Journal: Environmental Health Perspectives

Article Title: Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

doi: 10.1289/EHP9505

Figure Lengend Snippet: Evaluations of PQ 2 + or PQ + clearance in the brains of mice and in cultured astrocytes with or without DRP1 knockdown. (A,B) In vivo extracellular residue of the loading PQ 2 + ( PQ 2 + -LT ) was detected by microdialysis followed by HPLC in WT (A) and O c t 3 − / − (B) mice treated with either empty AAV control, neuronal DRP knockdown by AAV, astrocyte DRP1 knockdown by AAV, or non-selective DRP1 knockdown using mdivi-1. n = 5 mice per group. One-way ANOVA was followed by the Bonferroni multiple comparison test. Data are shown as mean ± SD . (C,D) PQ 2 + or PQ + uptake assays of GL261 astrocytes without or with SDT. PQ 2 + / PQ + concentration in the extracellular culture medium ranged from 0 to 800 μ M ( x -axis), and the uptake level of PQ 2 + / PQ + into astrocytes were detected in the cell lysate ( y -axis). Drp-1 siRNA was used to knockdown DRP1 in GL261 astrocytes. The average area under the curves were calculated and used for statistical analysis. n = 6 independent experiments per group. One-way ANOVA was followed by the Bonferroni multiple comparison test. Data are shown as mean ± SEM . **, p < 0.01 ; ***, p < 0.001 . The numeric data are shown in Excel Table S5. Note: AAV, adeno-associated virus; ANOVA, analysis of variance; Astro-DRP1 KD, DRP1 knockdown in astrocytes by AAV with gfaABC1D promoter; DRP1, dynamic related protein-1; HPLC, high-performance liquid chromatography; LT, loading treatment; mdivi-1, mitochondrial division inhibitor-1; Neuro-DRP1 KD, DRP1 knockdown in neuron by AAV with hsyn promoter; oct3, organic cation transporter-3; PQ, paraquat; Scramble, empty AAV for control; SD, standard deviation; SDT, sodium dithionite; SEM, standard error of the mean; siRNA, small interfering RNA; WT, wild-type.

Article Snippet: The HEK293 (Cat. No. h242), GL261 (Cat. No. m063), and Neuro-2a ( N 2 a ; Cat. No. m040) cell lines were purchased from iCell Co., Ltd.

Techniques: Cell Culture, Knockdown, In Vivo, Residue, Control, Comparison, Concentration Assay, Virus, High Performance Liquid Chromatography, Standard Deviation, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

doi: 10.1289/EHP9505

Figure Lengend Snippet: The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: 4F2hc, 4F2 heavy chain of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.

Article Snippet: The HEK293 (Cat. No. h242), GL261 (Cat. No. m063), and Neuro-2a ( N 2 a ; Cat. No. m040) cell lines were purchased from iCell Co., Ltd.

Techniques: Cell Culture, Knockdown, Control, Western Blot, Inhibition, Expressing, Comparison, Saline, Small Interfering RNA

Journal: Environmental Health Perspectives

Article Title: Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

doi: 10.1289/EHP9505

Figure Lengend Snippet: Transport of PQ 2 + or PQ + into the GL261 astrocyte cell line with and without specific inhibitors of ASCT2 and OCT3, with and without siRNA targeting Drp-1. PQ + / PQ 2 + (with or without SDT) transportation into GL261 astrocytes was evaluated using in vitro uptake assay. Specific inhibitors (Benser for ASCT2; D22 for OCT3) were used to evaluate the role of ASCT2 and OCT3, respectively. (A,B) The intake capacity of PQ 2 + by GL261 astrocytes without (A) or with (B) Drp1 siRNA. The average area under the curve was used for statistical analysis. n = 6 independent experiments. (C,D) The intake capacity of PQ + ( PQ 2 + pretreated with SDT) by GL261 astrocytes without (C) or with (D) Drp1 siRNA. Area under the curve was analyzed. n = 6 independent experiments. PQ 2 + / PQ + concentrations in extracellular culture medium ranged from 0 to 800 μ M ( x -axis). The intake PQ 2 + / PQ + was detected in the cell lysate ( y -axis). Data are shown as the mean ± SEM . One-way ANOVA was followed by the Bonferroni multiple comparison test. *, p < 0.01 ; ***, p < 0.001 . The numeric data are shown in Excel Table S8. Note: ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; Benser, benzylserine; D22, Decynium 22; DRP1, dynamic related protein-1; OCT3, organic cation transporter-3; PBS, phosphate-buffered saline; PQ, paraquat; SDT, sodium dithionite; SEM, standard error of the mean; siRNA, small interfering RNA.

Article Snippet: The HEK293 (Cat. No. h242), GL261 (Cat. No. m063), and Neuro-2a ( N 2 a ; Cat. No. m040) cell lines were purchased from iCell Co., Ltd.

Techniques: In Vitro, Comparison, Saline, Small Interfering RNA

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Barrier permeation enhancement effect of EVs and efficient delivery of aPD-L1/EV into brain. ( A ) Scheme of the in vitro barrier permeation test with C2BBe1 trans-well system (created from Biorender.com ). ( B ) Amounts of permeated aPD-L1 with free aPD-L1 or aPD-L1/EV administration through the C2BBe1 barrier. ( C ) Comparison of drug delivery efficiency with RO or IN administration of FITC-conjugated IgG or IgG/EV. The upper column displays the fluorescence of IgG within brain sections, and the lower column shows the cumulative fluorescence intensity of brain sections. ( D ) Distribution of aPD-L1 in GL261 tumor-bearing mice. Green fluorescence indicates aPD-L1, and nuclei were stained with DAPI. Statistics in ( B, C ) were conducted by one-way ANOVA with Bonferroni post hoc test. Asterisks represent a significant difference between indicated groups, with significance defined as p < 0.05. Data were presented as mean ± SD with n = 3.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: In Vitro, Comparison, Fluorescence, Staining

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Therapeutic effect of ICI/EVs in managing orthotopic GL261 tumors. ( A ) Schematic of experimental procedures for GL261 tumor management (created from Biorender.com ). ( B, C ) Tumor volumes were evaluated with IVIS, and representative images are shown in ( B ). Changes in tumor volume were calculated based on the total flux observed on day 0 ( C ). ( D ) The survival of orthotopic GL261 tumor-bearing mice post-treatment was analyzed. The aPD-L1/EV + aCTLA-4/EV group demonstrated an extended survival time compared to other treatment groups. ( E ) Ki67 staining of GL261 tumors. Scale bar: 50 μm. Statistics for ( C ) were obtained from GLM with Bonferroni post hoc test, and for ( D ) from the Kaplan-Meier survival analysis with log rank test. The asterisk in ( C ) indicates a significant difference from the PBS and aPD-L1 treatments. In ( D ), the asterisk and hash signs represent significant difference from all treatment groups and from the PBS group, respectively. Significance was defined as p < 0.05. Data are presented as mean ± SEM with n = 4 in ( C ) and in ( D ) with n = 8∼18.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: Staining

Journal: Materials Today Bio

Article Title: Delivery of extracellular vesicles loaded with immune checkpoint inhibitors for immunotherapeutic management of glioma

doi: 10.1016/j.mtbio.2024.101244

Figure Lengend Snippet: Immune cells in GL261 tumors post treatment. ( A ) IHC images of cytotoxic T cells within the tumor tissue. Green fluorescence indicates CD8 expression on T cells, while nuclei are counterstained with DAPI. Scale bar: 50 μm. ( B ) IHC images of M1 macrophages within tumor tissue. Green fluorescence indicates F4/80-stained macrophages, and red fluorescence indicates iNOS expression. Nuclei are counterstained with DAPI. Scale bar: 20 μm. ( C-E ) Flow cytometry analysis results for total T cells ( C ), cytotoxic T cells ( D ), and M1 macrophages ( E ). Statistics for ( C, D, E ) were obtained from one-way ANOVA with Bonferroni post hoc test, with significance defined as p < 0.05. Data are expressed as mean ± SEM with n = 5.

Article Snippet: The GL261-luc murine glioblastoma cell line was purchased from Creative Biolabs (Shirley, NY, USA).

Techniques: Fluorescence, Expressing, Staining, Flow Cytometry