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: Scientific pages of pulmonology

Article Title: Method for the Culture of Mouse Pulmonary Microvascular Endothelial Cells

doi:

Figure Lengend Snippet: Products and supplies for culture of mouse pulmonary microvascular endothelial cells.

Article Snippet: ECIS array , Applied BioPhysics , Troy, NY, USA , 8W1E PC.

Techniques: Modification, Saline

Journal: Investigative Ophthalmology & Visual Science

Article Title: Glucocorticoid Induction of Occludin Expression and Endothelial Barrier Requires Transcription Factor p54 NONO

doi: 10.1167/iovs.13-11980

Figure Lengend Snippet: p54 knockdown prevents the barrier induction properties of dexamethasone. (A) BRECs were transfected with siRNA targeting p54 and seeded on 0.4-μm Transwell filters. After 48 hours, cells were switched to stepdown media with or without dexamethasone for 24 hours. Permeability to 70-kDa RITC-dextran tracer over a 4-hour time period was measured. The reduction in endothelial permeability to the dextran after dexamethasone treatment was ablated by p54 knockdown (P < 0.05). (B) BRECs transfected with siRNA against p54 were seeded on 8W10E+ arrays and TER was measured continually on the ECIS Z-theta instrument. GC treatment increased electrical resistance and knockdown of p54 decreased the GC induction of electrical resistance. After 13 hours of dexamethasone treatment and until the end of the experiment, p54 knockdown significantly reduced the glucocorticoid-induction compared with control (P < 0.001). *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: Functional Permeability Measures BRECs were transfected with siRNA and seeded on Transwells (Corning, Corning, NY) or 8W10E+ arrays (Applied Biophysics) at 20k cells/cm 2 .

Techniques: Knockdown, Transfection, Permeability, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Full fabrication and application schematic diagram of GelMA-VEGF/ECM-PCSK9 composite hydrogel and the related signaling pathway of PCSK9 that promotes BMSC osteogenic differentiation.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques:

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Construction and characterization of GelMA-VEGF/ECM-PCSK9 composite hydrogel. A Schematic diagram showing the process of composite hydrogel construction; B) Photographs of GelMA-VEGF hydrogel and GelMA-VEGF/ECM-PCSK9 hydrogel formation after UV light respectively; C i) Electron microscopic image of pure GelMA hydrogel, with a scale of 100 μm; ii) Enlarged electron microscopic image of GelMA hydrogel, with a scale of 50 μm; D) i The electron microscope image of the combination of GelMA hydrogel and ECM, with a scale of 100 μm; ii Electron microscope magnified image of GelMA hydrogel combined with ECM, with a scale of 50 μm; E) The infrared spectrum (FITR) diagram of the acellular ECM, GelMA hydrogel and GelMA/ECM composite hydrogel contains common basic energy groups; F) Load rate of PCSK9 in ECM; G) Release rate of VEGF loaded with GelMA hydrogel and GelMA/ECM composite hydrogel respectively; H) Release rate of PCSK9 loaded with ECM and GelMA/ECM composite hydrogel respectively; I) Release rate of VEGF and PCSK9 loaded in GelMA and GelMA/ECM on different time points respectively; J) Release rate of VEGF and PCSK9 respectively when loaded in GelMA/ECM; K) The swelling rate of GelMA gel and GelMA/ECM composite gel dissolved in PBS (n = 6); L) Degradation rate of GelMA hydrogel and GelMA/ECM composite gel in vitro (n = 6).∗means that compared with the control group, p < 0.05; ∗means that compared with the control group, p < 0.01; ∗∗∗means that compared with the control group, p < 0.001.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Microscopy, In Vitro, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: Angiogenic capacity formulations of HUVECs in response to different composite biomaterial in vitro. A) Calcein/PI staining of HUVECs seeded on glass slides, showing the cell migration profiles of HUVECs treated with different material groups, scale bar = 200 μm; B) Quantitative analysis of the intercellular blank areas in each group, with the baseline group serving as the negative control; C) Angiogenic images of HUVECs co-cultured with different composite materials for 4 h and 8 h respectively, scale bar = 250 μm; D–G) Quantitative assessment of angiogenic capacity in each group via ImageJ software analysis of key angiogenic parameters. Abbreviations: NC = negative control group; V = exogenous VEGF protein-only group; GV=GelMA + exogenous VEGF protein group; GVE = GelMA + VEGF + ECM group; GVEP= GelMA/VEGF + ECM/PCSK9 group. Statistical notations: ∗∗means that compared with the control group, p < 0.01; ns = no significant difference between group.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Migration, Negative Control, Cell Culture, Software, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: The effect of different composite hydrogel on the osteogenic differentiation of BMMSC in vitro. Cultivate BMMSC for osteogenic differentiation in osteogenic medium with GelMA, GelMA-VEGF, GelMA-VEGF/ECM, ECM-PCSK9, and GelMA-VEGF/ECM-PCSK9 for 7 days respectively. A,B) The cell nucleus was stained with DAPI (blue), RUNX2 was stained with RUNX2 antibody (green), and COL1A1 was stained with COL1A1 antibody (red), with a scale bar of 200 μm. C,D) The quantitative analysis results of COL1A1 and RUNX2 immunofluorescence images; E,F) Quantitative analysis of ALP staining and ARS staining for BMMSC co-culture with different kinds of hydrogels; G) ALP staining result for BMMSC co-culture with different kinds of hydrogels for 7days, scale bar = 200 μm; F) ARS staining result for BMMSC co-culture with different kinds of hydrogels for 14days, scale bar = 200 μm; I, J) After 7 and 14 days of co-culture with different combinations of composite hydrogels and BMMSC for osteogenesis and differentiation, the PCR experiment results of osteogenesis related indicators suggest that compared with the control group. G = simple GelMA hydrogel group, GV=GelMA hydrogels + VEGF protein group, GV/E = GelMA + VEGF/ECM group, EP = ECM + PCSK9 protein group, GVEP=GelMA + VEGF/ECM + PCSK9 protein group, the significant differences between the groups are expressed as ∗ p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ns means there is no significant difference between the groups.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: In Vitro, Staining, Immunofluorescence, Co-Culture Assay, Control

Journal: Bioactive Materials

Article Title: A composite hydrogel enables the spatiotemporal delivery of distinct cytokines to drive the native vascularized bone regeneration

doi: 10.1016/j.bioactmat.2026.02.048

Figure Lengend Snippet: After adding different concentrations of PCSK9 to BMMSC for osteogenic induction, western blotting (WB) experiment was performed to evaluate the expression of phosphorylated proteins and total proteins among different osteogenic differentiation relevant signaling pathways. A) WB images of different signaling pathways that related to osteogenic differentiation after adding different concentrations of PCSK9; B-D) Quantitative analysis results of phosphorylated protein and total protein. Compared with the control group, ∗ means p < 0.05, ∗∗ means p < 0.01.

Article Snippet: VEGF, ELISA kit for VEGF and PCSK9 were purchased from Boster company (Wuhan, China).

Techniques: Western Blot, Expressing, Protein-Protein interactions, Control

Journal: bioRxiv

Article Title: A bivalent lysine-acetylated small-molecule binding site in MYC

doi: 10.64898/2026.03.02.707789

Figure Lengend Snippet: (A) Representative FISH image of PC3M cells. The MYC dual color breakapart probe (Abbott Park, Illinois) labeled the 5’MYC as red and the 3’MYC as green. The normal signal pattern shows two fusions (overlap with the green and red signals). Single green and/or red signals indicate MYC rearrangements. Nuclei are counter-stained with DAPI (blue). (B) Colony-formation assays were performed comparing PC3-MCas9 cells to PC3-MCas9 lines co trans-fected with pairs of enriched sgRNA guides: (A169/A361) and (A169/ A423) following 14 days of treatment with 6μM MYCi975. Data was normalized to DMSO control and represent the mean ± S.D. from biological replicates. (*p<0.05; **p<0.01; ***p<0.001). (C) Relative cell viability of PC3-MCas9 cells co-transfected with sgRNA pairs (A11/A362, A169/A423, and A11/A423) was measured following continuous exposure to MYCi975 at 6µM for 20 days, followed by 10µM until day 35. (D, E) Multiple-sequence alignment of the MYC emBII and bHLH-LZ regions across vertebrate species. Blue dots denote DNA-binding residues in BHLH; green dots mark leucine-zipper dimerization residues. The MYC mutations identified are indicated in red font. (F) Expression and BLI K d of recombinant MYC mutants WAL-QRA, WAL (emBII mutant), and QRA (bHLH mutant) with MYCi975. (G) Colony formation assay of HO15.19 fibroblast, 22Rv1 and PC3 cells stably expressing Flag-tagged WT MYC and WAL-QRA MYC after treatment with DMSO and varying concentrations of MYCi975 for 14 days. Representative wells of colony formation assay are shown. (H) Cell viability of HO15.19 MYC and WAL-QRA MYC mutant was measured by IncuCyte live-cell imag-ing for 4 days with varying concentrations of MYCi975.

Article Snippet: 22Rv1, PC3, MyC-CaP, HEK293, MV-4-11, PC12, A549, LLC1 and HL-60 cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Labeling, Staining, Control, Transfection, Sequencing, Binding Assay, Expressing, Recombinant, Mutagenesis, Colony Assay, Stable Transfection

Journal: bioRxiv

Article Title: A bivalent lysine-acetylated small-molecule binding site in MYC

doi: 10.64898/2026.03.02.707789

Figure Lengend Snippet: (A) Schematic of the CRISPR Cas9 suppressor screening strategy. A Cas9-expressing PC-3M cell pool was transduced with a pooled sgRNA library targeting the MYC locus. Following enrichment in 6 µM MYCi975, resistant clones were isolated and sequenced to identify mutations conferring MYCi975 resistance. (B) Volcano plot summarizing the enrichment of sgRNAs guides across two independent screens. sgRNA guides targeting residues A169, A361, and A423 were significantly enriched. Deep sequencing of resistant clones identified mutation hotspots clustering close to the eMBII region (amino acid residues 196–200) and the bHLH (amino acid residues 370–380) domains of MYC. Representative sequences are shown. (C) BLI K d of MYCi975 for recombinant WT MYC, WAL-QRA MYC mutants, and individual domain MYC mutants (WAL and QRA). (D) Quantification of colony formation assays in HO15.19 fibroblast, 22Rv1 and PC3 cells expressing Flag-tagged WT MYC and WAL-QRA MYC mutant after MYCi975 treatment for 14 days. Data was normalized to DMSO control and represent the mean ± S.D. from biological replicates. *Denote statistical significance (*p<0.05; **p<0.01; ***p<0.001). (E) Immunoblot analyses of FLAG-tagged MYC protein stability in HO15.19, 22Rv1 and PC3 cells express-ing Flag-MYC WT or Flag-WAL-QRA-MYC mutant, treated with 6µM MYCi975 for 24 hours. GAPDH serves as a loading control.

Article Snippet: 22Rv1, PC3, MyC-CaP, HEK293, MV-4-11, PC12, A549, LLC1 and HL-60 cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: CRISPR, Expressing, Transduction, Clone Assay, Isolation, Sequencing, Mutagenesis, Recombinant, Control, Western Blot

Journal: bioRxiv

Article Title: A bivalent lysine-acetylated small-molecule binding site in MYC

doi: 10.64898/2026.03.02.707789

Figure Lengend Snippet: (A) We analyzed high-resolution acetylome data from the Clinical Proteomic Tumor Analysis Consortium (CPTAC), including 8 cohorts totaling 928 tumors and 389 normal tissues. K148 MYC modification was significantly elevated in tumors compared to matched solid normal tissues in 5 of 8 cohorts examined: breast cancer (PDC000239; p<0.0001), lung squamous cell carcinoma (PDC000233; p<0.0001), lung adenocarci-noma (PDC000491; p=0.0184), glioblastoma (PDC000245; p=0.028), and a multi-cancer cohort (PDC000450; p=0.009) comprising breast cancer, epithelial and complex epithelial neoplasms, glioblastoma, lung cancer, meningiomas, skin cancer and uterine adenocarcinoma. One lung cancer cohort (PDC000224) and two uterine corpus endometrial carcinoma cohorts (PDC000226, PDC000443) did not follow this pattern. Each data point represents an individual patient sample. Statistical analysis was performed using a two-tailed Mann-Whitney U test. p-values: ****p<0.0001, *p<0.05. (Abbreviations: AM, Additional Metastatic; M, Metastatic; PT, Primary Tumor; RT, Recurrent Tumor; N, Normal). (B) Unsupervised clustering of MYC target genes based on responses to wild-type MYC versus acetylation-deficient MYC (K148R) identified four clusters comprising MYC-repressed (clusters 1, 4) and MYC-induced (clusters 2, 3) programs. MYCi975 selectively induced acetylation-dependent stress-response genes (cluster 1) and repressed acetylation-dependent canonical MYC targets (cluster 2), while minimally affecting acety-lation-independent programs (clusters 3, 4), indicating preferential disruption of acetylated MYC dependent transcription. (C) Functional enrichment analysis of each gene cluster highlighting the top 10 significantly enriched path-ways. (D) Inverse correlation between AcK148-MYC abundance and sensitivity to MYCi975. Scatter plot showing the inverse relationship between the relative abundance of the AcK148-MYC modification (AcK148-MYC:MYC ratio) and cellular sensitivity to the MYC inhibitor MYCi975 (IC50) across a panel of 12 cancer cell lines including leukemia (Kasumi-1, HL-60, THP-1, MV-4:11), Prostate (PC3 and 22Rv1), Liver (PLC/PRF/5, HepG2), lung (A549), breast (MDA-MB-231), ovarian (A2780) and colorectal (HT-29) cell lines. A higher ratio of AcK148-MYC is associated with a lower IC50, indicating increased sensitivity to MYCi975.

Article Snippet: 22Rv1, PC3, MyC-CaP, HEK293, MV-4-11, PC12, A549, LLC1 and HL-60 cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Modification, Two Tailed Test, MANN-WHITNEY, Disruption, Functional Assay

Journal: bioRxiv

Article Title: A bivalent lysine-acetylated small-molecule binding site in MYC

doi: 10.64898/2026.03.02.707789

Figure Lengend Snippet: (A) Chemical structure of new MYCI analog MYCi648. (B) Binding affinities K d of MYC inhibitors to WT MYC and acetylated MYC (AcK148/157-MYC), along with cellular activity measured by E-box luciferase inhibition, CMV luciferase control assays, and viability assays in PC3 and control PC12 cells. (C) Tumor growth inhibition (TGI) in the MycCaP/FVB/N prostate cancer model treated with vehicle (n=8) or MYCi648 (30 mg/kg/d; n=5) i.p. (***p<0.001). (D) Tumor growth inhibition in the LLC1 syngeneic model (C57BL/6 mice) treated with vehicle (n=6) and MYCi648 (50 mg/kg/d; n=6) i.p. (***p<0.001). (E) BLI K d of MYCi648 to WT MYC and the WAL-QRA MYC mutant, demonstrating reduced binding to the WAL-QRA MYC mutant protein. (F) Quantification of colony formation assays in 22Rv1 and PC3 cells expressing WT MYC and WAL-QRA MYC mutant after MYCi648 treatment for 14 days. Data was normalized to DMSO control and represent the mean ± S.D. from biological replicates. *Denote statistical significance (*p<0.05; **p<0.01; ***p<0.001).

Article Snippet: 22Rv1, PC3, MyC-CaP, HEK293, MV-4-11, PC12, A549, LLC1 and HL-60 cell lines were purchased from the American Type Culture Collection (ATCC).

Techniques: Binding Assay, Activity Assay, Luciferase, Inhibition, Control, Mutagenesis, Expressing