Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: ELISA platform for assessment of uEVs (A) Schematic illustration of sandwich ELISA using Tim4. (B) Expression of MGAM, MUC1, and CD9 in nephron segments. The expression pattern was visualized by kidney cell explorer and by fluorescent immunohistochemistry using human kidney tissue. The numbers above the figure are the same as in Figure 1 G. Scale bars, 100 μm. See also Figures S4 and .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA, Expressing, Immunohistochemistry

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet: Application of expression signature of uEVs for diagnosis of CKD (A) Comparison of MUC1 and MGAM levels (absorbance at 450 nm) measured by customized ELISA in samples from healthy controls and patients with CKD. G1 indicates CKD patients with eGFR ≥90 and G2-5 with <90 mL/min/1.73m 2 . (B and C) ROC curve for distinguishing patients with decreased renal function (eGFR <60 (B), eGFR <90 (C)) from healthy controls by logistic regression. (D) ROC curve for distinguishing patients with CKD having normal eGFR (≥90) from healthy controls by logistic regression. (E) Box and beeswarm plot of the assay value of MGAM/MUC1 (the expression of MGAM divided by that of MUC1) in patients with CKD having each renal function and healthy controls in the discovery and validation cohorts. (F) ROC curve for distinguishing patients with decreased renal function (eGFR <90) from healthy controls by logistic regression using combinations of MGAM/MUC1, urine creatinine (uCr), urine albumin (uAlb), and L-FABP. (G) The change in eGFR in follow-up period in two groups (MGAM/MUC1 below 0.35 and the others). Boxes in boxplots indicate the 25th and 75th percentiles, and the horizontal lines inside the boxes indicate the median. Bars indicate the 10th and 90th percentiles. The data were compared using the two-tailed Mann–Whitney U test. See also Figures S6–S8 .

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Expressing, Biomarker Discovery, Comparison, Enzyme-linked Immunosorbent Assay, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: Urinary extracellular vesicles signature for diagnosis of kidney disease

doi: 10.1016/j.isci.2022.105416

Figure Lengend Snippet:

Article Snippet: Antibodies used as capture reagents were as follows: anti-CD63 antibody contained in the abovementioned kit, and mouse monoclonal anti-CD9 antibody (clone12A12, Shionogi, Osaka, Japan), rabbit polyclonal anti-MGAM antibody (Cat# 22195-1-AP, Proteintech, Chicago, IL, USA), mouse monoclonal anti-MUC1 antibody (Cat# sc-7313, Santa Cruz, Dallas, TX, USA), mouse monoclonal anti-Poliovirus receptor (PVR) antibody (Cat# 66913-1-Ig, Proteintech), mouse monoclonal anti-PKD2 antibody (Cat# H00005311-M01, Abnova, Taipei, Taiwan), mouse monoclonal anti-CD133 (PROM1) antibody (Cat# 66666-1-lg, Proteintech), and rabbit monoclonal anti-CD90/Thy1 antibody (Cat# ab92574, Abcam, Cambridge, UK).

Techniques: Labeling, Enzyme-linked Immunosorbent Assay, Isolation, Software, Microplate Reader Absorbance Measurement, Mass Spectrometry, Transmission Assay, Electron Microscopy, Fluorescence, Microscopy

Journal: Cell Reports

Article Title: The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

doi: 10.1016/j.celrep.2021.110080

Figure Lengend Snippet:

Article Snippet: Rabbit anti-p97 , Proteintech , Cat#10736-1-AP; RRID: AB_2214635.

Techniques: Virus, Subcloning, Recombinant, Staining, Picogreen Assay, Proliferation Assay, Flow Cytometry, DNA Extraction, Sequencing, Luciferase, Software, Transfection, Modification, Magnetic Beads, Membrane

Journal: The Journal of Clinical Investigation

Article Title: N-cadherin upregulation mediates adaptive radioresistance in glioblastoma

doi: 10.1172/JCI136098

Figure Lengend Snippet: (A) qRT/PCR showing that mGSRR cells display increased Cdh2 and decreased Cdh1 mRNA expression compared with mGS. Two-tailed Student’s t test. (B) Western blot showing expression of Slug, Snail1, and Zeb1 are gradually increased upon repeated irradiation in mGS cells. (C) Western blot showing that Snail overexpression induces elevation of N-cad, Olig2, and Zeb1, and suppression of Tuj1 in mGS cells. (D) Wnt/β-catenin transcriptional activity is suppressed in mGSRR and mGS with Snail1 overexpression (OE) compared with mGS cells. ***P < 0.001, Tukey’s HSD test. (E) Clonogenic survival assay shows mGS Snail1 OE cells have a higher survival rate than mGS cells. Two-tailed Student’s t test. *P < 0.05, **P < 0.01. (F) Western blot showing increased N-cad, β-catenin, Slug, and Zeb1 expression 2 days after mouse recombinant IGF1 (100 ng/mL), but not TGF-β1 (10 ng/mL) treatment in mGS cells. (G) Western blot showing IGF1 overexpression increases N-cad, β-catenin, Zeb1, and IGF1R expression in mGS cells. (H) Survival curves for mice implanted with 1000 cells (GS with IGF1 expression vector) and subjected to whole-brain irradiation (2 Gy/day, days 3 to 7, 10 Gy total). (I) Left: schematic showing experimental design for clonogenic survival assay with repeated irradiation. Single mGS cells seeded in agarose medium were exposed to repeated irradiation (5 doses of 4 Gy, every 3 days) with or without drug rescue. IGF1R (AEW541 0.5 μM; PPP 0.2 μM) and TGF-β1 (LY2157299 10 μM, SB431542 10 μM) inhibitors were used. Right: quantification of percentage of surviving colonies shows that IGF1R inhibitors selectively decreased survival rate. Drugs alone had no effect on colony formation (data not shown). ***P < 0.001, Dunnett’s test. (J) Mice implanted orthotopically with mGSRR cells had a survival benefit after whole-brain irradiation (2 Gy × 5 days) with adjuvant PPP (15 mg/kg, i.p. twice a day from day 3–7) in contrast to vehicle control, only IR or PPP alone (8 mice/group; log-rank test). All blots show representative images (n = 3 or more).

Article Snippet: Cells were treated with recombinant mouse IGF1 (100 ng/mL, R&D Systems, #791-MG), recombinant mouse TGF-β1 (10 ng/mL, R&D Systems, #7666-MB), SB216763 (1–5 μM, Selleckchem, S1075), or CHIR99021 (1–5 μM, Selleckchem, S1263).

Techniques: Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Irradiation, Over Expression, Activity Assay, Clonogenic Cell Survival Assay, Recombinant, Plasmid Preparation, Adjuvant

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: (A) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. control (CTL) in HEK293-OATP1B3 cells without any pre-incubation (Co-incubation). (B) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in HEK293-OATP1B3 cells pretreated with bortezomib (Btz) vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE of [ 3 H]CCK-8 accumulation (1 μM, 3 min) in human SCH pretreated with bortezomib (Btz) (50 and 250 nM, 7 h) vs. vehicle CTL. After pretreatment, cells were washed three times with the HBSS buffer, and the [ 3 H]CCK-8 accumulation was determined in the absence of bortezomib. Model-estimated fold change and associated SE of OATP1B3-mediated [ 3 H]pitavastatin (1 μM, 1 min) (D) and [ 3 H]E 2 17βG accumulation (1 μM, 2 min) (E) in bortezomib pretreatment vs. vehicle CTL at each indicated pretreatment concentration and time (Pre-incubation). In D and E, HEK293-OATP1B3 and HEK293-Mock cells were pretreated with vehicle control (CTL) or bortezomib at the indicated concentrations and time. After washing with the HBSS buffer, OATP1B3-mediated [ 3 H]pitavastatin (D) and [ 3 H]E 2 17βG accumulation (E) was determined by subtracting the values determined in the HEK293-Mock cells from those in HEK293-OATP1B3 cells. (F) Model-estimated fold change and associated SE in [ 3 H]CCK-8 accumulation (1 μM, 3 min) vs. CTL. Cells were pre-incubated with bortezomib-free (CTL) or 50 nM bortezomib-containing media for 2 h. At the end of pre-incubation, the culture medium was removed. After washing, CTL- and bortezomib-pretreated cells were cultured in bortezomib-free medium for the indicated time duration. [ 3 H]CCK-8 (1 μM, 3 min) accumulation was determined at the indicated time points after washing three times ( n = 3 in triplicate). A generalized linear mixed model was fit to the data in A-F as described in the “Materials and Methods” (n = 3 for A, D-F; n = 6 for B; n = 5 for C; all experiments were performed in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p <0.05) vs. CTL.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: CCK-8 Assay, Control, Incubation, Concentration Assay, Cell Culture

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: Human SCH were cultured as described in the Materials and Methods section. (A) Immunoblot of OATP1B3 and OATP1B1 in whole cell lysates of human SCH that were treated with bortezomib (Btz) (50 and 250 nM) or vehicle control (CTL). β-actin served as the loading control. Representative images are shown from n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (B) Fold changes of OATP1B1 and OATP1B3 protein levels. Densitometry of OATP1B1 and OATP1B3 protein levels was normalized to that of β-actin. Fold changes of total protein levels of OATP1B1 and OATP1B3 in bortezomib-treated cells vs. CTL were expressed as mean ± SD n = 3 and 4 donors for 50 and 250 nM treatment, respectively. (C) Immunoblot of ubiquitin in whole cell lysates of human SCH treated with bortezomib (Btz) (50 nM, 7 h) or vehicle control. β-actin served as the loading control. Representative images are shown from n = 3 donors.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Western Blot, Control, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-OATP1B1 cells were seeded at a density of 1.2 x 10 5 cells/well in a 24-well plate and were cultured to confluence. Human SCH were cultured as described in the “Materials and Methods”. (A) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle control (CTL) in HEK293-OATP1B1 cells (Co-incubation). (B) Model-estimated fold change and associated SE in [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) vs. vehicle control (CTL) at each indicated time and concentration (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]pitavastatin accumulation was determined in the absence of bortezomib. (C) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation in the presence of 10–250 nM bortezomib (Btz) or 25 μM rifampicin (Rif) vs. vehicle CTL in HEK293-OATP1B1 cells (Co-incubation). (D) Model-estimated fold change and associated SE in [ 3 H]E 2 17βG accumulation (1 μM, 2 min) vs. vehicle CTL treatment in HEK293-OATP1B1 cells pretreated with bortezomib (Btz) for the indicated times and at the indicated concentrations (Pre-incubation). Following pretreatment, cells were washed three times with HBSS buffer, and the [ 3 H]E 2 17βG accumulation was determined in the absence of bortezomib. (E) Model-estimated fold change and associated SE of [ 3 H]pitavastatin accumulation (1 μM, 0.5 min) in human SCH pre-incubated with bortezomib (50 nM for 7 h) or co-incubated with positive control bromosulfophthalein (BSP) (100 μM) vs. vehicle CTL. Fold changes and SEs were estimated by linear mixed effects models, as described in the “Data Analysis” section (n = 3 hepatocyte donors in triplicate). To account for multiple comparisons, p-values were adjusted based on the Bonferroni method. * indicates a statistically significant difference (adjusted p <0.05) vs. CTL.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Control, Incubation, Concentration Assay, Positive Control

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-FLAG-OATP1B1 and–FLAG-OATP1B3 stable cell lines were seeded at a density of 3x10 6 cells per 100-mm 2 dish. After culturing for 48 h, cells were treated with bortezomib (Btz) (50 nM, 7 h) or vehicle control (CTL). Whole cell lysates (WCL) (500 μg) from HEK293-FLAG-OATP1B1 (A, left panel) and HEK293-FLAG-OATP1B3 (B, left panel) were subjected to immunoprecipitation (IP) with FLAG antibody, followed by immunoblotting with ubiquitin antibody. Immunoblots of FLAG (A and B, middle panels) and ubiquitin (right panels of A and B) were conducted using whole cell lysates (50 μg) of HEK293-FLAG-OATP1B1 (A) and HEK293-FLAG-OATP1B3 (B) cells treated with bortezomib (50 nM, 7 h) or vehicle control. β-actin served as the loading control. FLAG-OATP1B1 and FLAG-OATP1B3 protein levels determined by densitometry were normalized to levels of β-actin. Fold changes of total protein levels in bortezomib-treated cells vs. CTL were expressed as mean ± SD. Representative images from n = 3 independent experiments are shown.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Stable Transfection, Control, Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner

doi: 10.1371/journal.pone.0186924

Figure Lengend Snippet: HEK293-OATP1B3 cells were seeded at a density of 1.2 x 10 5 cells/well in 24-well plates, and were cultured for 48 h prior to performing the experiment. (A) The concentration-dependent accumulation of CCK-8 (0.1–40 μM, 3 min) was determined in HEK293-OATP1B3 cells pretreated with control (CTL) or bortezomib (Btz) (50 nM, 7 h). Solid and dashed lines represent the best fit lines of the Michaelis–Menten equation to the data of vehicle control (CTL) (closed circles) and bortezomib (Btz) pretreatment (open circles), respectively. A representative graph of three independent experiments performed in triplicate is shown. The student’s t-test was conducted to compare the V max and K m values between bortezomib and vehicle control pretreatment. * indicates a statistically significant difference ( p <0.05; bortezomib vs. CTL). (B) HEK293-OATP1B3 cells were pretreated with 50 nM bortezomib (Btz) or vehicle control (CTL) for 7 h. Surface levels of OATP1B3 were determined via biotinylation, followed by immunoblotting with OATP1B3 and Na-K-ATPase antibodies. GAPDH was used as a cytoplasmic protein marker. OATP1B3 surface protein levels were determined by densitometry and were normalized to those of Na-K-ATPase. Fold changes in the surface levels of OATP1B3 (bortezomib vs. CTL) were expressed as mean ± SD of three independent experiments.

Article Snippet: Bortezomib was procured from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Cell Culture, Concentration Assay, CCK-8 Assay, Control, Western Blot, Marker