Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 1. Processing, complexing, and localization of PC1 and PC2. (A) Immunoblot (IB) of endogenous human PC1 and PC2 derived from membrane fractions of a renal cortical tubule epithelial (RCTE) cell line. Samples were untreated (Un) or treated with EndoH (+E) or PNGase F (+P) and detected with an antibody against N-terminal PC1 (7e12; PC1 NT) or PC2 (YCE2). A nonspecific protein (Supplemental Figure 1, A and B) is indicated (n.s.). N-terminal glycoproducts, EndoH resistant (NTR) and EndoH sensitive (NTS), were resolved and were both reduced to the size of the aa backbone with PNGase F treatment (~330 kDa). All of PC2 was sensitive to EndoH. Representative blots are shown from 3 independent experiments. (B) IPs with a PC1 CT (BD3) or PC2 (YCE2) antibody from RCTE cells followed by deglycosylation detected with PC1 NT or YCE2 (PC2). PKD1–/– epithelial cells (9-12 cells; PKD1–/–) and IP with irrelevant antibody (IgG) were used as negative controls. The PC1 and PC2 complex was formed in the ER (EndoH sensitive), since PC2 coimmu- noprecipitated all PC1 glycoforms, including PC1-FL, even in high-salt (500 mM NaCl) conditions. Representative blots are shown from 3 independent experiments. (C) Maturation of PC1-NTR was affected by 2 μg/ml swainsonine (+Sw) treatment. A 72-hour swainsonine treatment reduced the PC1-NTR molecular weight but did not affect PC1-NTS, PC1-FL, or PC2, indicating that only PC1, but not PC2, traffics through the Golgi apparatus. Representa- tive blots are shown from 3 independent experiments. (D) Schematic of PC1 cleavage and glycosylation showing the size of the FL and the 2 GPS/GAIN N-terminal cleavage products, NTS and NTR.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Western Blot, Derivative Assay, Membrane, Molecular Weight, Glycoproteomics

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 2. Subcellular localization of PC1 glycoforms. (A) Labeling of RCTE cell surface proteins using alkoxyamine biotin (Alk. Biotin) and IP with neutra- vidin shows that PC1-NTR was the sole PC1 glycoform localized to the cell surface. PC2 was only detected with neutravidin IP after prolonged exposure and remained sensitive to EndoH digestion; compare level of input and surface protein. EGFR was used as a PM protein control. Representative blots are shown from 3 independent experiments. IF of streptavidin-488–labeled Alk. Biotin–treated and untreated cells shows efficient surface glycoprotein labeling. Scale bar: 20 μm. (B) Density gradient fractionation of RCTE cells shows that a portion of PC1-NTR cofractionated with markers of PM (ORAI/ SMO) and cilia (Arl13b/SMO), while most detectable PC2 was distributed in fractions overlapping with ER proteins, calnexin, and STIM1, with minor overlap with the cilia fraction. The relative signal intensity is plotted below. Gradient samples were loaded on 2 different SDS-PAGE gels that were run simultane- ously and transferred onto the same membrane for detection. Representative blots are shown from 3 independent experiments. (C) Coimmunoprecipita- tion of PC2 with PC1 from gradient fraction 12 (cilia enriched) followed by deglycosylation using EndoH (+E) or PNGase F (+P) or no enzyme (Un). Only the EndoH-resistant PC1-NTR glycoform along with EndoH-sensitive PC2 cofractionated with Arl13b.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Labeling, Control, Fractionation, SDS Page, Membrane

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 3. PM and cilia colocalization of PC1 and PC2. (A) Diagram of mCherry-PC1 and GFP-PC2 fusion proteins used in the IF experiments. (B) Confocal images of mCherry-PC1– and GFP-PC2–cotransfected RCTE cells showing prefixation surface labeling of PC1 (mCherry antibody) in live cells or all PC1 after permeabilization (total mCherry), with PC2 (GFP) and DAPI. Scale bar: 10 μm. Peripheral overlapping PC1/PC2 punctae are indicated with yellow arrows, with colocalization also seen in the ER. (C) Optical sectioning (z-stack, XZ plane) of confocal image of ciliated RCTE cells cotransfected with mCherry-PC1 and GFP-PC2 and subjected to prefixation PC1 labeling (mCherry antibodies). Surface PC1 and PC2 colocalized in primary cilia, while PC1 signal was also seen on the PM (red arrow). Scale bar: 10 μm. (D) Low-magnification image of surface-labeled RCTE cells cotransfected with mCherry-PC1 and GFP-PC2, showing mCherry-PC1 detected on the surface only in cells also expressing GFP-PC2 (arrows). Scale bar: 50 μm. (E) Deglycosylation analysis of RCTE cells expressing mCherry-PC1 alone or cotransfected with GFP-PC2. Mature mCherry-PC1 (PC1-NTR) was detected only in cells cotransfected with GFP-PC2, while cleaved ER-resident mCherry-PC1 (NTS) accumulated in the absence of PC2. Representative blots are shown from 3 independent experiments.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Labeling, Expressing

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 4. PC1 maturation and trafficking depend on PC2. (A) IB of membrane-purified proteins from MEFs derived from WT, Pkd2+/–, Pkd2–/–, and Pkd1–/– embryos detected with PC1 NT or PC2 antibodies. A Coomassie-stained loading control is shown. PC1- NTR was completely absent and PC1-NTS elevated in Pkd2–/– cells. Representative blots are shown from 3 independent experiments. (B) Glycosylation analysis of WT and Pkd2–/– MEFs showing that PC1- NTR was absent and PC1-NTS elevated in Pkd2–/– cells compared with WT MEFs. Representative blots are shown from 3 independent experiments. (C) IF detection of cilia (acetylated α-tubulin, Ac. tubulin) and PC2 (H280) in WT, Pkd2–/–, and Pkd1–/– MEFs (scale bar: 10 μm). (D) Quantification of these localizations (n = 50 cilia). PC2 was found on 30% of WT cilia but not on Pkd2–/– or Pkd1–/– cilia, indicating a crucial role for PC1 in PC2 cilia localization in MEFs (****P = 0.0001 by 2-tailed Fisher’s exact test).

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Membrane, Purification, Derivative Assay, Staining, Control, Glycoproteomics

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 5. Maturation of PC1 is associated with the dosage of PC2. (A) Membrane protein purified from P9 mouse kidneys of WT, Pkd2+/–, Pkd1+/–, and bigenic combinations with the Pkd1RC/RC genotype and Pkd2WS25 allele assayed by SDS-PAGE and probed with PC1 NT and PC2 antibodies. Densitometric profiles of the NT products are shown with a Coomassie-stained loading control. Reduction of Pkd2 reduced the level of PC1-NTR, while Pkd1 reduction lowered the level of both products. Note in Pkd1RC/RC animals that the PC1-FL product is more evident, consistent with the previously described partial cleavage defect (20). Representative blots are shown from 3 independent experiments. (B) IB of membrane-purified protein from WT, Pkd1RC/RC Pkd2+/+, and Pkd1RC/RC Pkd2+/– MEFs detected with PC1 NT or PC2 antibodies showing the PC1-FL, PC1-NTR, and PC1-NTS glycoforms, PC2, and control Coomassie band. Representative blots are shown from 3 independent experiments. (C) Quantification of PC1-NTR from MEFs with various Pkd1 and Pkd2 genotypes. Results were derived from a minimum of 3 independent IBs and biological replicates obtained from 2 separate crosses (numbers [n] indicated) and compared with the WT average from each group, with significance determined by the Student’s t test. (D) Relative ratio of PC1-NTR to NTS expression for indicated MEF geno- types (numbers [n] indicated). The significance of the difference between means was compared using the Student’s t test. For C and D, quartile box plots represent the median, quartiles, and minimum/maximum range, with means of each group in parentheses. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Membrane, Purification, SDS Page, Staining, Control, Derivative Assay, Expressing

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 6. Pkd2 depletion aggravates the Pkd1RC/RC cystic phenotype. (A) Masson trichrome–stained kidney cross sections of 4-month-old mice of the Pkd1RC/RC genotype with the addition of Pkd2WS25/+, Pkd2+/–, or Pkd2WS25/–, and Pkd2WS25/– mice with the Pkd1RC/+ genotype. PKD severity and fibrosis worsened in bigenic mice, with evidently more severe disease in the Pkd1RC/RC Pkd2+/– genotype, corresponding to PC1-NTR levels of about 30% (Figure 5C). However, the Pkd1RC/RC Pkd2WS25/– combination resulted in the most severe disease. Scale bar: 1 mm. (B–D) Graphical representations of %KW/BW (B), cystic index (C), and blood urea nitrogen (D) of the various genotypes quantify the increased disease severity with Pkd1/Pkd2 combined phenotypes (see Supplemental Table 1 for details). Error bars depict ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using a 2-way ANOVA with Student’s t test. F, female; M, male. +Note that 5 of 10 Pkd1RC/RC Pkd2WS25/– animals died before 4 months (F: P42, P74; M: P38, P51, P79).

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Staining

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 7. Analysis of maturation of endogenous PC1 truncation mutants. (A) Diagram showing approximate locations of the truncations in PC1 and the locations of the REJ domain, PKD repeats, and CT-CC. (B) Glycosylation analysis of MEFs isolated from WT and Pkd1del31/del31 embryos detected with the PC1 NT antibody. The del31 mutation truncated PC1 after the GPS/GAIN cleavage site, and since the truncated protein was cleaved, an NT as well as a truncated FL (tFL) product were observed. EndoH analysis shows that PC1-NTS, but not PC1-NTR, was generated. Representative blots are shown from 3 independent experiments. (C) IP of endogenous PC2 (H280) in WT and Pkd1del31/del31 (–/–) MEFs shows that PC2 did not coimmunoprecipitate with the del31 mutant PC1. Lysate control is shown above. Representative blots are shown from 3 independent experiments. (D–G) Pkd1del17/+ (WT/del17) adult mouse kid- ney (D and E) and human fibroblasts from a female ADPKD patient with an extracellular truncation due to a translocation in exon 15 (WT/tr15; 77-2) (F and G), disrupting PC1 extracellularly in the REJ domain or PKD1 repeats, respectively. (D, F, and G) Glycosylation analysis comparing untreated (Un), EndoH- digested (+E), and PNGase F–digested (+P) protein. In all cases, only the EndoH-sensitive truncated product (PC1-tNTS) was seen, with no PC1-tNTR glycoform. In del17 and tr15, the PC1 truncated product was expressed at a much higher level than that detected WT, and so longer exposures are shown (E and G) to visualize the WT allele/products. Representative blots are shown from 3 independent experiments.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Glycoproteomics, Isolation, Mutagenesis, Generated, Control, Translocation Assay

Journal: Journal of Clinical Investigation

Article Title: Polycystin-1 maturation requires polycystin-2 in a dose-dependent manner

doi: 10.1172/jci76972

Figure Lengend Snippet: Figure 8. Deletion of CC2 and missense mutations in PC2 influence PC1 maturation. (A) Diagram of WT and mutant human PC2 used in the cotransfec- tion experiments (B and C) showing the positions of domains, deletions, and aa substitutions. (B) mCherry-PC1 cotransfected with WT GFP-PC2 or PC2 lacking the C-terminal cytoplasmic tail (GFP-PC2-L703X) into RCTE cells. Detection of PC1 NT (mCherry) revealed EndoH-resistant PC1-NTR (arrow) only in WT-PC2, but not in PC2-L703X, cotransfected cells. Detection of PC2 (GFP) shows that a portion of PC2-L703X was EndoH resistant (R; arrow). Representa- tive blots are shown from 3 independent experiments. (C) Cotransfection of various GFP-PC2 mutants and mCherry-PC1 into RCTE cells showing the effect of PC2 mutations on the PC1 glycosylation pattern and PC2 products. Deletion of the EF hand and coiled coil 1 (delEF+CC1) did not disrupt PC1 maturation, but deletion of coiled coil 2 (delCC2) greatly reduced the level of the PC1-NTR product. Pathogenic missense substitutions in PC2, especially p.R322Q and p.W414G, also had a negative effect on PC1 maturation. Representative blots are shown from 3 independent experiments. (D) Quantification of the ratio of PC1-NTR to NTS glycoforms obtained from the cotransfection experiments with WT and mutant PC2 GFP constructs (C and Supplemental Figure 5A). Deletion mutants removing CC2 but not EF+CC1 largely disrupted PC1 maturation, while missense mutations also significantly disrupted maturation. n = 3 for all except R322W (n = 2) and D511V (n = 4). Quartile box plots represent the median, quartiles, and minimum/maximum range, with the mean of each group in parentheses. P values are shown as compared with GFP-PC2-WT (control) with the Student’s t test; ****P < 0.0001.

Article Snippet: The following antibodies were used: PC1 NT IgG1, 7e12 (70) (WB 1/1,000); PC1 CT Rb, BD3 (a gift of Oxana Beskrovnaya, Genzyme, Framingham, Massachusetts, USA) (IP 1/250); PC1 CT Gt, EB08670 (Everest Biotech) (IP 1/250); PC1 CT Rb, PKS-A (71); PC2 Rb, H280 (Santa Cruz Biotechnology Inc.) (WB 1/5,000); PC2 IgG2a, YCE2 (Santa Cruz) (WB 1/2,000, IF 1/500); EGFR Rb (BD Transduction Laboratories) (WB 1/1,000); acetylated α-tubulin, IgG2b (Invitrogen) (IF 1/5,000); anti-Tag(CGY)FP (Evrogen AB121) (1/5,000 WB); mCherry Rb (5993-100; BioVision) (Surface Labeling 1/1,000); mCherry IgG2a (Novus Biologicals NB196752 1C51) (WB 1/2,000); calnexin Rb (Novus Biologicals) (1/250 IF, 1/1,000 WB); ORAI-1 Rb (H-46; Santa Cruz Biotechnology Inc.) (1/1,000 WB); Arl13b (17711-1-AP; Proteintech) (1/1,000 WB); and STIM1 IgG2a (M01; Abnova) (1/2,000 WB).

Techniques: Mutagenesis, Cotransfection, Glycoproteomics, Construct, Control

Journal: Frontiers in immunology

Article Title: Persistence of Candida albicans in the Oral Mucosa Induces a Curbed Inflammatory Host Response That Is Independent of Immunosuppression.

doi: 10.3389/fimmu.2019.00330

Figure Lengend Snippet: FIGURE 3 | The persistent strain 101 does not suppress the antifungal host response. (A,B) Relative expression of Il10 (left) and Tgfb1 transcripts (right) in epithelial sheets (A) or in bulk tongue tissue (B) of mice that were infected with strain 101 or SC5314 for the indicated period of time. Each symbol represents a pool of epithelial sheets from three animals each (A) or a single mouse (B). The geomean of each group is indicated. Data are pooled from two independent experiments each. (C,D) % Foxp3+ Tregs within the viable CD45+CD3+CD4+ population in the tongue of mice that were infected with strain 101 or SC5314 for the indicated period of time. Representative FACS plots and the gating strategy are shown in C, summary plots showing the mean + SD of data pooled from two independent experiments with 3–4 animals per group are shown in D. (E,F) Mice were treated with anti-CD25 or isotype control antibody prior to infection with strain 101. Relative expression of Il17a (left), S100a8 (middle), and Cxcl1 (right) (E) and tongue fungal loads (F) in the bulk tongue tissue at the indicated time point after infection are shown. In (E), each symbol represents a single animal, The geomean of each group is indicated. The dotted line represents transcript levels in naïve animals (mean of 8 animals). In (F), each bar is the geomean + SD of 4 animals per group. (G,H) WT mice were infected sublingually with a 1:1 mixture of strain 101 and strain SC5314 or with each strain alone. Tongues were harvested on day 1 post-infection and analyzed for the infiltration of Ly6G+Ly6CloCD11b+ neutrophils by flow cytometry (G) and for the expression of S100a9 and Il17f (H) transcript by RT-qPCR. Data are the mean + SD of 3–4 mice per group. Graphs display data representative of one out of two independent experiments. The dotted line represents the detection limit. Statistics were calculated using one-way ANOVA. In (E), statistics compare infected to naïve groups. ***p < 0.001, ****p < 0.0001.

Article Snippet: Where indicated, 0.125mg anti-CD25 antibody (clone PC61.5.3, BioXCell) or the corresponding isotype control were administered i.p. per mouse 7 days prior to infection.

Techniques: Expressing, Infection, Control, Cytometry, Quantitative RT-PCR