Journal: JCI Insight

Article Title: Metalloproteinase PAPP-A regulation of IGF-1 contributes to polycystic kidney disease pathogenesis

doi: 10.1172/jci.insight.135700

Figure Lengend Snippet: (A) Relative mRNA expression of IGF-1 pathway components in kidneys of 7.5-month-old C57BL/6J (n = 4–6) and Pkd1RC/RC mice (n = 5–7). PCR data are expressed relative to Gapdh. (B) Correlation between kidney size (total kidney weight relative to heart weight) and renal Pappa mRNA expression in Pkd1RC/RC mice (n = 15). (C) Pappa mRNA levels in various tissues of WT (n = 3–5) and Pkd1RC/RC mice (n = 4–6). (D) Pappa mRNA levels in WT (n = 6) and Pkd2WS25/– (n = 5) mouse kidneys (16 weeks old). (E) ELISA analysis of PAPP-A protein levels in human ADPKD cystic fluid (n = 6) compared with normal serum reference. (F) Immunolocalization of PAPP-A in normal and ADPKD human kidneys. (G) Western blot analysis of PAPP-A protein levels in normal human RCTE and ADPKD cystic epithelial cells (9-12); graph shows quantification relative to tubulin. Scale bars: 200 μm. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed (for Igf1, 1-tailed) Student’s t test.

Article Snippet: Human ADPKD kidney tissue slides were produced in-house under an institutionally approved IRB protocol, and normal human kidney tissue slides were purchased from Novus Biologicals.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: HTLV-1 Rex hijacks UPF1 in a CRM1 dependent manner, leading to NMD inhibition and revealing unexpected proviral roles of UPF1

doi: 10.1101/2023.06.20.545693

Figure Lengend Snippet: a) Co-immunoprecipitation (coIP) experiments on 293T cells extracts. Cells express the indicated combinations of Rex and RNA containing the RxRE motif. IP were performed using rabbit polyclonal antibodies against CRM1 (middle panel) or from a pre-immune serum. Proteins revealed by western blot were indicated on the side. b) Same as A with an antibody targeting Rex. c) Same as a) with an antibody targeting UPF1. CoIP were incubated for 30 min with RNAse A (0.1mg/ml) (panel (ii)) or not (panel (i)). * indicates an aspecific signal. Rex specific signal corresponds to the lower band. d) coIP experiments in 293T cells transfected with plasmids coding the indicated forms of Rex and revealed by western blot. An empty vector is transfected in lane 1. IP was performed with a rabbit polyclonal antibody against UPF1. e) coIP experiments in 293T cells transfected with the indicated siRNA and revealed by western blot. IP was performed against Rex (middle panel) or UPF1 (lower panel). IP UPF1 after siUPF1 treatment define background signals of Rex and CRM1. f) Proximity Ligation Assay (PLA) carried out in 293T cells transiently transfected with a HA-REX coding plasmid. PLA combined antibodies specific of the HA tag and FTSJ1, eIF5A or UPF1 were used as indicated. FTSJ1 (ribosomal RNA methyltransferase 1) is a nucleolar protein used as a negative control (no described interaction with Rex). eIF5A, a known partner of Rex, was used as positive control. Each dot corresponds to the colocalization of the indicated protein with a resolution of 40nm. Magnification of one representative cell is framed in the merge. For each condition, the number of spots per cell and their localisation in the cytoplasmic or nuclear compartment are represented in a bar plot on the right. Scale bar: 10μm

Article Snippet: 50000 cells were cultivated per well into 4 well Chamber Slide™ Labtek® II in DMEM (for HeLa or 293T cells) or RPMI medium (for Jurkat, C91PL or C8166 cells) complemented with 10% SVF and Penicillin/Streptomycin.

Techniques: Immunoprecipitation, Western Blot, Incubation, Transfection, Plasmid Preparation, Proximity Ligation Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: HTLV-1 Rex hijacks UPF1 in a CRM1 dependent manner, leading to NMD inhibition and revealing unexpected proviral roles of UPF1

doi: 10.1101/2023.06.20.545693

Figure Lengend Snippet: a) coIP experiments in the indicated lymphocytes cells extracts. IP were performed using rabbit polyclonal antibodies against UPF1. Proteins revealed by western blot are indicated on the side. b) (i) Confocal microscopy experiments were performed in C91PL and C8166 lymphocytes. CRM1 and UPF1 were revealed. The arrow shows a representative example of the CRM1 and UPF1 super foci (surface >= 1% total surface). Images were acquired with a x63 objective. Scale bar: 10μm. (ii) Quantitative analysis of the relative number of cells with UPF1 nuclear retention (left) or with at least one CRM1 foci. c) RIP using a rabbit polyclonal antibody targeting UPF1 were performed with Jurkat, C8166 or C91PL lymphocytes. Immunoprecipitated unspliced viral RNA (vRNA) was quantified by RTqPCR. The relative enrichment of vRNA associated to UPF1 in Jurkat and C8166 compared to C91PL was displayed in the graph. The relative amount of vRNA in the total extract before RIP is indicated in Supplementary Fig 4D. d) RIP experiments were carried out with the immunoprecipitation of UPF1, Rex, UPF2 or CRM1 as indicated in C91PL and in C8166 cells. The relative enrichment in vRNA in C91PL compared to C8166 is represented for each immunoprecipitated protein. e) coIP experiments in the C91PL cells extracts. IP were performed from the same extract using UPF1, UPF2 or a control antibody (Ctr). Proteins revealed by western blot were indicated on the side. f) Schematic diagram of the double RIP experiment performed in 293T cells co-transfected with the HTLV-1 WT molecular clone and a HA-UPF1 coding plasmid (upper panel). Quantification of the relative enrichment in vRNA after HA RIP targeting UPF1 (RIP1) and Rex RIP (RIP2) (lower panel) compared to RIP Ctr. g) RIP targeting UPF1, Rex or using a control antibody (as indicated) were performed with 293T cells transfected with HTLV-1 R23L molecular clone complemented with either Rex NES or Rex WT (orange and grey bars respectively). As a negative control, cells were transfected with an empty vector (blue bars). Immunoprecipitated RNA (vRNA) was quantified by RTqPCR. The graph displays the relative enrichment of vRNA associated to UPF1 or Rex compared to control antibody.

Article Snippet: 50000 cells were cultivated per well into 4 well Chamber Slide™ Labtek® II in DMEM (for HeLa or 293T cells) or RPMI medium (for Jurkat, C91PL or C8166 cells) complemented with 10% SVF and Penicillin/Streptomycin.

Techniques: Western Blot, Confocal Microscopy, Immunoprecipitation, Transfection, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: HTLV-1 Rex hijacks UPF1 in a CRM1 dependent manner, leading to NMD inhibition and revealing unexpected proviral roles of UPF1

doi: 10.1101/2023.06.20.545693

Figure Lengend Snippet: a) (i) Schematic representation of the experiment. (ii) Protein expression in cell extracts used for dual luciferase assay was monitored by western blot. (iii) Dual luciferase assay was performed on shCtr or shUPF1 293T cells transfected with a Rex WT plasmid or an empty vector. Firefly luciferase as well as Renilla luciferase were quantified. Firefly (FLuc)/Renilla (RLuc) ratio was plotted in a graph. b) Confocal microscopy experiments were performed in C91PL and C8166 lymphocytes. Panel (i): CRM1, UPF1 and p19 were revealed by IF. The arrow shows a representative example of p19 and UPF1 colocalization in C91PL. Images were acquired with a x20 objective. Scale bar: 50μm. Panel (ii): same as panel (i) with acquisitions with a x63 objective (scale bar 10μm). White arrows show p19 foci. Red arrow shows the CRM1 superfoci. c) Extracellular fraction (ECF) containing the viral particles was collected from 15.10 6 of C91PL, C8166 and Jurkat cells and analysed (lanes 4, 8, 12). The presence of UPF1, RRM2 and p19 was evaluated by WB and total protein was monitored with stainless procedure (upper and lower panels respectively). Cells used to harvest the ECF were also lysed and monitored by WB after serial dilutions (equivalent to 4.10 6 , 2.10 6 and 10 6 cells; lanes 1, 5, 9; 2, 6, 10; 3, 7, 11 respectively). d) Distribution of all the proteins quantified by mass spectrometry in the ECF (y axis) and in the “free particles” fraction (x axis) produced by C91PL cultures (in blue). Each coordinate corresponds to the mean of 3 independent experiments. On each axis, the density of proteins is represented. UPF1 position is indicated in orange on the density plots. Specific proteins (RNA decay factors partners involved in UPF1 dependant decay pathways, helicases, viral proteins, UPF1, RRM2, and CRM1) are indicated. Viral proteins GAG and ENV were found in both fractions, confirming the presence of viral particles.

Article Snippet: 50000 cells were cultivated per well into 4 well Chamber Slide™ Labtek® II in DMEM (for HeLa or 293T cells) or RPMI medium (for Jurkat, C91PL or C8166 cells) complemented with 10% SVF and Penicillin/Streptomycin.

Techniques: Expressing, Luciferase, Western Blot, Transfection, Plasmid Preparation, Confocal Microscopy, Mass Spectrometry, Produced