Journal: International Journal of Molecular Sciences

Article Title: Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death

doi: 10.3390/ijms222413511

Figure Lengend Snippet: Less starvation-induced cell death in PC1KO mIMCDs. ( A ) Knockout of polycystin-1 (PC1KO) in mouse inner medullary collecting duct cells (mIMCDs) was confirmed by Western blot. ( B ) Reduced Pkd1 mRNA expression was confirmed by quantitative PCR (qPCR) (N = 7). ( C ) Representative microscopic brightfield images of wild-type (WT) and polycystin-1 knockout (PC1KO) mouse inner medullar collecting duct cells (mIMCDs) after 80 h of starvation by incubation in Hank’s balanced salt solution (HBSS). WT mIMCDs are less confluent and show more dark rounded detached cells. Scale bar = 300 µm. ( D ) Analysis of confluence of WT (black) and PC1KO (grey) mIMCDs during incubation in HBSS using the IncuCyte Live Cell Analyzer. Left: quantification of % confluence over time in HBSS with linear fit and 95% confidence interval (dashed line) superimposed; right: quantification of the slope of the linear fit of the confluence over time. Paired observations of each independent experiment are represented by the same symbol (N = 3). ( E ) Analysis of Cytotox Green signal increase during incubation in HBSS in WT (black) and PC1KO (grey) mIMCDs. Left: quantification of % of Cytotox Green signals over the total area of the image over time in HBSS; right: quantification of the slope of the linear fit over time (N = 3). ( F ) Percentage live cells over time in HBSS as analyzed by Trypan Blue exclusion in WT (black) and PC1KO (grey) mIMCDs (N = 4). ( G ) Apoptosis was analyzed by Western blotting of cleaved Caspase 3 in WT and PC1KO mIMCDs. Left: Representative Western blot of protein lysates following 0 h or 72 h of nutrient starvation; right: quantification of cleaved Caspase 3 levels over Actin (N = 3). ( H ) Percentage live cells following 72 h of incubation in HBSS as analyzed by Trypan Blue exclusion in WT and PC1KO mIMCDs, transfected either with Vehicle (+Veh) or human PC1 (+hPC1). Paired observations of each independent experiment are represented by the same symbol (N = 3). ( I ) Analysis of confluence of WT (black) and PC1KO (grey) mIMCDs during incubation in HBSS, followed by recovery in normal medium (DMEM) using the IncuCyte Live Cell Analyzer. Left: quantification of confluence (normalized to the initial time of recovery), with the linear fit in DMEM superimposed. The dashed line is the 95% interval of the linear fit; right: quantification of the slope of the linear fit in DMEM during recovery. Paired observations of each independent experiment are represented by the same symbol (N = 3). ( J ) Analysis of phosphorylated S6 (pS6) levels in WT and PC1KO mIMCDs in basal conditions. Left: representative Western blot; Right: quantification of pS6 levels over total S6 (N = 9); ( K ) Analysis of pS6 levels in WT and PC1KO mIMCDs following 48 h of HBSS and 24 h of recovery in DMEM. Left: representative Western blot; Right: quantification of pS6 levels over total S6 (N = 4). NS: not significant, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: For other mRNA, we used primer and probe sets from Thermo Fischer Scientific: Mm00465434_m1 (mPKD1; spanning exon 1–2), Mm00435829_m1 (mPKD2; spanning exon 1–2), Mm01187303_m1 (mATG5), Mm00503201_m1 (mATG12), Mm01265461_m1 (mBECN1), Mm00448091_m1 (mSQSTM1) and mHif1a (Mm00468869_m1). qPCR was performed with the corresponding StepOnePlus Real-Time PCR System Software and analyzed with the 2 −ΔΔCt method [ ].

Techniques: Knock-Out, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Incubation, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death

doi: 10.3390/ijms222413511

Figure Lengend Snippet: Altered regulation of PC1 expression in PC2KO mIMCDs is linked with enhanced autophagy. ( A ) Western blot analysis of PC1 protein levels in WT, PC1KO and PC2KO mIMCDs incubated in full medium (0 h) or subjected for 72 h to nutrient starvation (72 h). Left: representative Western blot; right: quantification of PC1 levels over Actin in WT and PC2KO cells, normalized to WT levels (N = 8). ( B ) Pkd1 mRNA expression in basal conditions (0 h) or following 48 h of nutrient starvation (48 h) was evaluated by quantitative PCR (qPCR). Two-way ANOVA revealed a significant difference between 0 h and 48 h, and between WT and PC2KO mIMCDs (N = 6). ( C ) Representative Western blot of PC1 in WT and PC2KO mIMCDs subjected to 72 h of nutrient starvation. In the final 24 h before harvest, 100 nM Bafilomycin A1 (Baf A1), 1 µM MG132 or DMSO was added. (N = 3). ( D ) Western blot analysis of LC3-II in WT or PC2KO mIMCDs transfected with Vehicle (+Veh) or human PC1 (+hPC1) followed by 48 h of starvation. Left: representative Western blot; right: quantification of LC3-II levels over actin, normalized to WT levels. Paired observations of each independent experiment are represented by the same symbol (N = 4). ( E ) Percentage live cells following 72 h of incubation in HBSS as analyzed by Trypan Blue exclusion in WT and PC2KO mIMCDs, transfected either with Vehicle (+Veh) or human PC1 (+hPC1). Paired observations of each independent experiment are represented by the same symbol (N = 4). NS: not significant, ∗ p < 0.05, ∗∗ p < 0.01.

Article Snippet: For other mRNA, we used primer and probe sets from Thermo Fischer Scientific: Mm00465434_m1 (mPKD1; spanning exon 1–2), Mm00435829_m1 (mPKD2; spanning exon 1–2), Mm01187303_m1 (mATG5), Mm00503201_m1 (mATG12), Mm01265461_m1 (mBECN1), Mm00448091_m1 (mSQSTM1) and mHif1a (Mm00468869_m1). qPCR was performed with the corresponding StepOnePlus Real-Time PCR System Software and analyzed with the 2 −ΔΔCt method [ ].

Techniques: Expressing, Western Blot, Incubation, Real-time Polymerase Chain Reaction, Transfection

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Deletion of PC1 results in a switch from tubulogenesis to cyst formation and is accompanied by an increase of cAMP and a decrease in cell motility (A and B) PC1-competent clonal cells (PKD1 +/+ ) mainly formed tubule-like structures within a collagen matrix when incubated with control medium (Ctrl). In contrast, PC1-deficient cells (PKD1 −/− ; shown for two different clones #1 and #2) primarily formed cysts within the collagen matrix in the presence of control medium (Ctrl). Application of forskolin (FSK; 10μM) to PKD1 +/+ cells turned them into cyst-forming cells resembling the phenotype obtained by PC1-deletion under control condition. Incubation of PKD1 −/− cells with forskolin (FSK; 10μM) had no impact on their morphology. (A) provides the analysis of the ratio of cystic to tubule-like formed structures (analysis of 128 images from n = 4 individual experiments). (B) shows representative images after five days of culture within a collagen I matrix. (C) Measurements of cAMP concentrations revealed similar values in clonal PC1-competent cells (PKD1 +/+ ) compared to wild type cells (wt). PC1-deletion resulted in significantly elevated cAMP levels in PC1-deficient cells (PKD1 −/− #1 and #2) with PKD1 +/+ set as 100% (n = 6 individual experiments). Incubation of the cells with forskolin (FSK; 10μM) resulted in a significant increase of cAMP in PC1-competent and PC1-deficient cells to a similar level (n = 3 individual experiments). (D and E) Movement of spheroids of PC1-competent and PC1-deficent cells (shown for PKD1 −/− #1) within the collagen matrix was captured by live imaging of 30 spheroids per condition and cell type from n = 3 individual experiments for 48 h. Deletion of PC1 resulted in decreased motility. Incubation with forskolin (FSK; 10μM) also led to a reduction of motility in PC1-competent cells to a similar degree obtained from PC1-defcient cell under control condition. Application of forskolin to PC1-deficient spheroids had no additional effect. (D) Analysis of movements of spheroids within the matrix. (E) Representative traces of single spheroids within 48 h of imaging. ∗significant compared to PKD1 +/+ -Ctrl; § significant compared to PKD1 −/− #1-FSK and PKD1 −/− #2-FSK, respectively. Data are represented as mean ± SEM. See also Figures S1–S , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Incubation, Control, Clone Assay, Imaging

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: cAMP leads to impaired capacity for tubule formation but is more pronounced in PC1-deficient cells PC1-competent (PKD1 +/+ ) and PC1-deficient cells (PKD1 −/− ) were cultured within a collagen matrix in the presence of forskolin (FSK; 10μM) where they formed cysts within four days. Thereafter PC1-competent cysts (n = 47 from three individual experiments) and PC1-deficient cysts (n = 75 from four individual experiments) were punctured with a pulled glass capillary microneedle and the use of a micromanipulator. Punctured cysts were imaged for 70 h as depicted in (A). (B) Analysis of the ratio of cysts forming tubules after injury. (C) Analysis of the length of the formed tubules within 70 h. (D) Outgrowth speed of the formed tubules by tracking the distance within 1000 min (measured from minute 100 to 1100 after injury). (E) Representative images showing tubular outgrowth in PC1-competent and PC1-deficient cysts. ∗significant compared PKD1 +/+ . Data are represented as mean ± SEM. See also Figure S9 , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Cell Culture

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Lack of PC1 along with increase of cAMP leads to defective cell migration (A) Tracks of individual cells (50 cells per condition from n = 3 individual experiments) imaged for 6 h in wound healing assays starting 2 h after wounding the cell monolayer. Deletion of PC1 (PKD1 −/− ; red) resulted in reduced migration. The same was obtained when incubating PC1-competent cells (PKD1 +/+ ; blue) with forskolin. (B) Representative images of wound healing assays showing the invaded area of cells within 6 h. (C) Quantification of the euclidean distance of the individually tracked cells (left) and the ratio of euclidean distance in relation to the accumulated distance (right) indicating significant impairment of directed cell migration upon loss of PC1 or application of forskolin in PC1-competent cells. ∗significant compared to PKD1 +/+ -Ctrl. Data are represented as mean ± SEM. See also Figure S6 , and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Migration

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: Loss of PC1 and increase of cAMP lead to dense cell aggregation and reduction of disseminating single cells PC1-competent cells (PKD1 +/+ ) and PC1-deleted cells (PKD1 −/− ) were cultured at low density (3200 cells/cm 2 ) and analyzed with regard to their growing pattern. (A) PC1-competent cells kept growing in a disseminated pattern forming small colonies with a significant amount of single cluster-independent cells. PC1-deleted cells resulted in dense and evenly bordered cell aggregations with only a few single cells being detectable outside these cell formations. Forskolin (FSK; 10μM) significantly reduced the number of single PC1-competent and PC1-deficient cells (analysis of 72 images from n = 3 individual experiments). (B) Voronoi-based analysis of cell distance between clustering cells (defined as > two cells). Cell distance was significantly higher in PC1-competent cells compared to PC1-deficient cells and PC1-competent cells in the presence of forskolin. (C) Analysis of cell density within bigger cell aggregates (comprising 10 - 50 cells/cluster). Cells were significantly less dense within the clusters of PC1-competent cells under control condition compared to PC1-deficient cells or PC1-competent cells in the presence of forskolin. (D) Representative images after 48 h of culture of PC1-competent cells (PKD1 +/+ ) and PC1-deficient cells (PKD1 −/− ) under control condition (Ctrl) or in the presence of forskolin (FSK; 10μM). Row 1 shows representative images of DAPI-stained cells. In each image, one cell cluster was marked by a yellow outline in order to facilitate finding of the corresponding parts in the Voroni-based animations and heat map analyses. Row 2 shows Voronoi-based animation of cell clusters (defined as > two cells; white) and single cells (red). Row 3 shows heatmap analysis of cells visualizing cell density defined as blue = low cell density and red = high cell density. ∗significant compared to PKD1 +/+ -Ctrl. # significant compared to PKD1 −/− -Ctrl. Data are represented as mean ± SEM. See also Figures S7 and .

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Cell Culture, Control, Staining

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet: PC1 deletion and cAMP increase lead to impaired epithelial tubular cell orientation in metanephric mouse kidneys Metanephric kidneys (n = 3 per condition) from KspCreER T2 : Pkd1 lox;lox mice were dissected at embryonic day 13.5 and either incubated with control medium in order to preserve PC1 expression ( Pkd1 +/+ ) or medium supplemented with hydroxytamoxifen (500 nM) resulting in tubule-specific deletion of PC1 ( Pkd1 −/− ). Thereafter, kidneys were cultured ex vivo for five days either incubated with control medium (Ctrl) or in the presence of forskolin (FSK; 10μM). (A) Violin plots illustrating the deviation of the tubule cell’s longitudinal axis from the longitudinal tubular axis with a reference angle set = 90° (n = 30 tubules per condition with a mean of 10 analyzed cells per tubule). (B and C) Representative images of metanephric kidneys stained for E-Cadherin illustrating tubule epithelial cell borders and binarized images with depicted longitudinal cell axes. ∗significant compared to PKD1 +/+ -Ctrl. Data are represented as mean ± SEM.

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Incubation, Control, Expressing, Cell Culture, Ex Vivo, Staining

Journal: iScience

Article Title: Loss of Polycystin-1 causes cAMP-dependent switch from tubule to cyst formation

doi: 10.1016/j.isci.2022.104359

Figure Lengend Snippet:

Article Snippet: Guide RNAs targeting Exon 2 of the Pkd1 gene were ligated with a pSpCas9(BB)-2A-puro-vector (PX459) V2.0 from Addgene (ID 62988; Watertown, MA, USA).

Techniques: Recombinant, Luciferase, Plasmid Preparation, Cloning, Software, Chemotaxis Assay, Migration, Sequencing, Cell Culture