range, to optimize visualization of the relevant bands (original images are presented in Supplementary Fig. S6A ). The samples in panels “c” and “e” were run on the same gel for each experiment, with the middle, irrelevant lanes removed for clarity (see original images, Supplementary Fig. S6B and C ). ( d , e ) 24 h treatment with protease inhibitors ( d ) or γ-secretase inhibitor ( e ) had no effect on PC1-CTT detection.

    Techniques Used: Inhibition, Expressing, Molecular Weight

    Related Articles

    Transduction:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: At the time of inactivation, a corresponding control was generated using viral transduction with plasmid LV-Lac (Addgene 12108 ). .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Amplification:

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method. .. 2.6 Statistical Analysis Comparison of kidney/body weight (KBW) mutant male vs. female curves was performed using generalized linear model in R ( ) and the effect size was estimated calculating Cohen's d with the compute.es package in R. Analysis of KBW in mutants fed NIH31 or NIH37 was done using Student's t test.

    In Vitro:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: Kidney cell lines derived from Pkd1 cko/cko animals were immortalized and inactivated in vitro as previously described . .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Polymerase Chain Reaction:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    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). .. Cells were grown in DMEM/F12 media (Life cat. no. 21041-025) with 2% FBS (GEMINI Bio-Products cat. no. 100-106), 1 × Insulin-Transferrin-Selenium (Thermo Fisher Scientific, cat. no. 41400-045), 5 µM dexamethasone (SIGMA, cat. no. D1756), 10 ng/ml EGF (SIGMA, cat. no. SRP3196), 1 nM 3,3′,5-Triiodo-L-thyronine (SIGMA, cat. no. T6397) and 10 mM HEPES (CORNING, cat. no. 25-060-CI).

    Mutagenesis:

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: 2.5 mRNA Expression Studies Eighty mouse kidneys with induced deletion of Pkd1 at P40 were harvested between 102 and 210 days of age (14 control females, 21 mutant females, 19 control males and 26 mutant males). .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method.

    Isolation:

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: Total RNA was isolated using Trizol (Life, cat. no. 15596-018) followed by RNeasy plus kit (Qiagen, cat. no. 74136). .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method.

    Cell Culture:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    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). .. Whole embryos were minced, washed in PBS and cultured in six-well tissue culture plates in Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS).

    Mouse Assay:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: Briefly, kidneys from several mice (121112-C: male, < 12-days-old; 94414: male, 463-days-old; 96784: male, 61-days-old) were harvested, minced and digested using a collagenase/hyaluronidase solution (Stemcell technologies, cat. no. 07912) followed by proximal or collecting/distal tubule cell enrichment using, respectively, biotinylated Lotus Tetragonolobus Lectin (LTL) (Vector Laboratories, cat. no. B-1325) or biotinylated Dolichos Biflorus Agglutinin (DBA) (Vector Laboratories, ca. no. B-1035) and Cellection Biotin Binder kit (ThermoFischer, cat. no. 11533D). .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Microarray:

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: Further sample processing for microarray analysis was performed by the University of Chicago Genomics Facility, Knapp Center for Biomedical Discovery, following the facility's protocols, and hybridized to Illumina's MouseRef-8 v2.0 BeadChip expression arrays. .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method.

    Generated:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: At the time of inactivation, a corresponding control was generated using viral transduction with plasmid LV-Lac (Addgene 12108 ). .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method. .. 2.6 Statistical Analysis Comparison of kidney/body weight (KBW) mutant male vs. female curves was performed using generalized linear model in R ( ) and the effect size was estimated calculating Cohen's d with the compute.es package in R. Analysis of KBW in mutants fed NIH31 or NIH37 was done using Student's t test.

    Plasmid Preparation:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: At the time of inactivation, a corresponding control was generated using viral transduction with plasmid LV-Lac (Addgene 12108 ). .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

    Expressing:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    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). .. Cells were grown in DMEM/F12 media (Life cat. no. 21041-025) with 2% FBS (GEMINI Bio-Products cat. no. 100-106), 1 × Insulin-Transferrin-Selenium (Thermo Fisher Scientific, cat. no. 41400-045), 5 µM dexamethasone (SIGMA, cat. no. D1756), 10 ng/ml EGF (SIGMA, cat. no. SRP3196), 1 nM 3,3′,5-Triiodo-L-thyronine (SIGMA, cat. no. T6397) and 10 mM HEPES (CORNING, cat. no. 25-060-CI).

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method. .. 2.6 Statistical Analysis Comparison of kidney/body weight (KBW) mutant male vs. female curves was performed using generalized linear model in R ( ) and the effect size was estimated calculating Cohen's d with the compute.es package in R. Analysis of KBW in mutants fed NIH31 or NIH37 was done using Student's t test.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Fatty Acid Oxidation is Impaired in An Orthologous Mouse Model of Autosomal Dominant Polycystic Kidney Disease
    Article Snippet: .. For RT-PCR, cDNA was generated using Superscript Vilo (Life, cat. no. 11755050) amplified with the expression assay Mm00465436_g1 (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372), normalized to Gapdh and analyzed using the delta–delta-Ct method. .. 2.6 Statistical Analysis Comparison of kidney/body weight (KBW) mutant male vs. female curves was performed using generalized linear model in R ( ) and the effect size was estimated calculating Cohen's d with the compute.es package in R. Analysis of KBW in mutants fed NIH31 or NIH37 was done using Student's t test.

    BAC Assay:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    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). .. Mouse embryonic fibroblasts (MEFs) were obtained from E12.5 wild type or Pkd1 F/H BAC mice (expressing mouse Pkd1 with three C-terminal HA tags ).

    Derivative Assay:

    Article Title: A cleavage product of Polycystin-1 is a mitochondrial matrix protein that affects mitochondria morphology and function when heterologously expressed
    Article Snippet: Kidney cell lines derived from Pkd1 cko/cko animals were immortalized and inactivated in vitro as previously described . .. Pkd1 inactivation was confirmed using genomic PCR and/or reverse-transcriptase PCR (TaqMan gene expression assay, Applied Biosystems, cat. no. 4351372, Mm00465436_g1).

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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: 92/100, based on 1 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

    Recombinant PC1 localizes to mitochondria in a subset of cells. ( a ) Schematics showing PC1 domains and predicted topology. Full-length PC1 (FL) is a 4,302-aa protein with 11 predicted transmembrane (TM) domains 3 . N-terminal to the first TM, a G-protein-coupled receptor cleavage site (GPS) undergoes autocatalytic cleavage to produce 3,048-aa N-terminal (NFT; ~325 kDa) and 1,254-aa C-terminal fragments (CTF; ~150 kDa) that remain non-covalently associated 4 . 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 ) Schematics showing PC1 domains and predicted topology. Full-length PC1 (FL) is a 4,302-aa protein with 11 predicted transmembrane (TM) domains 3 . N-terminal to the first TM, a G-protein-coupled receptor cleavage site (GPS) undergoes autocatalytic cleavage to produce 3,048-aa N-terminal (NFT; ~325 kDa) and 1,254-aa C-terminal fragments (CTF; ~150 kDa) that remain non-covalently associated 4 . 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

    Model of how PC1 signaling could alter nephron architecture. PC1 could respond to ligand binding or mechanical stimuli (from cilia, cell-cell/matrix) by modulating the amount of PC1-CTT that is released and traffics to mitochondria. In mitochondria, PC1-CTT could impact fatty acid oxidation (FAO) either by regulating fatty acid uptake or mitochondrial function. Altered FAO could change the pool of acetyl-CoA (and possibly ROS and NAD+/NADH), with effects in tubulin acetylation (affecting trafficking and cytoskeleton) and histone acetylation (possibly with global gene expression reprogramming), both previously linked to PKD 77 , 78 . 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 function 45 , 79 , 80 . 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: Model of how PC1 signaling could alter nephron architecture. PC1 could respond to ligand binding or mechanical stimuli (from cilia, cell-cell/matrix) by modulating the amount of PC1-CTT that is released and traffics to mitochondria. In mitochondria, PC1-CTT could impact fatty acid oxidation (FAO) either by regulating fatty acid uptake or mitochondrial function. Altered FAO could change the pool of acetyl-CoA (and possibly ROS and NAD+/NADH), with effects in tubulin acetylation (affecting trafficking and cytoskeleton) and histone acetylation (possibly with global gene expression reprogramming), both previously linked to PKD 77 , 78 . 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 function 45 , 79 , 80 . 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: Ligand Binding Assay, Expressing

    PC1 CTT is enriched in mitochondria. ( a ) Immunoblot showing total cell lysates (“mito−”) and mitochondrial enriched fractions (“mito+”) of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5 and of MEFs obtained from wild type or transgenic Pkd1 F/H BAC mice expressing PKD1-HA. While Full-length (FL) and CTF (CTF) are detected in both total lysates and in the mitochondrial fractions, only the PC1 C-terminal fragment (CTT, molecular weight between 15~20 KDa) is consistently enriched in the mitochondrial fraction. The ratio of the lower two bands varied from experiment to experiment (see also Fig. 6a and e , and Supplementary Fig. S3 ). The cropped blots at the bottom of each panel show subsequent staining of the same membrane with nuclear (HP1β) and mitochondrial (Tim23) markers. ( b ) Constructs of mouse PC1 CTT used in panel “c” to map the putative MTS, a sequence which it is highly conserved. A previously reported nuclear localization sequence (NLS) 7 is shown in red. ( c ) Fluorescence imaging in live cells expressing eGFP or mPC1-CTT truncation constructs described in panel “b” (Mito: MitoTracker Deep Red). ( d ) NIH3T3 cells transiently transfected with various constructs. Previously published constructs 8 that express hPC1-CTT either fused to a membrane anchor (CD16.7-h-PC1) or as a smaller truncated form, both including the MTS and NLS sequences. The top panel shows the predominant membrane-anchored pattern of CD16.7-hPC1; the middle panel shows that in some cells this construct is presumably cleaved, releasing a fragment that localizes to mitochondria; in the lower panel the truncated hPC1-CTT is seen predominantly in mitochondria, with diffuse expression elsewhere in the cell but without nuclear enrichment. ( e ) Live cell imaging of human MTS sequence variants fused to eGFP (in green) co-localized with mitotracker (magenta). L4137P and L4139P are predicted to be pathogenic while V4146I is predicted to be a normal variant.

    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 is enriched in mitochondria. ( a ) Immunoblot showing total cell lysates (“mito−”) and mitochondrial enriched fractions (“mito+”) of MDCK cells with stable, inducible expression of PKD1 (eGFP-PKD1-HA) or pcDNA5 and of MEFs obtained from wild type or transgenic Pkd1 F/H BAC mice expressing PKD1-HA. While Full-length (FL) and CTF (CTF) are detected in both total lysates and in the mitochondrial fractions, only the PC1 C-terminal fragment (CTT, molecular weight between 15~20 KDa) is consistently enriched in the mitochondrial fraction. The ratio of the lower two bands varied from experiment to experiment (see also Fig. 6a and e , and Supplementary Fig. S3 ). The cropped blots at the bottom of each panel show subsequent staining of the same membrane with nuclear (HP1β) and mitochondrial (Tim23) markers. ( b ) Constructs of mouse PC1 CTT used in panel “c” to map the putative MTS, a sequence which it is highly conserved. A previously reported nuclear localization sequence (NLS) 7 is shown in red. ( c ) Fluorescence imaging in live cells expressing eGFP or mPC1-CTT truncation constructs described in panel “b” (Mito: MitoTracker Deep Red). ( d ) NIH3T3 cells transiently transfected with various constructs. Previously published constructs 8 that express hPC1-CTT either fused to a membrane anchor (CD16.7-h-PC1) or as a smaller truncated form, both including the MTS and NLS sequences. The top panel shows the predominant membrane-anchored pattern of CD16.7-hPC1; the middle panel shows that in some cells this construct is presumably cleaved, releasing a fragment that localizes to mitochondria; in the lower panel the truncated hPC1-CTT is seen predominantly in mitochondria, with diffuse expression elsewhere in the cell but without nuclear enrichment. ( e ) Live cell imaging of human MTS sequence variants fused to eGFP (in green) co-localized with mitotracker (magenta). L4137P and L4139P are predicted to be pathogenic while V4146I is predicted to be a normal variant.

    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: Expressing, Transgenic Assay, BAC Assay, Mouse Assay, Molecular Weight, Staining, Construct, Sequencing, Fluorescence, Imaging, Transfection, Live Cell Imaging, Variant Assay

    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 ) 48 h serum starvation. The bottom panel in “b” confirms Hif1α induction in the total lysate of the corresponding samples. In panel “b”, the same blot is shown at low signal intensity in the high molecular weight range, and at high signal intensity in the low molecular weight range, to optimize visualization of the relevant bands (original images are presented in Supplementary Fig. S6A ). The samples in panels “c” and “e” were run on the same gel for each experiment, with the middle, irrelevant lanes removed for clarity (see original images, Supplementary Fig. S6B and 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 ) 48 h serum starvation. The bottom panel in “b” confirms Hif1α induction in the total lysate of the corresponding samples. In panel “b”, the same blot is shown at low signal intensity in the high molecular weight range, and at high signal intensity in the low molecular weight range, to optimize visualization of the relevant bands (original images are presented in Supplementary Fig. S6A ). The samples in panels “c” and “e” were run on the same gel for each experiment, with the middle, irrelevant lanes removed for clarity (see original images, Supplementary Fig. S6B and 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, Molecular Weight