Journal: Aging Cell

Article Title: The Proinflammatory Secretome of Senescent Cells Can Be Controlled by a HIF2A ‐Dependent Upregulation and a FURIN ‐Dependent Cleavage of the ANGPTL4 Secreted Factor

doi: 10.1111/acel.70307

Figure Lengend Snippet: Cleavage of ANGPTL4 by FURIN is required for its ability to promote the proinflammatory SASP. (A‐C) MRC5/RAF:ER cells were transfected with control (siCTRL) or FURIN (siFURIN) siRNA and treated (+) or not (−) with 4‐OHT to induce senescence (OIS). (A) RT‐qPCR analysis against the indicated genes 3 days after 4‐OHT treatment. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test results are shown. (B) Western blot analysis of FURIN, ANGPTL4: Full length in whole cell extracts (ANGPTL4) and secreted in the supernatant (cANGPTL4) and IL6. Representative picture of n = 3 independent experiments. (C) RT‐qPCR analysis of the indicated SASP encoding genes. Mean ± SEM of n = 3 independent experiments. Paired one‐way ANOVA test values are shown. (D) Relative mRNA expression of FURIN , ANGPTL4 and proinflammatory SASP encoding genes measured by RT‐qPCR in MRC5 cells overexpressing or not ANGPTL4 (ANGPTL4 OE) and transfected with control (siCTRL) or FURIN (siFURIN) siRNA. Mean ± SEM of n = 4 independent experiments. Paired one‐way ANOVA test results are shown.

Article Snippet: The membranes were then blocked for 1 h with 5% milk in TBS‐T 0.05% and subsequently incubated overnight at 4°C with primary antibodies in TBS‐T with 5% milk: α‐ANGPTL4 (sc‐373761, Santa Cruz, 1/250), α‐IL6 (sc‐28343, Santa Cruz, 1/500), α‐FURIN (18413‐1‐AP, Proteintech, 1/1000), α‐TUBULIN (T6199‐100, Sigma, 1/5000). α‐HIF1A (610958, BD Biosciences, 1/1000), HIF2A (NB100‐132, Novus Biologicals, 1/1000), INHBA (GTX108405, GeneTex 1/1000).

Techniques: Transfection, Control, Quantitative RT-PCR, Western Blot, Expressing

Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Upregulation of the UII system in the kidneys of neonatal pigs following IR injury. Quantitative RT-PCR analysis showing relative mRNA expression levels of (A) furin, (B) UII, (C) URP, and (D) UT in whole kidney tissue, and (E) UT in intrarenal arteries from sham and IR piglets ( n = 4 per group). (F–G) Representative Western blot and densitometric analysis of UT protein expression in intrarenal arteries ( n = 4 per group). (H–I) Western blot and quantification of furin protein expression in kidney tissues ( n = 4 per group). (J–K) representative immunohistochemistry (IHC) images and quantification of UII immunostaining intensity in renal tubules from sham and IR piglets (4 kidney sections per group). * p < 0.05, unpaired t -test.

Article Snippet: For Western blot analysis, the primary antibodies included a rabbit polyclonal anti-furin antibody (18413-1-AP, Proteintech, Rosemont, IL, USA) at a 1:500 dilution and a rabbit polyclonal anti-GPR14 (UT receptor) antibody (TA358466, OriGene Technologies, Rockville, MD, USA) at a 1:100 dilution.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunohistochemistry, Immunostaining

Journal: Renal Failure

Article Title: Urotensin II system contributes to ischemic acute kidney injury in neonatal pigs

doi: 10.1080/0886022X.2025.2534018

Figure Lengend Snippet: Furin inhibition reduces cIR-driven UII production, and UT and furin blockade attenuate cIR-induced cytotoxicity in primary neonatal pig proximal tubule epithelial cells. (A) Representative fluorescence microscopy images showing furin and UII immunostaining and their colocalization in primary neonatal pig proximal tubule epithelial cells (PTECs; 3 biological replicates). (B) Quantification of intracellular furin levels in control (ctrl) and chemical ischemia-reperfusion (cIR)–treated cells ( n = 4). (C) Secreted UII levels in cIR-treated cells in the absence or presence of the furin inhibitor SSM3 trifluoroacetate (SSM3; n = 4). (D) cIR-induced cytotoxicity was significantly reduced by pharmacological inhibition of furin (SSM3) and by UT inhibition with urantide (URTD). * p < 0.05 ctrl vs. cIR; p < 0.05 cIR vs. SSM3 + cIR; $ p < 0.05 cIR vs. both SSM3 + cIR and URTD + cIR. Unpaired t -test (B); One-way ANOVA and Holm-Šídák’s multiple comparisons tests (C–D); n = 4, each. Scale bar = 50 µm.

Article Snippet: For Western blot analysis, the primary antibodies included a rabbit polyclonal anti-furin antibody (18413-1-AP, Proteintech, Rosemont, IL, USA) at a 1:500 dilution and a rabbit polyclonal anti-GPR14 (UT receptor) antibody (TA358466, OriGene Technologies, Rockville, MD, USA) at a 1:100 dilution.

Techniques: Inhibition, Fluorescence, Microscopy, Immunostaining, Control

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: HEK293 FRT furin knockout. HEK293 FRT cells were transfected with plasmids containing genes coding for Cas9 and three furin sgRNAs. Surviving cells were clonally selected by serial dilution in a 96-well plate. Lysates from HEK293 FRT (WT) and four mutant clones were evaluated for furin expression by western blot. Clones 1, 2, and 4 showed no discernable furin expression, and clone 1 (boxed) was selected for further study as ΔFur293.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Knock-Out, Transfection, Serial Dilution, Mutagenesis, Clone Assay, Expressing, Western Blot

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage assays. HEK293 FRT cells, ΔFur293 cells, and ΔFur293 cells stably expressing transgenic wild-type furin (ΔFur293/Fur) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry (panel B) as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR with (+) and without (−) furin treatment in vitro , and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two separate assays of each cell line are shown.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Complementation with mutant furin. ΔFur293 cells were stably transfected with genes for furin that contained mutations designed to impair its intracellular trafficking or catalytic function. Mutations S773A/S775A (ADA) and S773D/S775D (DDD) alter furin trafficking, while the N295A mutant (Ala-295) inhibits catalytic activity. Two separate clonal lineages stably transfected with mutant or wild-type furin were treated with the anti-transferrin receptor/PE RIT HB21-LR to assess cytotoxicity. The EC50 (pM) values from at least four separate assays for each line were normalized for furin expression levels and plotted. Dashed lines denote the average value for each clone and error bars indicate the standard error. The largest significant P -values ( P <0.05) between sets of clones from a one-way ANOVA performed as described are indicated. All P -values are reported in Table S3 .

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Mutagenesis, Stable Transfection, Transfection, Activity Assay, Expressing, Clone Assay

Journal: Biology Open

Article Title: Intracellular trafficking of furin enhances cellular intoxication by recombinant immunotoxins based on Pseudomonas exotoxin A

doi: 10.1242/bio.061792

Figure Lengend Snippet: Cleavage by mutant furin . ΔFur293 cells stably expressing transgenic furin mutants (FurADA, FurDDD, and FurAla-295) were incubated for various time intervals from 0.5 to 8 h in culture with the anti-transferrin receptor/PE24 RIT HB21-LR. Whole cell lysates were evaluated for full length and cleaved HB21-LR by western blot (panel A) and densitometry as described. Also shown are untreated (U) cell lysates for each cell line, HB21-LR in vitro with (+) and without (−) furin treatment, and the β-actin loading control. The ratio between the furin-cleaved band intensity and the total intensity of all RIT bands at each time point is plotted in panel B. The individual densitometric analysis values (points) and mean (bar) for at least two internalization and cleavage assays in each cell line are shown.

Article Snippet: For furin expression level analysis, mouse anti-furin antibody (mAb, Santa Cruz Biotechnology, sc-133141), mouse anti-β-actin (mAb, Thermo Fisher Scientific, BA3R), and goat anti-mouse IgG alkaline phosphatase conjugated antibody (mAb, Santa Cruz Biotechnology: sc-2058) were used.

Techniques: Mutagenesis, Stable Transfection, Expressing, Transgenic Assay, Incubation, Western Blot, In Vitro, Control