Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Active Fraction Combination from Liuwei Dihuang Decoction (LW-AFC) Alleviated the LPS-Induced Long-Term Potentiation Impairment and Glial Cells Activation in Hippocampus of Mice by Modulating Immune Responses

doi: 10.1155/2019/3040972

Figure Lengend Snippet: Effect of LW-AFC on cytokine secretion in the (a) hippocampus and (b) plasma of LPS-treated mice. ∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05 vs. con and Student's t -test; ### p < 0.001, ## p < 0.01, and # p < 0.05 vs . mod, one-way ANOVA, and Dunnett's test, mean ± SD, n = 5–8. A, IL-1 β ; B, IL-6; C, MCP-1; D, G-CSF; E, IL-10; F, TNF- α ; G, IL-6; H, IFN γ ; I, IL-12; J, IL-10. con, control; mod, model; INDO, indomethacin.

Article Snippet: The secretion of TNF- α in the cell supernatant was determined using an AlphaLISA mouse TNF- α Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer's protocols using an EnVisionTM 2104 instrument (PerkinElmer, Waltham, MA, USA).

Techniques: Clinical Proteomics, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Active Fraction Combination from Liuwei Dihuang Decoction (LW-AFC) Alleviated the LPS-Induced Long-Term Potentiation Impairment and Glial Cells Activation in Hippocampus of Mice by Modulating Immune Responses

doi: 10.1155/2019/3040972

Figure Lengend Snippet: Effect of compounds from LW-AFC on cell viability and secretion of TNF- α in LPS-stimulated and non-LPS-stimulated BV-2 cells: (a) the cell viability of non-LPS-stimulated BV-2 cells; (b) the cell viability of LPS-stimulated BV-2 cells; (c) secretion of TNF- α in non-LPS-stimulated BV-2 cells; (d) secretion of TNF- α in LPS-stimulated BV-2 cells. ∗∗∗ p < 0.001 and ∗∗ p < 0.01 vs . con and Student's t -test; ### p < 0.001, ## p < 0.01, and # p < 0.05, vs . mod, one-way ANOVA, and Dunnett's test. Mean ± SD n = 3. con, control; mod, model; INDO, indomethacin; DEX, dexamethasone.

Article Snippet: The secretion of TNF- α in the cell supernatant was determined using an AlphaLISA mouse TNF- α Kit (PerkinElmer, Waltham, MA, USA) according to the manufacturer's protocols using an EnVisionTM 2104 instrument (PerkinElmer, Waltham, MA, USA).

Techniques: Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583 enhances cytokine secretion of activated anti-CD133 CAR T cells. Anti-CD133 CAR T cells were co-cultured with U251-CD133OE cells, the effector-to-target cell (E:T) ratio was 4:1. Vehicle and eight small molecules targeting adenosine receptors were added at different final concentrations. After 16 h, the medium was collected and secretion of INF-γ (A) and GM-CSF (B) was detected using AlphaLISA kits. Data were normalized to vehicle control and are represented as the mean ± SD of triplicates from a representative experiment of n = 3 experiments.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Cell Culture, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583-mediated CAR T cell enhancement requires T cell activation. Anti-CD133 CAR T cells (1 × 10 6 ) were activated by co-incubation with 2.5 × 10 5 U251-CD133OE cells (A) or CD3/CD28 beads (B) ; unstimulated CAR T cells (C) were used as control. Vehicle and BAY 60-6583 were added at different final concentrations. After 16 h, the medium was collected and the secretion of GM-CSF, INF-γ, and TNF-α was detected using AlphaLISA kits. Data were normalized to vehicle control and are represented as the mean ± SD of triplicates from a representative experiment of n = 4 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1-way ANOVA; ns, not significant.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Activation Assay, Incubation, Control

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: BAY 60-6583 specifically improved activities of CD133-CAR T cells. (A–D) Anti-CD133 CAR T cells were co-cultured with U251-CD133OE luc cells or U251 WT luc cells at different E:T ratios. Co-cultures were performed in the presence of vehicle or BAY 60-6583. After 16 h, the medium was collected, and the secretion of INF-γ (A) , TNF-α (B) , and GM-CSF (C) was detected using AlphaLISA kits (results for the E:T ratio of 4:1 are shown). (D) At 48 h after co-culture, cytotoxicity was determined by detecting the bioluminescence signal. Data are represented as the mean ± SD of triplicates from a representative experiment of n = 5 experiments. **** p < 0.0001 by 2-way ANOVA; ns, not significant (E) CFSE-labeled CAR T cells were co-cultured with irradiated U251-CD133OE cells in the presence of BAY 60-6583 or vehicle control. After 120 h, flow cytometry was used to analyze cell proliferation. Results shown are from a representative experiment of n = 4 experiments.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Cell Culture, Co-Culture Assay, Labeling, Irradiation, Control, Flow Cytometry

Journal: Frontiers in Pharmacology

Article Title: BAY 60-6583 Enhances the Antitumor Function of Chimeric Antigen Receptor-Modified T Cells Independent of the Adenosine A2b Receptor

doi: 10.3389/fphar.2021.619800

Figure Lengend Snippet: The photo-affinity probe enhanced CAR T cell effects similarly to unmodified BAY 60-6583 (A) The structure of the photo-affinity probe (B–D) Anti-HER2 CAR T cells (1 × 10 5 ) were activated by TransAct (B) or 4 × 10 5 MDA-MB-453 luc cells (C, D) . Probe, BAY 60-6583, or vehicle control was added at the beginning. After 16 h, the medium was collected, and the secretion of INF-γ, TNF-α, and GM-CSF was detected using AlphaLISA kits (B, C) ; 48 h later, cytotoxicity was assessed (D) . Data are represented as the mean ± SD from a representative experiment of n = 3 experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 by 1-way ANOVA; ns, not significant.

Article Snippet: After 16 h, the supernatants of the cultures were collected, and cytokines (TNF-α, IFN-γ, and GM-CSF) were detected using AlphaLISA detection kits (TNF-α, AL208C; IFN-γ, AL217C; and GM-CSF, AL216C) from PerkinElmer, Waltham, MA, United States.

Techniques: Control

Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a Left, immunostaining of PD-L1 (green) and TOM20-labeled mitochondria (red) in BT549 human TNBC cells with or without ATAD3A-knockdown (shATAD3A#1 and shATAD3A#2). Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, t -test). b Immunoblot of PD-L1 in the cytoplasm and mitochondria of control and ATAD3A-knockdown BT549 cells. TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls, respectively. Cyto, cytoplasm; mito, mitochondria. c Flow cytometry (left) and quantification (right) of surface PD-L1 in control and ATAD3A-knockdown BT549 cells ( n = 3, one-way ANOVA). d Venn diagram depicting overlapped genes for the interaction protein of ATAD3A set (BioGRID, RP5-832C2.1), the protein localization to mitochondrion set (GOBP 0070585) and the intrinsic component of mitochondrial membrane (GOCC 0098573). e Immunoblot of PINK1 in control and ATAD3A-knockdown BT549 cells. f Immunoblot of PD-L1 and PINK1 in HEK293T cells overexpressing PD-L1 (OE-PD-L1) and control cells (OE-Control), assessed after immunoprecipitation with immunoglobulin G (IgG) or antibody to PINK1. g Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and PINK1 in vitro. Purified Flag-labeled full-length PINK1 was incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. h Schematic diagram of Flag-labeled full-length (FL) and truncated mutants with indicated domains (amino acids 1–155, amino acids 156–320, amino acids 321–509, amino acids 510–581) of PINK1. MTS, mitochondrial targeting sequence; N-lobe, kinase domain N; C-lobe, kinase domain C; CTD, C-terminal domain. i Protein direct interaction analysis of the intracellular domain of PD-L1 (ICD) and truncated PINK1 mutants in vitro. Purified Flag-labeled full-length and truncated PINK1 were incubated with Biotin-labeled PD-L1 ICD domain, followed by streptavidin pull-down and immunoblot. The estimated size of PINK1-4 (amino acids 510–581) which did not express in HEK293T cells was labeled with asterisk. j Immunoblot of PD-L1 in the cytoplasm and mitochondria of MDA-MB-231 cells with or without PINK1-knockdown (shPINK1#1 and shPINK1#2). TOM20 and Tubulin were used as mitochondria and cytoplasm protein controls. Cyto, cytoplasm; mito, mitochondria. k Left, co-localization of PD-L1 (green) and TOM20 (red) in control, ATAD3A knockdown, PINK1 knockdown or ATAD3A and PINK1 double knockdown BT549 cells. Scale bars, 20 μm and 2 μm (inset). Right, the percentage of PD-L1 co-localized with TOM20 ( n = 5 fields, one-way ANOVA). l Immunoblot of PD-L1 in the cytoplasm and mitochondria of control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. m Immunoblot of indicated proteins in PD-L1-transfected HEK293T cells with or without PINK1 overexpression. n Immunoblot of PD-L1 in control and PINK1-knockdown (shPINK1#1 and shPINK1#2) BT549 cells. o Immunoblot of PD-L1 in BT549 cells transfected with control shRNA or shATAD3A (shATAD3A#1 and shATAD3A#2). p Immunoblot of total PD-L1 in control, ATAD3A-knockdown, PINK1-knockdown or ATAD3A and PINK1 double knockdown BT549 cells. q Immunoblot of PD-L1 in control and ATAD3A-knockdown BT549 cells treated with 20 μM CHX for indicated times. h, hours. r Quantification of PD-L1 intensity in immunoblot in control and ATAD3A-knockdown BT549 cells. s Immunoblot of PD-L1 in control and ATAD3A-knockdown MDA-MB-231 cells incubated with 20 nM BafA1 for indicated times. h, hours. Data are representative of at least two independent experiments and are shown as means ± SD. See also Supplementary information, Figs. and .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Immunostaining, Labeling, Knockdown, Western Blot, Control, Flow Cytometry, Membrane, Immunoprecipitation, In Vitro, Purification, Incubation, Sequencing, Transfection, Over Expression, shRNA

Journal: Cell Research

Article Title: Targeting ATAD3A-PINK1-mitophagy axis overcomes chemoimmunotherapy resistance by redirecting PD-L1 to mitochondria

doi: 10.1038/s41422-022-00766-z

Figure Lengend Snippet: a – h BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA for Atad3a (shAtad3a#1 and shAtad3a#2). a , b The endpoint tumor images ( a ) and volume ( b ) of tumors formed by control and Atad3a-knockdown cells in BALB/c mice ( n = 6, one-way ANOVA). c Left, IHC staining of Atad3a and PD-L1 on serial sections of tumors formed by control and Atad3a-knockdown cells. Scale bars, 50 μm. Right, IHC score of Atad3a in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). d IHC score of PD-L1 in control and Atad3a-knockdown tumors ( n = 6 fields, t -test). e Quantification of the percentage of tumor-infiltrating CD8 + T cells in tumors formed by control and Atad3a-knockdown cells by flow cytometry ( n = 5, t -test). f Quantification of the percentages of tumor-infiltrating IFNγ + CD8 + T cells and IFNγ + CD4 + T cells by flow cytometry ( n = 5, t -test). g Ratio of CD8 + cytotoxic T lymphocytes to CD4 + CD25 + Foxp3 + T reg cells ( n = 5, t -test). h Quantification of the percentages of PD-1 + TIM-3 + CD8 + T cells (left) and PD-1 + TIM-3 + CD4 + T cells (right) by flow cytometry ( n = 5, t -test). i – m BALB/c mice were inoculated orthotopically with 5 × 10 4 4T1 cells transfected with control shRNA (shControl) or shRNA specific for Atad3a (shAtad3a), Pink1 (shPink1) or both (shAtad3a + shPink1). i , Left, the endpoint images of tumors formed by control, Atad3a-knockdown, Pink1-knockdown or Atad3a and Pink1 double knockdown 4T1 cells in BALB/c mice. Right, immunoblot of Atad3a and Pink1 in these 4T1 cells. j The volume of tumors mentioned above ( n = 6, one-way ANOVA). k Quantification of the percentage of tumor-infiltrating CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). l Quantification of the percentage of tumor-infiltrating IFNγ + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). m Quantification of the percentage of PD-1 + TIM-3 + CD8 + T cells by flow cytometry ( n = 5, one-way ANOVA). n – s 4T1 tumors formed by control and Atad3a-knockdown cells were established orthotopically in BALB/c mice and received vehicle, anti-PD-L1 antibody (PD-L1 mAb), paclitaxel (PTX) or combined anti-PD-L1 antibody with paclitaxel treatment (PD-L1 mAb + PTX). IgG2b and saline were used as controls. n Experimental protocol. o , p The endpoint tumor images ( o ) and the volume ( p ) of tumors ( n = 6, one-way ANOVA). q – s Quantification of the percentages of tumor-infiltrating CD8 + T cells ( q ), IFNγ + CD8 + T cells ( r ) and PD-1 + TIM-3 + CD8 + T cells ( s ) in 4T1 tumors formed by control and Atad3a-knockdown cells received treatments as described above, determined by flow cytometry ( n = 5, one-way ANOVA). t Schematic model. Patients with PD-L1-positive TNBC could be divided into two groups based on ATAD3A expression. Patients with ATAD3A-high tumors might respond more poorly to ICIs plus paclitaxel therapy, and inhibition of ATAD3A is required to improve clinical outcome. Patients with ATAD3A-low tumors might benefit significantly from ICIs plus paclitaxel combination therapy. See also Supplementary information, Figs. – .

Article Snippet: Mouse IgG2b (APC, clone MPC-11, BioLegend) and rat IgG2b (APC, clone RTK4530, BioLegend) were used as controls respectively.

Techniques: Transfection, Control, shRNA, Knockdown, Immunohistochemistry, Flow Cytometry, Western Blot, Saline, Expressing, Inhibition