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

Journal: Frontiers in Aging Neuroscience

Article Title: Western diet-induced visceral adipose tissue inflammation promotes Alzheimer’s disease pathology via microglial activation in a mouse model

doi: 10.3389/fnagi.2025.1644988

Figure Lengend Snippet: Identification of epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines using AB array and QuantSeq 3′-mRNA sequencing. (A) Heatmap of cytokine expression in eWAT using the AB array. A total of 115 cytokines were significantly upregulated in the WD group compared with those in the control group, as indicated by the color scale representing relative expression levels (blue: downregulation, red: upregulation). (B) Heatmap of cytokine expression in eWAT determined using QuantSeq 3′-mRNA sequencing. In this analysis, 132 cytokines showed significant upregulation in the WD group compared with those in the control group, with similar color coding for relative expression changes. (C) A subset of five cytokines, including CCL8, CCL9, CXCL13, and IL-18, were consistently increased across both datasets (AB array for eWAT and QuantSeq for plasma) and were identified as eWAT-associated pro-inflammatory cytokines contributing to systemic and neuroinflammation in the WD group. (D) Protein–protein interaction (PPI) network analysis of differentially expressed genes (DEGs) was constructed using known interaction data. The network highlights significant interactions among key pro-inflammatory cytokines, illustrating the interconnected roles of these factors in inflammatory signaling. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the DEGs, with significant pathways related to inflammation, such as cytokine–cytokine receptor interaction and the chemokine signaling pathway. The x -axis denotes the number of genes associated with each pathway, while the size and color of the dots represent gene count and statistical significance, respectively.

Article Snippet: Recombinant mouse cytokines CCL8 (PMC1104) and CXCL13 (PMC1614) were purchased from Peprotech (Rocky Hill, NJ, USA), and CCL9 (463-MG) and IL-18 (9139) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Ab Array, Sequencing, Expressing, Control, Clinical Proteomics, Construct

Journal: Frontiers in Aging Neuroscience

Article Title: Western diet-induced visceral adipose tissue inflammation promotes Alzheimer’s disease pathology via microglial activation in a mouse model

doi: 10.3389/fnagi.2025.1644988

Figure Lengend Snippet: Validation of epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines. A Western diet (WD) significantly increased the levels of eWAT-associated pro-inflammatory cytokines in both eWAT and plasma. (A–E) Western blot analysis validated a marked increase in the expression of eWAT-associated pro-inflammatory cytokines in the WD group compared with that in the controls. Specifically, the levels of CCL8, CCL9, CXCL13, and IL-18 were significantly upregulated in the eWAT of WD group, indicating heightened inflammatory activity in this tissue. (F–I) Corresponding ELISA analysis of plasma samples showed increased concentrations of these cytokines in the WD group, with significant increases observed for CCL8, CCL9, CXCL13, and IL-18. Importantly, in the eWAT-removal group, plasma levels of these cytokines were reduced to levels comparable to the sham, confirming that eWAT is a primary source of the increased circulating cytokines observed in the WD group. Both the control (normal diet-fed) and the WD group underwent the same surgical procedure without eWAT removal. The results are presented as mean ± SD with a sample size of n = 9 for western blot quantification and n = 5 for plasma ELISA assay. Asterisks denote statistically significant differences compared with the control group, with * p < 0.05 and *** p < 0.001, ns = no significant difference, One-way analysis of variance followed by Dunnett’s post hoc test.

Article Snippet: Recombinant mouse cytokines CCL8 (PMC1104) and CXCL13 (PMC1614) were purchased from Peprotech (Rocky Hill, NJ, USA), and CCL9 (463-MG) and IL-18 (9139) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Biomarker Discovery, Western Blot, Clinical Proteomics, Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Aging Neuroscience

Article Title: Western diet-induced visceral adipose tissue inflammation promotes Alzheimer’s disease pathology via microglial activation in a mouse model

doi: 10.3389/fnagi.2025.1644988

Figure Lengend Snippet: Epididymal white adipose tissue (eWAT)-associated pro-inflammatory cytokines induce significant neurotoxicity in HT22 cells. (A) The viability of HT22 hippocampal neuronal cells was not affected after 24 h of direct culture with eWAT-associated cytokines (CCL8, CCL9, CXCL13, and IL-18) at varying concentrations (25, 50, and 100 ng/mL). The results indicate no direct cytotoxic effects of these cytokines on hippocampal neurons under these conditions. (B) To evaluate the cytotoxicity of eWAT-associated cytokine in BV2 microglia, each cytokine was treated at a concentration of 50, 100, and 200 ng/ml. eWAT-associated cytokines did not induce cytotoxicity in BV2 microglial cells after 24 h of exposure, but they significantly increased microglial proliferation. (C) Treatment with each cytokine at a concentration of 100 ng/mL and analysis by ELISA showed that eWAT-associated pro-inflammatory cytokines significantly increased IL-6 secretion in BV2 microglial cells compared to those of untreated controls. (D) Intracellular reactive oxygen species (ROS) levels were measured in BV2 cells using a DCFDA fluorescence assay. Treatment with eWAT-associated cytokines led to a significant increase in ROS production, indicating oxidative stress–mediated microglial activation. (E) Treatment of HT22 hippocampal neurons with conditioned medium (CM) containing 100 ng/mL of each eWAT-associated cytokine to BV2 microglia significantly decreased cell viability, demonstrating the neurotoxic effect of cytokine-activated microglia on neurons. The data are presented as mean ± SD, with sample sizes ranging from n = 3 to n = 6 per group. Statistical significance is indicated by asterisks (*, compared with the control at 24 h): * p < 0.05, ** p < 0.01, and **** p < 0.0001, ns = no significant difference, One-way analysis of variance followed by Dunnett’s post hoc test.

Article Snippet: Recombinant mouse cytokines CCL8 (PMC1104) and CXCL13 (PMC1614) were purchased from Peprotech (Rocky Hill, NJ, USA), and CCL9 (463-MG) and IL-18 (9139) were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Activation Assay, Control