Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: (A) Graph representing the distribution of FUS-P525L protein in nucleus and cytoplasm. The fluorescent intensities of the nucleus and cytoplasm were calculated from the experiment as performed in , and the ratio is plotted here. A student-paired t-test was used to calculate the significance value (n=6). (B) Incucyte images (real-time cell death analysis) representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. (C) Graph representing the number of propidium iodide (PI) positive cells (dead cells) from the experiment as performed in B. The time on the x-axis reflects the time-point after transfection in hours. The significance was calculated using 2-way ANOVA with multiple comparisons (n=4). Error bars in all graphs represent mean ±SEM, and the same color points in A depict the data from a single experimental set. *, **, ***, and **** denote p-value≤0.05, ≤0.01, ≤0.001, and ≤0.0001, respectively.

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques: Transfection

Journal: bioRxiv

Article Title: RGG peptide induces the disassembly of disease-relevant FUS and TDP43 condensates

doi: 10.1101/2025.03.19.643735

Figure Lengend Snippet: Incucyte images representing the cellular uptake of propidium iodide (PI) in different conditions in HeLa cells. Scale bar=100um. The images are part of and .

Article Snippet: The cell death analysis was performed using the IncuCyte S3 live-cell analysis instrument (Sartorius), and the change in the number of PI-positive cells (dead cells) in different conditions was plotted in the graph.

Techniques:

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: Cells were seeded in 6-well plates and incubated in the IncuCyte S3 Live-Cell analysis system (Sartorius, Göttingen, Germany) to monitor cell growth.

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: Enhanced starvation-induced autophagy in proximal tubular cell lines derived from ADPKD patients. ( A ) Western blot analysis of the annotated proteins in proximal tubular epithelial cells (PTECs) from 3 young healthy individuals (Control 1–3) and ADPKD patients (ADPKD 1–3). For each individual, 2 monoclonal cell lines were analyzed (a,b). Human embryonic kidney (HEK) cell lysate and lysate of human renal tissue (kidney) were used as controls. ( B ) Quantification of polycystin-1 (PC1) and -2 (PC2) over Actin, normalized to the mean of the control group, in the 6 Control (white) and 6 ADPKD (grey) PTECs. ( C ) LC3 Western blot analysis in 3 PTECs from 3 young individuals (1a, 2a and 3a; white) and 4 cell lines from 4 young ADPKD patients (1b, 2b, 3b and 4; grey). Cells were treated with DMSO (−Baf A1) or 100 nM Baf A1 (+Baf A1) for 3 h before harvest. Upper: representative Western blot. Lower left: quantification of the average LC3-II levels over Actin, normalized to the mean of the control group. Lower right: quantification of the average LC3-I levels over Actin, normalized to the mean of the control group. ( D ) LC3 mRNA expression was evaluated by quantitative PCR (qPCR) in 3 PTECs from 3 young healthy individuals (Control 1a, 2a, 3a; white + closed symbols) and 3 young ADPKD patients (1b, 2b, 3b; grey + open symbols). The symbols represent the 2 independent experiments in each cell line. ( E ) FoxO1 protein levels in 3 PTECs from 3 young healthy individuals (Control 1a, 2a and 3a) and 3 PTECs from 3 young ADPKD patients (1b, 2b and 3b). Left: representative Western blot; right: quantification of FoxO1 levels over Actin (N = 2). ( F ) LC3 Western blot analysis in 3 PTECs from 3 young healthy individuals (1a, 2a and 3a; white + closed symbols) and 4 PTECs from 3 young ADPKD patients (1a, 1b, 2b and 3b; grey + open symbols), subjected to 48 h of starvation. Cells were treated with 100 nM Baf A1 (+Baf A1) for 3 h before harvest. Upper: representative Western blot. Lower: quantification of the average LC3-II levels over Actin of 3 independent experiments in 7 cell lines. Each symbol represents an independent experiment. ( G ) Analysis of confluence of 4 control (black) and 4 ADPKD (grey) PTECs during incubation in HBSS using the IncuCyte Live Cell Analyzer. The curve of each cell line is plotted with the average of each group represented by the superimposed symbols. Linear regression analysis revealed significantly different slopes between Control and ADPKD group. ( H ) Percentage live cells following 96 h of incubation in HBSS as analyzed by Trypan blue exclusion in 4 PTECs from 3 young healthy individuals (1a, 1b, 2a and 3a; white + closed symbols) and 3 PTECs from 3 young ADPKD patients (1b, 2b and 3b; grey + open symbols). Each symbol represents an independent experiment. Throughout this figure, the same individuals/patients are assigned with the numbers 1 to 4, while monoclonal cell lines with letters a and b. NS: not significant, ∗ p < 0.05, ∗∗∗ p < 0.001.

Article Snippet: Cells were seeded in 6-well plates and incubated in the IncuCyte S3 Live-Cell analysis system (Sartorius, Göttingen, Germany) to monitor cell growth.

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