Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of FXIIa attenuates kidney fibrosis in mice with unilateral ureteral obstruction

doi: 10.1007/s00018-025-05988-z

Figure Lengend Snippet: FXIIa and FXII trigger p21 expression in renal epithelial cells. ( a ) Phosphorylated (P) and total protein levels of EGFR, Akt, ERK1/2, and PCNA in UUO and CTR kidneys of animals injected either with IgG or 3F7. The animals were sacrificed at day 3 post ligation. Representative western blots are shown. ( b ) Densitometric evaluation of western blots shown in (a) visualized in a heatmap. n = 5/group. *IgG CTR vs IgG UUO ( p < 0.05), †IgG UUO vs 3F7 UUO ( p < 0.05). ( c ) Expression of p21 and p16 in HEK293 cells stimulated with FXIIa or FXII. β-actin was used as a loading control. Data are representative of three independent experiments. ( d ) p21 staining in FXIIa or FXII-stimulated HEK293 cells. ( e ) Percentage of p21. + HEK293 cells upon stimulation with FXIIa or FXII; calculated from five independent experiments. ( f ) Expression of p21 in HEK293 cells stimulated with FXIIa/FXII in the absence or presence of 3F7 or IgG. β-actin was used as a loading control. Data are representative of three independent experiments. ( g ) Phosphorylated and total protein levels of Akt and ERK1/2 in HEK293 cells treated for the indicated time points with FXIIa or FXII. Phosphoproteins were detected with phospho-specific antibodies. Equal loading was confirmed with pan-specific antibodies. Data are representative of three independent experiments. ( h , i ) Expression of p21 in HEK293 cells stimulated with FXIIa or FXII in the absence or presence of MEK inhibitor PD98059 (PD) or PI3K/Akt inhibitor LY294002 (LY). β-actin was used as a loading control. Data are representative of three independent experiments. ( j , k ) Western blots showing P-Akt and P-ERK1/2 in HEK293 cells stimulated with FXIIa or FXII in the absence or presence of PD or LY. Phosphoproteins were detected with phospho-specific antibodies. Equal loading was confirmed with pan-specific antibodies. Data are representative of three independent experiments. ( l ) Stability of FXII during 90 min incubation at the HEK293T cell monolayer. Data are representative of three independent experiments. FXII HC , FXII heavy chain; FXII LC , FXII light chain. ( m ) FXIIa amidolytic activity measured at the HEK293T cell monolayer. The hydrolysis of S2302 by FXIIa was measured spectrophotometrically at 405 nm (A 405 ). One representative experiment out of three is shown. ( n ) Expression of p21 in HEK293T cells stimulated with FXII, FXIIa, or catalytically dead FXII (FXII CD ). β-actin was used as a loading control. Data are representative of three independent experiments. *** p < 0.001; * p < 0.05

Article Snippet: Afterwards, the cells were washed with TBS (25 mM TRIS, 137 mM NaCl, 2.7 mM KCl, pH 7.4), fixed with 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 (T) for 1 h, and blocked with 1% BSA in TBS for 1 h. Subsequently, the cells were incubated overnight at 4 °C with a rabbit anti-p21 antibody (Cell Signaling, Danvers, MA; cat. no.: 2947; 1:300 in 1% BSA in TBS-T).

Techniques: Expressing, Injection, Ligation, Western Blot, Control, Staining, Incubation, Activity Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Inhibition of FXIIa attenuates kidney fibrosis in mice with unilateral ureteral obstruction

doi: 10.1007/s00018-025-05988-z

Figure Lengend Snippet: 3F7 reduces the number of p21. + cells in UUO kidneys. ( a ) Western blots showing expression of p21 in HRPTEpC cells stimulated with FXII, FXIIa, or vehicle (V). β-actin was used as a loading control. Data are representative of three independent experiments. ( b ) mRNA expression of Cdkn1a in UUO and CTR kidneys of mice treated either with IgG or 3F7 at day 10 post ligation. The qPCR data are presented as a ΔCt using Actb (β-actin) as a reference gene. ( c ) Western blot showing p21 in UUO and CTR kidneys of animals injected either with IgG or 3F7 at day 10 post obstruction. β-actin was used as a loading control. Representative western blots are shown. ( d ) Densitometric evaluation of western blots shown in (c). ( e ) p21 staining in representative UUO and CTR kidney sections of mice treated either with IgG or 3F7 at day 10 post ligation. Scale bar 50 µm. ( f ) p21 staining quantification. ( g ) FXII immunostaining in human renal sections from patients suffering from hydronephrosis ( n = 2) and normal kidney tissue (donor; n = 6). An asterisk depicts the localization of FXII in the glomerular mesangium, an arrow indicates tubular epithelial cells, and an arrowhead points to peritubular vessels. The red asterisk shows a fibrotic lesion in the renal interstitium. Scale bar 100 µm. n = 5–10/group **** p < 0.0001, *** p < 0.001; * p < 0.05

Article Snippet: Afterwards, the cells were washed with TBS (25 mM TRIS, 137 mM NaCl, 2.7 mM KCl, pH 7.4), fixed with 4% PFA for 20 min, permeabilized with 0.1% Triton X-100 (T) for 1 h, and blocked with 1% BSA in TBS for 1 h. Subsequently, the cells were incubated overnight at 4 °C with a rabbit anti-p21 antibody (Cell Signaling, Danvers, MA; cat. no.: 2947; 1:300 in 1% BSA in TBS-T).

Techniques: Western Blot, Expressing, Control, Ligation, Injection, Staining, Immunostaining

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) Schematic highlighting the DNA damage-dependent and independent p53 pathways functioning during the cell cycle. ( B ) p53 WT and p53 KO hTERT-RPE1 FUCCI cells were treated with the indicated doses of NCS for 1 h. NCS was removed by washing and cells allowed to proliferate for 5 days before fixing and staining with crystal violet. Relative cell proliferation is quantified underneath. Low dose (12.5 ng/ml) and high dose (100 ng/ml) NCS doses used in subsequent experiments are highlighted in green and red, respectively (mean ± SEM; n = 3 independent experiments). ( C ) p53 WT hTERT-RPE1 FUCCI cells were treated with the indicated doses of NCS for 1 h. NCS was removed by washing and cells were either fixed and stained immediately for the number of 53BP1 foci or after 4 h treatment with 25 ng/ml nocodazole to determine the percentage of mitotic entry (mean ± SD; n = 3 independent experiments). ( D ) p53 WT hTERT-RPE1 FUCCI cells were treated with either low dose or high dose NCS for 1 h. NCS was removed by washing and Late S/G2 cells were imaged continuously for 2 days. Cell cycle fate is plotted for individual cells. Untreated cells with no DNA damage were imaged and plotted as a control (left). The analysis was repeated in p53 KO and p21 KO hTERT-RPE1 FUCCI cells (right). Pooled analyses are shown from 3 independent experiments. ( E ) The percentage of mitotic entry and G2 to G1 cell-cycle collapse is plotted from ( D ). ATM and ATR inhibitor treated cells (ATMRi) are included as a control (mean ± SD; n = 3 independent experiments). Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( F ) G2 length in cells treated as in ( D ) is plotted for individual cells in a box and whiskers plot (median, 25th and 75th percentiles and whiskers extending to minimum and maximum values; mean (+) for the different conditions). ATM and ATR inhibitor treated cells (ATMRi) are included as a control. Pooled analyses are shown from three independent experiments. Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( G ) G1 length following mitosis in cells that entered mitosis following treatments as in ( D ) is plotted for individual cells in a box and whiskers plot (median, 25th and 75th percentiles and whiskers extending to minimum and maximum values, mean (+) for the different conditions; pooled analyses shown from three independent experiments). n.d.: no data points could be obtained due to no cells entering mitosis. Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( H ) Schematic detailing possible cell cycle fates of cells after DNA damage (left). DNA damage was induced in asynchronous (AS) p53 WT , p53 KO and p21 KO hTERT-RPE1 FUCCI cells by a high dose of 100 ng/ml NCS (AS Hi) for 24 h. DMSO (AS No) was used as a negative control. Cells held in mitosis (M) for 18 h and released into G1 for 24 h were used to test the outcome of mitotic timer pathway induced G1 arrest (M → G1). Cells trapped in G1 with the CDK4/6 inhibitor Palbociclib (CDK4/6i) for 24 h were used as a control for normal G1 arrest. The percentage of S/G2 and 2C/4C G1 cells is plotted (mean ± SEM, n = 3 independent experiments) (middle). Representative images of some key conditions are shown. Scale bar: 20 µm (right). Significance for all experiments: ** P < 0.01; **** P < 0.0001; ns not significant. All P values are listed in Dataset EV .

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Staining, Control, Negative Control

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) Western blot analysis of the indicated p53 KO , p21 KO , WIP1 KO , and Cdh1 KO hTERT-RPE1 FUCCI cell lines (blots are representative of 3 independent experiments). ( B ) Representative images of cells stained for DNA damage markers 53BP1 and γ-H2A.X pS139 analysed in Fig. ( n = 3 independent experiments). Scale bars: 50 µm and 10 µm (inset). ( C ) p53 WT hTERT-RPE1 cells treated with the indicated doses of NCS for 1 h were Western blotted with the antibodies indicated ( n = 3 independent experiments). ( D ) Levels of γ-H2A-X pS139, p53 and p53 pSer15 from panel ( C ) are plotted (mean ± SEM; n = 3 independent experiments). ( E ) Representative images for the cells described in Fig. ( n = 3 independent experiments). Scale bar: 10 µm.

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Western Blot, Staining

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) Wild-type (WT), WIP1 ΔCT , p21 KO , and p53 KO hTERT-RPE1 FUCCI cells synchronised in G2 with CDK1 inhibitor for 18 h were treated with either low dose (LD) or high dose (HD) NCS for 8 h to trigger DNA damage, then Western blotted for cell cycle and DNA damage markers ( n = 4 independent experiments). ( B – D ) The levels of cyclin A2 and cyclin B2 ( B ), cyclin D1 and phosphorylated Rb pS807/S811 ( C ), or p21 ( D ) are plotted as mean ± SEM ( n = 4 independent experiments) for cell lines treated as in ( A ). Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. Significance for all experiments: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns not significant. All P values are listed in Dataset EV .

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Western Blot

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) p53 WT and p53 KO hTERT-RPE1 FUCCI cells were treated with low or high dose NCS for 1 h. NCS was removed by washing and cells given Wee1 inhibitor (Wee1i). Late S/G2 cells were imaged continuously for 2 days. Cell cycle fate is plotted for individual cells. Pooled analyses are shown from three independent experiments. ( B ) The percentage of mitotic entry and G2 to G1 cell-cycle collapse is plotted from ( A ) (mean ± SD; n = 3 independent experiments). Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( C ) G2 length in cells treated as in ( A ) is plotted for individual cells in a box and whiskers plot (median, 25th and 75th percentiles and whiskers extending to minimum and maximum values; mean (+) for the different conditions). Arrows indicate the mean G2 length for p53 WT cells treated with low or high dose NCS without Wee1 inhibitor. Pooled analyses are shown from three independent experiments. Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. ( D ) Asynchronous (AS) p53 WT hTERT-RPE1 FUCCI cells were treated with DMSO (AS control), CDK4/6 inhibitor (G1 arrest) or CDK1 inhibitor (G2 arrest) for 24 h before fixation. p53 WT cells arrested in G2 were treated with DMSO, high dose NCS or Nutlin-3A for a further 24 h (G2 arrest + DMSO/NCS/N3A) prior to fixation. The percentage of S/G2 and 2C/4C G1 cells is plotted (mean ± SEM, n = 3 independent experiments). ( E ) Asynchronous (AS) p53 WT hTERT-RPE1 FUCCI cells were treated with Nutlin-3A and imaged continuously for 2.5 days. Cell cycle fate is plotted for individual cells, tracking from the phase they were in at the time of Nutlin-3A treatment. Pooled analyses are shown from three independent experiments. ( F ) p21-mTagBFP2 hTERT-RPE1 FUCCI cells were treated with high dose NCS for 1 h, then washed and imaged (High DNA damage), or treated with Nutlin-3A and imaged (+Nutlin-3A). Representative images are shown, and mean p21-BFP intensity along with FUCCI marker intensities are plotted (mean ± SEM; n = 5 cells). Asynchronous cells are included as a control (No DNA damage). Scale bars: 10 µm. ( G ) Wild-type (WT), WIP1 ΔCT , p21 KO and Cdh1 KO hTERT-RPE1 cells were arrested in G2 by treatment with CDK1i for 18 h, and the G2-arrested cells were treated with Nutlin-3A for the time points indicated. Samples were subjected to immunoblotting for cell cycle markers, using the indicated antibodies ( n = 3 independent experiments). ( H ) The behaviour of cyclin A2 and cyclin B1 from cells treated as in ( G ) is plotted (mean ± SEM; n = 3 independent experiments). ( I ) The levels of cyclin A2 and cyclin B1 remaining after 8 h treatment in the different cell lines treated as in ( G ) are plotted (mean ± SEM; n = 3 independent experiments). ( J , K ) The behaviour of p53 and p21 ( J ), and cyclin D1 and Rb phosphorylation (Ser807/Ser811) ( K ) are plotted for each cell line treated as in ( G ) (mean ± SEM; n = 3 independent experiments). For all experiments, significance: *** P < 0.001; **** P < 0.0001; ns not significant. All P values are listed in Dataset EV .

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Control, Marker, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) Wild-type and p21-GFP hTERT-RPE1 cells were arrested in G1 with CDK4/6i or in G2 with CDK1i for 18 h. Asynchronous (AS) cells were included as a control. Cells were treated with or without Nutlin-3A for 6 h prior to lysis, and proteins were isolated from the lysates by GFP-TRAP beads (GFP IP). Input and IP extracts were subjected to immunoblotting with the indicated antibodies ( n = 2 independent experiments). ( B ) Wild-type hTERT-RPE1 FUCCI cells were synchronised in G2 with CDK1 inhibitor for 18 h, treated with 100 nM of the CDK2 inhibitor PF-06873600 (CDK2i), 500 nM, 1 µM or 2 µM (left to right) of the alternative CDK2 inhibitor INX-315 (CDK2i-2), or 5 µM of the pan CDK inhibitor Flavopiridol (panCDKi) or DMSO (–) for 8 h, and Western blotted for cell cycle markers. ( C ) Cyclin A2 and B1 levels after 8 h treatment as in ( B ) are plotted for each condition (mean ± SEM; n = 3 independent experiments). Statistical significance was analysed using a one-way ANOVA with Dunnett’s multiple comparisons test (**** P < 0.0001). ( D ) Wild-type hTERT-RPE1 FUCCI cells were treated with the CDK4/6 inhibitor Palbociclib (CDK4/6i) for 1 h before late S/G2 cells were imaged continuously for 2 days. Cell cycle fate is plotted for individual cells. Pooled analyses are shown from 3 independent experiments. All P values are listed in Dataset EV .

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Control, Lysis, Isolation, Western Blot

Journal: The EMBO Journal

Article Title: WIP1 mutations suppress DNA damage triggered bypass of the mitotic timer

doi: 10.1038/s44318-025-00495-0

Figure Lengend Snippet: ( A ) Wild-type hTERT-RPE1 FUCCI cells were synchronised in G2 with a CDK1 inhibitor for 18 h. G2-arrested cells were treated with Nutlin 3 A (N3A), CDK2 inhibitor (CDK2i), a pan-CDK inhibitor (pan CDKi) or DMSO (–) for 1 or 8 h and then Western blotted ( n = 3 independent experiments). ( B ) Cyclins A2, B1, D1 and phospho-Rb (Ser807/Ser811) levels after 8 h treatment as in ( A ) are plotted (mean ± SEM; n = 3 independent experiments). ( C ) Wild-type, WIP1 ΔCT , p21 KO , and p53 KO hTERT-RPE1 FUCCI cells were synchronised in G2 with CDK1 inhibitor for 18 h, treated with N3A, CDK2i or DMSO (–) for 8 h, and Western blotted for cell cycle markers. ( D ) Cyclin A2 and B1 levels after 8 h treatment as in ( C ) are plotted (mean ± SEM; n = 3 independent experiments) (left). A schematic detailing the targets of the drugs used in the experiment (right). Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test where each condition is compared to DMSO control. ( E ) Wild-type, p53 KO , and p21 KO hTERT-RPE1 FUCCI cells were treated with CDK2i, and Late S/G2 cells were imaged. Representative images are shown ( n = 3 independent experiments). Scale bar: 10 µm. ( F ) Cell cycle fates are plotted for the cells in ( E ). Pooled analyses are shown from three independent experiments. ( G ) The percentage of G2 to G1 collapse is plotted for the cells described in ( E ) (mean ± SD; n = 3 independent experiments). Statistical significance was analysed using an ordinary one-way ANOVA with Tukey’s multiple comparisons test. For all experiments, significance: **** P < 0.0001; ns not significant. All P values are listed in Dataset EV .

Article Snippet: p21 Rabbit mAb (WB 1:1000) , Cell Signaling Technology , Cat: #2947.

Techniques: Western Blot, Control

Journal: EMBO Reports

Article Title: Differential contribution of TFE3 isoforms to cell motility and invasion

doi: 10.1038/s44319-025-00659-3

Figure Lengend Snippet: ( A ) KEGG pathway enrichment analysis of genes downregulated by TFE3-L-Myc or TFE3-S-Myc. Circle size represents the number of genes, while the color scale indicates the -log10 of the false discovery rate (FDR). ( B ) Flow cytometry analysis of 5-ethynyl-2’-deoxyuridine (EdU) incorporation in ARPE19 cells infected with control adenovirus (Null) or adenovirus expressing recombinant TFE3-L-Myc and TFE3-S-Myc for 30 h. ( C ) Quantification of the percentage of infected ARPE19 cells with incorporated EdU as shown in ( B ). Data are presented as mean ± SD of three independent experiments. ***(a) P = 0.0003; ***(b) P = 0.0005 (one-way ANOVA followed by Dunnett’s multiple comparison post-test). ( D ) Relative quantitative RT-PCR analysis of the mRNA expression of CDKN1C gene in ARPE19 cells infected with adenovirus expressing recombinant TFE3-L-Myc, TFE3-S-Myc or Control for 30 h. Data are presented as mean ± SD of three independent experiments. **(a) P = 0.0052; ***(b) P = 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparison post-test). ( E ) Immunoblot analysis of protein lysates from ARPE19 cells infected with adenovirus expressing recombinant TFE3-L-Myc, TFE3-S-Myc, or control adenovirus (Null) for 30 h. ( F ) Quantification of protein levels showing p21/GAPDH ratio expressed as fold change as shown in ( E ). Data are presented as mean ± SD of three independent experiments. *(a) P = 0.0234; *(b) P = 0.0262 (one-way ANOVA followed by Dunnett’s multiple comparison post-test). ( G ) Immunoblot analysis of protein lysates from ARPE19 cells infected with adenovirus expressing recombinant TFE3-L-Myc, TFE3-S-Myc, or control adenovirus (Null) for 30 h. ( H ) Quantification of protein levels showing phospho-Rb/Rb ratio expressed as fold change as shown in ( G ). Data are presented as mean ± SD of three independent experiments. (ns) not significant (a) P = 0.2001; ***(b) P = 0.0001; ***(c) P = 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparison post-test). ( I ) Immunofluorescence confocal microscopy of ARPE19 cells infected with adenovirus expressing recombinant TFE3-L-Myc, TFE3-S-Myc, or control adenovirus (Null) for 30 h, showing the cellular distribution of phospho-Rb (green) and recombinant TFE3-L-Myc or TFE3-S-Myc (red). Scale bars: 10 μm. ( J ) Relative quantitative RT-PCR analysis of the mRNA expression of JUP in ARPE19 cells infected with adenovirus expressing recombinant TFE3-L-Myc, TFE3-S-Myc, or control adenovirus (Null) for 30 h. Data are presented as mean ± SD of three independent experiments. ****(a) P < 0.0001; ****(b) P < 0.0001 (one-way ANOVA followed by Dunnett’s multiple comparison post-test). ( K ) Immunofluorescence confocal microscopy of ARPE19 cells infected with the indicated adenovirus for 30 h, showing cellular distribution of β-Catenin (pseudo-color magenta) and recombinant TFE3-L-Myc or TFE3-S-Myc (green). Scale bars: 10 μm.

Article Snippet: p21 Waf1/Cip1 (12D1) Rabbit mAb , Cell Signaling Technology , Cat# 2947, RRID:AB_823586.

Techniques: Flow Cytometry, Infection, Control, Expressing, Recombinant, Comparison, Quantitative RT-PCR, Western Blot, Immunofluorescence, Confocal Microscopy