low molecular weight rna marker  (Thermo Fisher)


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    Structured Review

    Thermo Fisher low molecular weight rna marker
    Low Molecular Weight Rna Marker, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 87/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/low molecular weight rna marker/product/Thermo Fisher
    Average 87 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    low molecular weight rna marker - by Bioz Stars, 2020-01
    87/100 stars

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    In Vitro:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: Paragraph title: In vitro transcription and RNAs substrate preparation ... Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min.

    Labeling:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: An RrrR duplex (dsRrrR) was produced by hybridizing the [α 32 P]-ATP labeled RrrR ssRNA sense transcript with the non-labeled RrrR ssRNA antisense transcript. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min.

    Produced:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: An RrrR duplex (dsRrrR) was produced by hybridizing the [α 32 P]-ATP labeled RrrR ssRNA sense transcript with the non-labeled RrrR ssRNA antisense transcript. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min.

    Incubation:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: Both RNA molecules were mixed in DEPC water in a 1:10 ratio (sense:antisense), incubated at 95°C during 5 min and subsequently cooled down to room temperature. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: RNA in vitro transcripts were separated by denaturing PAGE (8 M urea; 1× TBE; 10% polyacrylamide), and full-length bands were cut out using sterile scalpels after brief autoradiographic exposure. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min.

    Marker:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: Both RNA molecules were mixed in DEPC water in a 1:10 ratio (sense:antisense), incubated at 95°C during 5 min and subsequently cooled down to room temperature. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min. .. Semi-dry electrophoretic transfer was used to transfer RNA that was separated on denaturing polyacrylamide gels onto a positively charged nylon membrane (Roti® -Nylon plus, pore size 0.45 μm).

    Molecular Weight:

    Article Title: A regulatory RNA is involved in RNA duplex formation and biofilm regulation in Sulfolobus acidocaldarius
    Article Snippet: Both RNA molecules were mixed in DEPC water in a 1:10 ratio (sense:antisense), incubated at 95°C during 5 min and subsequently cooled down to room temperature. .. Low Molecular Weight RNA Marker, 10–100 nt (Affymetrix, cat# 76410) was 5′- radiolabeled in a 20 μl reaction volume as following: 10 μl of the RNA, 2 μl 10× T4 Polynucleotide Kinase (PNK) buffer (New England Biolabs (NEB)), 25 U T4 PNK (NEB) and 2.0 μl of [γ 32 P]-ATP (800 Ci/mmol, 10 μCi/μl) at 37°C for 30 min. .. Semi-dry electrophoretic transfer was used to transfer RNA that was separated on denaturing polyacrylamide gels onto a positively charged nylon membrane (Roti® -Nylon plus, pore size 0.45 μm).

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    Thermo Fisher gene exp pkd1 mm00465436 g1
    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 <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 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp pkd1 mm00465436 g1/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    gene exp pkd1 mm00465436 g1 - by Bioz Stars, 2020-01
    99/100 stars
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    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, Hemagglutination Assay, 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: Gene Knockout, 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, Hemagglutination Assay, 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: Gene Knockout, 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, Hemagglutination Assay, Molecular Weight