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Addgene inc pc1 hyper c199s
Pc1 Hyper C199s, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pc1 hyper c199s - by Bioz Stars, 2026-05

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Full-length and deletion myc-tagged constructs of mBicc1 were co-expressed with either full-length HA-tagged PC1 or PC2 in HEK-293 cells and tested for their ability to interact by co-IP. ( a ) Schematic diagram of the constructs used in this experiment. ( b ) Western blot analysis following co-IP experiments, using GST tagged constructs as bait, identified protein interactions between PC1 or PC2 domains and mBicc1. pcDNA3 was included as a negative control. CT = C-terminus, NT = N-terminus, GST = glutathione S-Transferase. Co-IP experiments (n=3) were quantified in ( e ). ( c ) Western blot showing expression of recombinant myc-tagged mBicc1 generated by in vitro translation or myc-tagged mBicc1 transfected in HEK-293 cells. ( d ) Western blot analysis following in vitro pulldown experiments, using purified GST tagged constructs as bait, identified direct protein interactions between PC1 or PC2 domains and in vitro translated myc-Bicc1. In vitro binding experiments (n=3) were quantified in ( f ). ( g ) Western blot analysis following co-IP experiments, using a rabbit PC1 antibody (2b7) as bait, identified protein interactions between endogenous PC1 and BICC1 in UCL93 cells. A non-immune rabbit IgG antibody or no antibody was included as a negative control; * denotes a non-specific IgG band which is not present in the no antibody control lane. ( h ) Western blot analysis following co-IP experiments, using an anti-BICC1 or anti-PC2 antibody as bait, identified protein interactions between endogenous PC2 and BICC1 in UCL93 cells. Non-immune goat and mouse IgG was included as a negative control. Figure 1—source data 1. Original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot displayed in .

Journal: eLife

Article Title: BICC1 interacts with PKD1 and PKD2 to drive cystogenesis in ADPKD

doi: 10.7554/eLife.106342

Figure Lengend Snippet: Full-length and deletion myc-tagged constructs of mBicc1 were co-expressed with either full-length HA-tagged PC1 or PC2 in HEK-293 cells and tested for their ability to interact by co-IP. ( a ) Schematic diagram of the constructs used in this experiment. ( b ) Western blot analysis following co-IP experiments, using GST tagged constructs as bait, identified protein interactions between PC1 or PC2 domains and mBicc1. pcDNA3 was included as a negative control. CT = C-terminus, NT = N-terminus, GST = glutathione S-Transferase. Co-IP experiments (n=3) were quantified in ( e ). ( c ) Western blot showing expression of recombinant myc-tagged mBicc1 generated by in vitro translation or myc-tagged mBicc1 transfected in HEK-293 cells. ( d ) Western blot analysis following in vitro pulldown experiments, using purified GST tagged constructs as bait, identified direct protein interactions between PC1 or PC2 domains and in vitro translated myc-Bicc1. In vitro binding experiments (n=3) were quantified in ( f ). ( g ) Western blot analysis following co-IP experiments, using a rabbit PC1 antibody (2b7) as bait, identified protein interactions between endogenous PC1 and BICC1 in UCL93 cells. A non-immune rabbit IgG antibody or no antibody was included as a negative control; * denotes a non-specific IgG band which is not present in the no antibody control lane. ( h ) Western blot analysis following co-IP experiments, using an anti-BICC1 or anti-PC2 antibody as bait, identified protein interactions between endogenous PC2 and BICC1 in UCL93 cells. Non-immune goat and mouse IgG was included as a negative control. Figure 1—source data 1. Original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot displayed in .

Article Snippet: Primary antibodies used in this study were mouse anti-BICC1 mAb (clone A12, Santa Cruz Biotechnologies, sc-514846), rabbit anti-BICC1 (Sigma-Aldrich, HPA045212, RRID: AB_10959667 ), mouse anti-PC1 mAb (clone 7e12, Santa Cruz Biotechnologies, sc-130554, RRID: AB_2163355 ) , rabbit anti-PC1 (clone 2b7) , goat anti-PC2 (sc-10376, Santa Cruz), rabbit anti-PC2 Ab (YCC2, a kind gift from Dr. S. Somlo or Santa Cruz Biotech, SC-28331, RRID: AB_672377 ), rat anti-HA (clone 3F10, Roche, 11867423001, RRID: AB_390918 ), mouse anti-GST mAb (Santa Cruz Biotechnologies, sc-138, RRID: AB_627677 ), rat anti-Myc (clone JAC6, Bio-Rad, MCA1929, RRID: AB_322203 ), mouse anti-V5-Tag mAb (clone SV5-Pk1, Biorad, MCA1360, RRID: AB_322378 ), rabbit anti-GAPDH mAb (clone 14C10, Cell Signaling, 2118, RRID: AB_561053 ) and mouse anti-γ-Tubulin mAb (clone GTU-88, Sigma-Aldrich, T6557, RRID: AB_477584 ).

Techniques: Construct, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Expressing, Recombinant, Generated, In Vitro, Transfection, Purification, Binding Assay, Control

Journal: eLife

Article Title: BICC1 interacts with PKD1 and PKD2 to drive cystogenesis in ADPKD

doi: 10.7554/eLife.106342

Techniques: Construct, Co-Immunoprecipitation Assay, Western Blot, Negative Control, Expressing, Recombinant, Generated, In Vitro, Transfection, Purification, Binding Assay, Control

Journal: eLife

Article Title: BICC1 interacts with PKD1 and PKD2 to drive cystogenesis in ADPKD

doi: 10.7554/eLife.106342

Figure Lengend Snippet: Full-length and deletion myc-tagged constructs of mBicc1 were co-expressed with either full-length HA-tagged PC1 or PC2 in HEK-293 cells and tested for their ability to interact by co-IP. ( a ) Schematic diagram of the constructs used in this set of experiments with the amino acid positions of full-length mBicc1 or the different deletions indicated. ( b, c ) Western blot analysis following co-IP experiments, using a PC1-HA-tagged construct as bait, identified protein interactions between PC1 and mBicc1 domains. pcDNA3 was included as a negative control ( b ). co-IP experiments (n=3) were quantified in ( c ). ( d, e ) Western blot analysis following co-IP experiments, using a PC2-HA tagged construct as bait, identified protein interactions between PC2 and mBicc1 domains ( d ). pcDNA3 was included as a negative control. Quantification of the co-IP experiments (n=3) is shown in ( e ). ( f, g ) Western blot analysis following co-IP experiments, using a PC1-HA-tagged construct as bait. The interaction between PC1 and PC2 was not altered in the presence of either full-length mBicc1 or its deletion domains. pcDNA3 was included as a negative control. Asterix represents non-specific interaction with mouse IgG. ( f ). co-IP experiments (n=3) were quantified in ( g ). One-way ANOVA comparisons were performed to assess significance, and p values are indicated. Error bars represent standard error of the mean. Figure 2—source data 1. Original western blots for , indicating the relevant bands. Figure 2—source data 2. Original files for western blot displayed in .

Techniques: Construct, Co-Immunoprecipitation Assay, Western Blot, Negative Control

( a–c ) BICC1 p.G821E/ PKD2 p.C632R patient with pedigree and the electropherograms ( a ), the structural analysis of the PKD2 showing the local structure around the cysteine at position 632 (indicated in red) and its putative impact in the variant including the REVEL score ( b ) as well as its location within the global PC2 structure highlighting the potential of the variant impacting the PC2 ion channel function ( c ). ( d, e ) Western blot analysis for PC2 comparing wildtype HEK293T, HEK293T BICC1 p.Gly821Glu (BICC1-G821E), HEK293T BICC1 p.Ser240Pro (BICC1-S240P) and HEK293T BICC1 knockout (BICC1-KO) cells and quantification thereof. γ-Tubulin was used as loading control. ( f–i ) BICC1 c.1179+1G>T/PKD1 p.Ala3981Val patient with pedigree and the electropherograms ( f ), the ultrasound analysis of the left and right kidneys ( g, h ) and the structural analysis of the PC1 showing the local structure around the alanine at position 3981 (indicated in red) and its putative impact in the variant including the REVEL score ( i ). Figure 5—source data 1. Original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot displayed in .

Journal: eLife

Article Title: BICC1 interacts with PKD1 and PKD2 to drive cystogenesis in ADPKD

doi: 10.7554/eLife.106342

Figure Lengend Snippet: ( a–c ) BICC1 p.G821E/ PKD2 p.C632R patient with pedigree and the electropherograms ( a ), the structural analysis of the PKD2 showing the local structure around the cysteine at position 632 (indicated in red) and its putative impact in the variant including the REVEL score ( b ) as well as its location within the global PC2 structure highlighting the potential of the variant impacting the PC2 ion channel function ( c ). ( d, e ) Western blot analysis for PC2 comparing wildtype HEK293T, HEK293T BICC1 p.Gly821Glu (BICC1-G821E), HEK293T BICC1 p.Ser240Pro (BICC1-S240P) and HEK293T BICC1 knockout (BICC1-KO) cells and quantification thereof. γ-Tubulin was used as loading control. ( f–i ) BICC1 c.1179+1G>T/PKD1 p.Ala3981Val patient with pedigree and the electropherograms ( f ), the ultrasound analysis of the left and right kidneys ( g, h ) and the structural analysis of the PC1 showing the local structure around the alanine at position 3981 (indicated in red) and its putative impact in the variant including the REVEL score ( i ). Figure 5—source data 1. Original western blots for , indicating the relevant bands. Figure 5—source data 2. Original files for western blot displayed in .

Techniques: Variant Assay, Western Blot, Knock-Out, Control

Substitution of six nucleotides within the miR-17 motif stabilizes Pkd1 mRNA. ( A ) Schematic illustrates the CRISPR/Cas9 editing + HDR template DNA approach utilized to precisely switch six nucleotides within the miR-17 motif in the Pkd1 RC 3′UTR. ( B ) Sanger sequencing of tail DNA from Pkd1 RC/RC*17 mouse demonstrates successful 3′UTR editing. The edited allele with disrupted miR-17 motif is denoted in pink, and the unedited allele with intact miR-17 motif is shown in blue. ( C ) Allele-specific DNA genotyping primers selectively detected edited [primer positions indicated as pink and black arrows in panel (B)] and unedited [primer positions indicated blue and black arrows in panel (B)] Pkd1 RC alleles. ( D ) Allele-specific qRT-PCR primers were used to compare Pkd1 RC and Pkd1 RC*17 transcript abundance within primary kidney epithelial cells derived from Pkd1 RC/RC*17 mice. qRT-PCR demonstrated higher abundance of Pkd1 RC*17 mRNA compared to Pkd1 RC mRNA. ( E ) Primary Pkd1 RC/RC*17 cells were treated with actinomycin and harvested at 2-h intervals to measure abundance of Pkd1 mRNA transcripts. qRT-PCR analysis demonstrated slower degradation of Pkd1 RC*17 mRNA compared to Pkd1 RC mRNA. c-Myc and Gapdh mRNA degradation was measured as internal controls. ( F ) Pkd1 F/F or Pkd1 −/− cells (Top panel) and Pkd1 RC/− or Pkd1 RC*17/− cells (lower panel) were transfected with the CRISPR-based Pkd1 exon-4 mRNA GFP sensor (green). Live-cell images and the quantification of percentage of cells exhibiting the GFP-positive puncta marking Pkd1 mRNA is shown in the lower left corner. The essential lack of detection of GFP puncta in Pkd1 −/− cells indicates specificity of Pkd1 mRNA sensor. A 78% increase in Pkd1 mRNA detection in Pkd1 RC*17/− cells compared to Pkd1 RC/− cells implies increased Pkd1 mRNA transcript in cells lacking the miR-17 motif. ( G ) Western blot demonstrates PC1 expression is increased in Pkd1 RC*17/− cells compared to Pkd1 RC/− cells, whereas PC2 and AQP2 expression remain unchanged. Error bars indicated SEM. Statistics: paired t -test (D); nonlinear regression (E); unpaired two-tailed t- test (F).

Journal: Nucleic Acids Research

Article Title: Disruption of a six-nucleotide miRNA motif improves PKD1 dosage and ameliorates polycystic kidney disease

doi: 10.1093/nar/gkaf1538

Figure Lengend Snippet: Substitution of six nucleotides within the miR-17 motif stabilizes Pkd1 mRNA. ( A ) Schematic illustrates the CRISPR/Cas9 editing + HDR template DNA approach utilized to precisely switch six nucleotides within the miR-17 motif in the Pkd1 RC 3′UTR. ( B ) Sanger sequencing of tail DNA from Pkd1 RC/RC*17 mouse demonstrates successful 3′UTR editing. The edited allele with disrupted miR-17 motif is denoted in pink, and the unedited allele with intact miR-17 motif is shown in blue. ( C ) Allele-specific DNA genotyping primers selectively detected edited [primer positions indicated as pink and black arrows in panel (B)] and unedited [primer positions indicated blue and black arrows in panel (B)] Pkd1 RC alleles. ( D ) Allele-specific qRT-PCR primers were used to compare Pkd1 RC and Pkd1 RC*17 transcript abundance within primary kidney epithelial cells derived from Pkd1 RC/RC*17 mice. qRT-PCR demonstrated higher abundance of Pkd1 RC*17 mRNA compared to Pkd1 RC mRNA. ( E ) Primary Pkd1 RC/RC*17 cells were treated with actinomycin and harvested at 2-h intervals to measure abundance of Pkd1 mRNA transcripts. qRT-PCR analysis demonstrated slower degradation of Pkd1 RC*17 mRNA compared to Pkd1 RC mRNA. c-Myc and Gapdh mRNA degradation was measured as internal controls. ( F ) Pkd1 F/F or Pkd1 −/− cells (Top panel) and Pkd1 RC/− or Pkd1 RC*17/− cells (lower panel) were transfected with the CRISPR-based Pkd1 exon-4 mRNA GFP sensor (green). Live-cell images and the quantification of percentage of cells exhibiting the GFP-positive puncta marking Pkd1 mRNA is shown in the lower left corner. The essential lack of detection of GFP puncta in Pkd1 −/− cells indicates specificity of Pkd1 mRNA sensor. A 78% increase in Pkd1 mRNA detection in Pkd1 RC*17/− cells compared to Pkd1 RC/− cells implies increased Pkd1 mRNA transcript in cells lacking the miR-17 motif. ( G ) Western blot demonstrates PC1 expression is increased in Pkd1 RC*17/− cells compared to Pkd1 RC/− cells, whereas PC2 and AQP2 expression remain unchanged. Error bars indicated SEM. Statistics: paired t -test (D); nonlinear regression (E); unpaired two-tailed t- test (F).

Article Snippet: Primary antibodies used: PC1 (7E12 Santa Cruz, #sc-130554), PC2 (PKD-RRC), AQP2 (Sigma–Aldrich, #A7310), Beta-Actin (Sigma–Aldrich, #A3854), Mannose Receptor (Abcam, #ab64693), Alpha Smooth Muscle (Sigma–Aldrich, #F377), Phospho-mTOR (Cell Signaling, #2971), pCREB (Cell Signaling, #9198), Total OXPHOS Rodent WB Antibody Cocktail (Abcam, #ab110413), and c-Myc (Abcam, #ab185656).

Techniques: CRISPR, Sequencing, Quantitative RT-PCR, Derivative Assay, Transfection, Western Blot, Expressing, Two Tailed Test

Substitution of six nucleotides within the miR-17 motif ameliorates PKD. Three independent Pkd1 RC*17/RC founder mouse lines were crossed with Ksp Cre ; Pkd1 F/F mice to produce littermate Ksp Cre ; Pkd1 RC*17/F ( Pkd1 RC*17/− , red circles) and Ksp Cre ; Pkd1 RC/F ( Pkd1 RC/− , black circles) mice. Representative gross kidney images and H&E-stained kidney sections ( A ), kidney-weight-to-body-weight ratios (KW/BW) ( B ), serum creatinine levels ( C ), and PC1 immunoblot with quantification ( D ) are shown for 18-day-old Pkd1 RC*17/− and Pkd1 RC/− progeny derived from founder 1. Equivalent analyses for progeny from founder 2 ( E – H ) and founder 3 ( I – L ) also revealed reduced cystic burden and increased PC1 expression in Pkd1 RC*17/− mice compared to Pkd1 RC/− mice. Error bars indicate SEM. Statistical analysis: Unpaired two-tailed t -test. N indicates biological replicates.

Journal: Nucleic Acids Research

Article Title: Disruption of a six-nucleotide miRNA motif improves PKD1 dosage and ameliorates polycystic kidney disease

doi: 10.1093/nar/gkaf1538

Figure Lengend Snippet: Substitution of six nucleotides within the miR-17 motif ameliorates PKD. Three independent Pkd1 RC*17/RC founder mouse lines were crossed with Ksp Cre ; Pkd1 F/F mice to produce littermate Ksp Cre ; Pkd1 RC*17/F ( Pkd1 RC*17/− , red circles) and Ksp Cre ; Pkd1 RC/F ( Pkd1 RC/− , black circles) mice. Representative gross kidney images and H&E-stained kidney sections ( A ), kidney-weight-to-body-weight ratios (KW/BW) ( B ), serum creatinine levels ( C ), and PC1 immunoblot with quantification ( D ) are shown for 18-day-old Pkd1 RC*17/− and Pkd1 RC/− progeny derived from founder 1. Equivalent analyses for progeny from founder 2 ( E – H ) and founder 3 ( I – L ) also revealed reduced cystic burden and increased PC1 expression in Pkd1 RC*17/− mice compared to Pkd1 RC/− mice. Error bars indicate SEM. Statistical analysis: Unpaired two-tailed t -test. N indicates biological replicates.

Techniques: Staining, Western Blot, Derivative Assay, Expressing, Two Tailed Test

Pkd1-oligonucleotide increases PC1 expression and improves cyst parameters in mouse cellular PKD model. Pkd1 RC/− cells were treated with 40 nM control oligonucleotide (Ctl oligo) or Pkd1-oligonucleotide (P1) and analyzed 48 h later. ( A ) qRT-PCR analysis was performed using a primer pair that amplifies the Pkd1 3′UTR. P1 prevented amplification of the Pkd1 3′UTR region it targets, while control oligo had no effect, indicating specific binding of P1 to the intended motif. ( B, C ) Pkd1 RC/− cells were treated with 40 nM Ctl or P1 for 48 h and then co-treated with actinomycin for 4 h to halt de novo transcription. qRT-PCR analysis using a primer pair that amplifies Pkd1 exon-4 showed that 68% more Pkd1 mRNA remained in P1-treated compared to Ctl-oligo-treated Pkd1 RC/− cells whereas cMyc transcript degraded equivalently. ( D ) (Top panel) Pkd1 RC/+ or Pkd1 RC/− cells were transfected with the CRISPR-based Pkd1 exon-4 mRNA GFP sensor (green). (Lower panel) Pkd1 RC/− cells treated with 40 nM Ctl or P1 oligo and co-transfected with the CRISPR-based sensor. Live cell imaging and quantification of percentage of cells exhibiting the GFP signal is shown. Pkd1 mRNA detection was reduced by 44% in Pkd1 RC/− cells compared to Pkd1 RC/+ cells. Treatment with P1 oligo improved Pkd1 mRNA detection by 50% compared to Ctl-oligo treatment. ( E ) Immunoblot showing increased PC1 expression in Pkd1 RC/− cells 48 h after P1 treatment compared to Ctl treatment. Actin serves as the loading control. ( F ) Pkd1 RC/− cells were transfected with 40 nM Ctl or P1 for 48 h and were then cultured in 3D Matrigel for 5 days to facilitate cyst formation. Quantification showing that cyst size was reduced in P1-oligo compared to Ctl-oligo-treated Pkd1 RC/− cells. ( G ) Immunofluorescence images showing live-cell MitoTracker labeling (MT, red) or pCreb (green) expression in P1-treated compared to Ctl oligo-treated Pkd1 RC/− cells. P1 treatment increased Mitotracker signal and reduced pCreb expression, implying improved mitochondrial function and reduced cAMP signaling. N = 3 biological replicates for each condition. Error bars indicate SEM. Statistical analysis: Unpaired, two-tailed, t -test [(A–D) and (F)]. N = 4 biological replicates(A). N = 3 biological replicates (B–D).

Journal: Nucleic Acids Research

Article Title: Disruption of a six-nucleotide miRNA motif improves PKD1 dosage and ameliorates polycystic kidney disease

doi: 10.1093/nar/gkaf1538

Figure Lengend Snippet: Pkd1-oligonucleotide increases PC1 expression and improves cyst parameters in mouse cellular PKD model. Pkd1 RC/− cells were treated with 40 nM control oligonucleotide (Ctl oligo) or Pkd1-oligonucleotide (P1) and analyzed 48 h later. ( A ) qRT-PCR analysis was performed using a primer pair that amplifies the Pkd1 3′UTR. P1 prevented amplification of the Pkd1 3′UTR region it targets, while control oligo had no effect, indicating specific binding of P1 to the intended motif. ( B, C ) Pkd1 RC/− cells were treated with 40 nM Ctl or P1 for 48 h and then co-treated with actinomycin for 4 h to halt de novo transcription. qRT-PCR analysis using a primer pair that amplifies Pkd1 exon-4 showed that 68% more Pkd1 mRNA remained in P1-treated compared to Ctl-oligo-treated Pkd1 RC/− cells whereas cMyc transcript degraded equivalently. ( D ) (Top panel) Pkd1 RC/+ or Pkd1 RC/− cells were transfected with the CRISPR-based Pkd1 exon-4 mRNA GFP sensor (green). (Lower panel) Pkd1 RC/− cells treated with 40 nM Ctl or P1 oligo and co-transfected with the CRISPR-based sensor. Live cell imaging and quantification of percentage of cells exhibiting the GFP signal is shown. Pkd1 mRNA detection was reduced by 44% in Pkd1 RC/− cells compared to Pkd1 RC/+ cells. Treatment with P1 oligo improved Pkd1 mRNA detection by 50% compared to Ctl-oligo treatment. ( E ) Immunoblot showing increased PC1 expression in Pkd1 RC/− cells 48 h after P1 treatment compared to Ctl treatment. Actin serves as the loading control. ( F ) Pkd1 RC/− cells were transfected with 40 nM Ctl or P1 for 48 h and were then cultured in 3D Matrigel for 5 days to facilitate cyst formation. Quantification showing that cyst size was reduced in P1-oligo compared to Ctl-oligo-treated Pkd1 RC/− cells. ( G ) Immunofluorescence images showing live-cell MitoTracker labeling (MT, red) or pCreb (green) expression in P1-treated compared to Ctl oligo-treated Pkd1 RC/− cells. P1 treatment increased Mitotracker signal and reduced pCreb expression, implying improved mitochondrial function and reduced cAMP signaling. N = 3 biological replicates for each condition. Error bars indicate SEM. Statistical analysis: Unpaired, two-tailed, t -test [(A–D) and (F)]. N = 4 biological replicates(A). N = 3 biological replicates (B–D).

Techniques: Expressing, Control, Quantitative RT-PCR, Amplification, Binding Assay, Transfection, CRISPR, Live Cell Imaging, Western Blot, Cell Culture, Immunofluorescence, Labeling, Two Tailed Test

PKD1-oligonucleotides raise PC1 protein levels in patient-derived ADPKD cyst cells. Three independent patient-derived ADPKD cyst cell lines ( PKD1 Q2556X, PKD1 Q2641*, and PKD1 pR3926Afs*34) were treated with 40 nM control oligonucleotide (Ctl), the mouse-specific Pkd1 -targeting oligo (P1), or the human-specific PKD1-targeting oligo (P2). The P1 and P2 oligos were also tested in the mouse Pkd1 RC/− cell line. ( A ) Immunoblot analysis revealed that both P1 and P2 increased PC1 protein expression across all three patient-derived ADPKD cell lines, as well as in the Pkd1 RC/– mouse line. ( B ) qRT-PCR using primers that amplify the PKD1 3′UTR miR-17 motif demonstrated that P2 selectively interfered with amplification of the human 3′UTR region, while P1 selectively inhibited the mouse 3′UTR. Each oligonucleotide showed partial cross-reactivity in the non-cognate species, whereas the control oligo had no effect. ( C ) Human ADPKD cell lines were treated with 40 nM ctl, P1, or P2 oligos for 48 h and then cultured in 3D Matrigel. Quantification revealed that treatment with either P1 or P2 oligo reduced 3D cyst size compared to Ctl oligo. Error bars indicate SEM. Statistical analysis: ANOVA with Tukey’s (B and C). N = 3 biological replicates for each cell line (B). N = 101–150 cysts were measured for each condition (C).

Journal: Nucleic Acids Research

Article Title: Disruption of a six-nucleotide miRNA motif improves PKD1 dosage and ameliorates polycystic kidney disease

doi: 10.1093/nar/gkaf1538

Figure Lengend Snippet: PKD1-oligonucleotides raise PC1 protein levels in patient-derived ADPKD cyst cells. Three independent patient-derived ADPKD cyst cell lines ( PKD1 Q2556X, PKD1 Q2641*, and PKD1 pR3926Afs*34) were treated with 40 nM control oligonucleotide (Ctl), the mouse-specific Pkd1 -targeting oligo (P1), or the human-specific PKD1-targeting oligo (P2). The P1 and P2 oligos were also tested in the mouse Pkd1 RC/− cell line. ( A ) Immunoblot analysis revealed that both P1 and P2 increased PC1 protein expression across all three patient-derived ADPKD cell lines, as well as in the Pkd1 RC/– mouse line. ( B ) qRT-PCR using primers that amplify the PKD1 3′UTR miR-17 motif demonstrated that P2 selectively interfered with amplification of the human 3′UTR region, while P1 selectively inhibited the mouse 3′UTR. Each oligonucleotide showed partial cross-reactivity in the non-cognate species, whereas the control oligo had no effect. ( C ) Human ADPKD cell lines were treated with 40 nM ctl, P1, or P2 oligos for 48 h and then cultured in 3D Matrigel. Quantification revealed that treatment with either P1 or P2 oligo reduced 3D cyst size compared to Ctl oligo. Error bars indicate SEM. Statistical analysis: ANOVA with Tukey’s (B and C). N = 3 biological replicates for each cell line (B). N = 101–150 cysts were measured for each condition (C).

Techniques: Derivative Assay, Control, Western Blot, Expressing, Quantitative RT-PCR, Amplification, Cell Culture

A Ratio of cleaved M to uncleaved prM quantified by western blot in LGTV-infected cell lysates, treated as indicated, at 24 h p.i. Each dot, one biological replicate. B Percentage mature virions in tomograms of LGTV-infected cells that were untreated ( N = 29), supplemented with Furin Inhibitor I ( N = 23), or NH 4 Cl ( N = 14). Each dot, one tomogram. C Polyprotein of a chimeric LGTV, rLGTV T:prME , with prM and ecto-E from TBEV strain 93/783. Protease sites are shown in the structural protein, and the furin site sequences from TBEV strains 93/783 and Torö are shown highlighting the difference at position 86 (., identical sequence). D Percentage survival of Ips1 -/- mice infected intraperitoneally with rLGTV T:prME R86 ( N = 5) or Q86 ( N = 10). E As ( D ), but for mice infected intracranially with rLGTV T:prME R86 ( N = 9) or Q86 ( N = 10). F , G Schematic ( F ) and result ( G ) of enzymatic cleavage assay using furin or PC1/3 with peptides covering furin site sequences in ( C ) (“RTRR”), or peptides with impaired furin sites (“RTRA”). Four independent experiments performed in duplicates are shown. H prM and M protein levels in cell lysates and supernatant 48 h p.i. by immunoblotting using an anti-M antibody. Viral NS3 and cellular tubulin included as infection and loading control. Representative blots are shown. I Percent prM intensity of total prM+M quantified in supernatant western blots at 48 and 72 h p.i. Four independent experiments performed in duplicates. J Slice through tomogram of rLGTV T:prME Q86-infected cell. Orange arrows, immature virions. Scale bar 100 nm. K segmentation of the tomogram in ( J ). L Percentage mature virions in tomograms of cells infected with rLGTV T:prME R86 ( N = 7) and Q86 ( N = 7). Each dot, one tomogram. A , B , G , I , L Bars indicate average±standard deviation. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. P values by unpaired two-tailed Student’s t test: A untreated vs inhibitor (20 µM; p = 0.0753, 40 µM; p = 0.0178, 60 µM; p = 0.0293), vs NH 4 Cl p = 0.00060), B untreated vs inhibitor p = 0.0037, vs NH 4 Cl p = 0.0028, G RTRR (R86 vs Q86) for furin p = 0.0184, for PC1/3 p = 2.91 × 10 −6 , I 48 h: p = 0.0085, 72 h: p = 0.2403, L p = 0.0290. In ( D , E ), Q86 vs R86 were assessed by log-rank (Mantel–Cox) test p = 0.2567.

Journal: Nature Communications

Article Title: Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

doi: 10.1038/s41467-026-68483-4

Figure Lengend Snippet: A Ratio of cleaved M to uncleaved prM quantified by western blot in LGTV-infected cell lysates, treated as indicated, at 24 h p.i. Each dot, one biological replicate. B Percentage mature virions in tomograms of LGTV-infected cells that were untreated ( N = 29), supplemented with Furin Inhibitor I ( N = 23), or NH 4 Cl ( N = 14). Each dot, one tomogram. C Polyprotein of a chimeric LGTV, rLGTV T:prME , with prM and ecto-E from TBEV strain 93/783. Protease sites are shown in the structural protein, and the furin site sequences from TBEV strains 93/783 and Torö are shown highlighting the difference at position 86 (., identical sequence). D Percentage survival of Ips1 -/- mice infected intraperitoneally with rLGTV T:prME R86 ( N = 5) or Q86 ( N = 10). E As ( D ), but for mice infected intracranially with rLGTV T:prME R86 ( N = 9) or Q86 ( N = 10). F , G Schematic ( F ) and result ( G ) of enzymatic cleavage assay using furin or PC1/3 with peptides covering furin site sequences in ( C ) (“RTRR”), or peptides with impaired furin sites (“RTRA”). Four independent experiments performed in duplicates are shown. H prM and M protein levels in cell lysates and supernatant 48 h p.i. by immunoblotting using an anti-M antibody. Viral NS3 and cellular tubulin included as infection and loading control. Representative blots are shown. I Percent prM intensity of total prM+M quantified in supernatant western blots at 48 and 72 h p.i. Four independent experiments performed in duplicates. J Slice through tomogram of rLGTV T:prME Q86-infected cell. Orange arrows, immature virions. Scale bar 100 nm. K segmentation of the tomogram in ( J ). L Percentage mature virions in tomograms of cells infected with rLGTV T:prME R86 ( N = 7) and Q86 ( N = 7). Each dot, one tomogram. A , B , G , I , L Bars indicate average±standard deviation. ns not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. P values by unpaired two-tailed Student’s t test: A untreated vs inhibitor (20 µM; p = 0.0753, 40 µM; p = 0.0178, 60 µM; p = 0.0293), vs NH 4 Cl p = 0.00060), B untreated vs inhibitor p = 0.0037, vs NH 4 Cl p = 0.0028, G RTRR (R86 vs Q86) for furin p = 0.0184, for PC1/3 p = 2.91 × 10 −6 , I 48 h: p = 0.0085, 72 h: p = 0.2403, L p = 0.0290. In ( D , E ), Q86 vs R86 were assessed by log-rank (Mantel–Cox) test p = 0.2567.

Article Snippet: For furin, 3 U furin (Thermo Fisher, Waltham, MA, USA) was mixed with 100 μM of substrate in a total volume of 100 μl reaction buffer (100 mM HEPES pH7.5 + 1 mM CaCl2 + 0.5% Triton ×-100) at 30 °C for 3 h. For proprotein convertase 1/3 (PC1/3), 1 μg recombinant human PC1/3 (R&D Systems, Minneapolis, MN, USA) was mixed with 100 μM of substrate in a total volume of 100 μl reaction buffer (25 mM MES pH 6.0 + 5 mM CaCl2 + 1% (w/v) Brij-35) at 37 °C for 1 h. The emission at 490 nm measured every 3 min and the average rate calculated by linear regression.

Techniques: Western Blot, Infection, Sequencing, Cleavage Assay, Control, Standard Deviation, Two Tailed Test

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