Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: The levels of TXNIP are elevated in GFAP-positive reactive astrocytes of cortex from patients with AD. (A) Volcano plots from the cerebral cortex region of brain tissues from patients with AD (AD) (n = 3) or non-AD (normal) (n = 3) in nanostring nCounter gene expression analysis. Volcano plots showing log2 fold change (x-axis) and -log10 p -values (y-axis) comparing patients with AD (AD) and non-AD (normal). Red dot represents TXNIP gene. Blue dots represent differentially expressed genes with p- value <0.05. Gray dots represent differentially expressed genes with p -value higher than 0.05. (B) The mRNA levels of TXNIP (log2 counts) in patients with AD (AD) and non-AD (normal). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test. (C) Representative immunofluorescence images of TXNIP protein expression in GFAP-positive astrocytes of the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group) showing TXNIP (green) and GFAP (red) around molecular layer (ML). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate TXNIP and GFAP-double positive cells. Symbols, which are expressed by white dotted line, indicate distinct area among OS, ML, and EGL. (D) Quantification of intensity for TXNIP-positive staining in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 10 images per individual subject). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test and analysis of variance (ANOVA). (E) Quantification of TXNIP and GFAP-double positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 10 images per individual subject). Data are presented as mean ± SD. ***, p < 0.001 by Student’s two-tailed t -test and analysis of variance (ANOVA). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Expressing, Two Tailed Test, Immunofluorescence, Staining

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: The levels of TXNIP are elevated in GFAP-positive astrocytes of cortex from APP/PS1 mice. (A) Representative immunofluorescence images of TXNIP protein expression in GFAP- positive astrocytes of cerebral cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group) showing TXNIP (green) and GFAP (red). DAPI-stained nuclei are shown in blue. Scale bar, 20 μm. White arrows indicate TXNIP and GFAP-double positive cells. OS, Outer surface. (B) Quantification of intensity for TXNIP-positive staining in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (total 10 images from n = 5 per group). Data are presented as mean ± SD. *, p < 0.05 by Student’s two-tailed t -test. (C) Quantification of TXNIP and GFAP-double positive astrocytes from immunofluorescence images in the cerebral cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (total 10 images from n = 5 per group). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Immunofluorescence, Expressing, Staining, Two Tailed Test

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: TXNIP increases pro-inflammatory phenotype in human astrocytes. (A) Volcano plots from control (Control) (n = 3) and TXNIP overexpressing (TXNIP) (n = 3) human astrocytes in nanostring nCounter gene expression analysis. Volcano plots showing log2 fold change (x-axis) and -log10 p -values (y-axis) comparing control (Control) and TXNIP overexpressing (TXNIP) human astrocytes in nanostring nCounter gene expression analysis. Red dots represent TXNIP, SERPING1, PSMB8, HLA-A, HLA-E, CXCL10, IL-1α, IL-1β, IL-6, CCL5, CCL20, CXCL2 and CXCL8 gene. Blue dots represent differentially expressed genes with p- value <0.05. Gray dots represent differentially expressed genes with p -value higher than 0.05. (B) The mRNA levels of TXNIP, SERPING1, PSMB8, HLA-A, HLA-E, CXCL10, IL-1α, IL-1β, IL-6, CCL5, CCL20, CXCL2 and CXCL8 (log2 counts) in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Data are presented as mean ± SD. **, p < 0.01; ***, p < 0.001 by Student’s two-tailed t -test. (C) Representative images of multiple cytokines, chemokine, growth factors, and other soluble proteins in supernatants from control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. The location of targets was indicated by the number of x-axis and the capital character of y-axis. The name of targets was indicated by a to t in right. (D) Quantification of pixel density for up-regulated targets (CD147, IL-6, CXCL10, CXCL11, LCN2, MIF, and CCL5) or down-regulated targets (Angiogenin, Dkk-1, GDF-15, CXCL1, IGFBP-2, IFGBP-3, CCL2, OPN, PDGF-AA, PTX3, TSP-1, uPAR, and VEGF) from D. Data are presented as mean ± SD. **, p < 0.01; *, p < 0.05 by Student’s two-tailed t -test. (E) Representative immunoblot analysis for TXNIP and phosphorylated NF-kB in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Quantification for protein levels of TXNIP and phosphorylated NF-kB in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. For immunoblots, β-actin and NF-kB were used as a loading control. Data are presented as mean ± SD. *, p < 0.05 using Student’s two-tailed t -test. Data are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Control, Expressing, Two Tailed Test, Western Blot

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: TXNIP increases mtROS production and decreases cellular antioxidant process in human astrocytes. (A) Quantification of mtROS levels using MitoSOX staining in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Data are presented as mean ± SD. *, p < 0.05 using two-tailed Student’s t-test. (B–D) Quantification of (B) reduced GSH levels, (C) ratio of GSH 2 /GSSG, and (D) GSSG levels in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test. (E) Representative immunoblot analysis for nuclear NRF2 in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. For immunoblots, histone H3 was used as a loading control. Data are presented as mean ± SD. *, p < 0.05 using two-tailed Student’s t-test. (F–I) Quantification of (F) HO-1 protein levels, (G) HO-1 activity, (H) GCLC protein levels, and (I) GCL activity in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (n = 3 replicates per group). Data are presented as mean ± SD. *, p < 0.05 by Student’s two-tailed t -test. Data are representative of three independent experiments.

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Staining, Control, Two Tailed Test, Western Blot, Activity Assay

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: TXNIP induces mitochondrial oxidative stress by reducing mitochondrial respiration and ATP production in human astrocytes. (A–B) Levels of oxygen consumption rate (OCR) (A) and quantification of OCR levels (B) in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (n = 3 replicates per group). Data are presented as mean ± SEM. **, p < 0.01 using two-tailed Student’s t-test. (C) Quantification of mitochondrial ATP production rate in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (n = 3 replicates per group). Data are presented as mean ± SD. **, p < 0.01 using a two-tailed Student’s t-test. (D) Representative immunoblot analysis for five mitochondrial ETC protein levels (left) including NDUFB8 for Complex I (C I (NDUFB8)), SDHB for Complex II (C II (SDHB)), UQCRC2 for Complex III (C III (UQCRC2)), MTCO1 for Complex IV (C IV (MTCO1)), and ATP5F1A for Complex V (C V (ATP5F1A)) in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Quantification for protein levels of C I (NDUFB8), C II (SDHB), C III (UQCRC2), C IV (MTCO1), and C V (ATP5F1A) (right) in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. For immunoblots, Tomm20 was used as a loading control for mitochondrial protein. Data are presented as mean ± SD. *, p < 0.05 using two-tailed Student’s t-test. Data are representative of three independent experiments.

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Control, Two Tailed Test, Western Blot

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: High levels of TXNIP are correlated with caspase-3 activation in GFAP-positive astrocytes of cortex from patients with AD and APP/PS1 mice. (A) Representative immunofluorescence images of TXNIP and cleaved-caspase-3 protein expression in the cerebral cortex tissues from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group) showing TXNIP (green) and cleaved-caspase-3 (purple) in GFAP (red)-positive astrocytes around molecular layer (ML). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer. Scale bars, 10 μm. White arrows indicate TXNIP and cleaved-caspase-3-double positive cells in GFAP-positive astrocytes. Symbols, which are expressed by white dotted line, indicate distinct area among OS and ML. (B) Quantification of intensity for cleaved-caspase-3-positive staining in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD) or non-AD (normal) (n = 10 images per individual subject). Data are presented as mean ± SD. *, p < 0.05 by Student’s two-tailed t -test and analysis of variance (ANOVA). (C) Quantification of TXNIP and cleaved-caspase-3-double positive cells in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD) or non-AD (normal) (n = 10 images per individual subject). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test and analysis of variance (ANOVA). (D) Representative immunofluorescence images of TXNIP and cleaved-caspase-3 protein expression in the cerebral cortex region of brain from APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing TXNIP (green) and cleaved-caspase-3 (purple) in GFAP (red)-positive astrocytes (n = 5 per group). DAPI-stained nuclei are shown in blue. Scale bar, 10 μm. White arrows indicate TXNIP and cleaved-caspase-3-double positive cells in GFAP-positive astrocytes. OS, Outer surface. Symbols, which are expressed by white dotted line indicates OS. (E) Quantification of intensity for cleaved-caspase-3-positive staining in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (total n = 10 images from n = 5 per group). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test. (F) Quantification of TXNIP and cleaved-caspase-3-double positive cells in GFAP-positive astrocytes from immunofluorescence images in the cerebral cortex region of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (total n = 10 images from n = 5 per group). Data are presented as mean ± SD. **, p < 0.01 by Student’s two-tailed t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Immunofluorescence, Expressing, Staining, Two Tailed Test

Journal: Redox Biology

Article Title: TXNIP contributes to induction of pro-inflammatory phenotype and caspase-3 activation in astrocytes during Alzheimer’s diseases

doi: 10.1016/j.redox.2023.102735

Figure Lengend Snippet: TXNIP induces caspase-3 activation and mitochondria-dependent apoptosis in human astrocytes. (A) Representative immunofluorescence images of mitochondrial morphology for mitochondria fragmentation by outer mitochondrial membrane protein Tomm20 staining in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes showing Tomm20 (green). DAPI-stained nuclei are shown in blue. Fragmentation of mitochondria is indicated (white arrows). Scale bar, 20 μm. Magnified views of selected regions (upper right): scale bar, 5 μm. (B) Quantification of cells with mitochondrial fragmentation from immunofluorescence images of mitochondrial morphology in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (The percent of morphological dead cells in a total of 100 cells from total n = 10 images per group from n = 3 replicates was calculated). Data are presented as mean ± SD. **, p < 0.01 using the two-tailed Student’s t-test. (C) Representative 2D (top) and 3D (bottom) images of control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (n = 10 images per group). Morphological features of cytotoxicity were indicated (white arrows). Scale bar, 20 μm. (D) Quantification of morphologically dead cells in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes (The percent of morphologically dead cells in total 100 cells from total n = 10 images per group from n = 3 replicates was calculated). Data are presented as mean ± SD. **, p < 0.01 using two-tailed Student’s t-test. (E) Cytotoxicity in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes was determined based on LDH level. (F) Quantification of cytochrome c in cytosolic and mitochondrial fractions from control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Data are presented as mean ± SD. **, p < 0.01 using two-tailed Student’s t-test. (G) Quantification of caspase-9 activity in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. Data are presented as mean ± SD. **, p < 0.01 using a two-tailed Student’s t-test. (H) Quantification of caspase-3 activity in control (Control) and TXNIP overexpressing (TXNIP) human astrocytes. (n = 3 replicates per group). Data are presented as mean ± SD. **, p < 0.01 using the two-tailed Student’s t-test. Data are representative of three independent experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For overexpression of human TXNIP, human astrocytes were seeded and transduced with pCMV6-AC-GFP construct of human TXNIP (NM_006472) as TXNIP over-expressed astrocytes (TXNIP over-expressed cells) (RG210804, Origene, Rockville, MD, USA) or pCMV6-AC-GFP vector (PS100010, Origene) as the control astrocytes (the control cells) using lipofectamine LTX with Plus reagent (15338100, Thermo Fisher Scientific) according to the manufacturer’s instructions.

Techniques: Activation Assay, Immunofluorescence, Membrane, Staining, Control, Two Tailed Test, Activity Assay

Journal: Cancer Research Communications

Article Title: Targeting NEDDylation is a Novel Strategy to Attenuate Cisplatin-induced Nephrotoxicity

doi: 10.1158/2767-9764.CRC-22-0340

Figure Lengend Snippet: Loss of TXNIP protects RPTECs from cisplatin-induced ROS and toxicity. A, RPTECs were transfected with lentiviral vector control or TXNIP shRNA particles. Puromycin was used to select for successful transfection. Immunoblotting was used to determine knockdown efficiency. β-Actin was used as a loading control. B, shRNA TXNIP and vector control RPTECs were treated with the indicated concentrations of cisplatin for 48 hours. Cells were stained with CellROX Green and imaged. C, ImageJ was used to determine the average CellROX Green signal intensity. Mean ± SEM, n = 45 cells; *, P < 0.05. D, shRNA TXNIP and vector control RPTECs were subjected to 72-hour treatment with the indicated cisplatin concentrations. MTT assay was used to determine cell viability. Mean ± SD, n = 3; *, P < 0.05.

Article Snippet: RPTECs were infected with lentiviral particles containing nontargeted scramble (vector) or target-specific short hairpin RNA (shRNA) directed at TXNIP (sc-270490-V) or NFE2L2 (sc-37030-V) according to the manufacturer's protocol (Santa Cruz Biotechnology).

Techniques: Transfection, Plasmid Preparation, Control, shRNA, Western Blot, Knockdown, Staining, MTT Assay

Journal: NeuroSci

Article Title: Localization of Thioredoxin-Interacting Protein in Aging and Alzheimer’s Disease Brains

doi: 10.3390/neurosci3020013

Figure Lengend Snippet: The primary antibodies used in this study.

Article Snippet: TXNIP , TXNIP , Bethyl , Rabbit/Poly , A303-229A , WB , 1:10,000.

Techniques:

Journal: NeuroSci

Article Title: Localization of Thioredoxin-Interacting Protein in Aging and Alzheimer’s Disease Brains

doi: 10.3390/neurosci3020013

Figure Lengend Snippet: Comparison of TXNIP recognition patterns with antibodies from different sources in different brain-derived cells. Parallel immunoblot membranes with the same amounts of cellular proteins were probed with optimal dilution of four commercial TXNIP antibodies. The results show varying recognition patterns. Equivalent loading between cellular samples is demonstrated with reprobing blots for β-actin. The blot showing results of using AbCAM TXNIP antibody had previously been probed with TXNIP Bethyl antibody Abbreviations: MG, extracts from control (Cont) and Aβ treated microglial extracts from different cases (1,2,3); EC, extracts from primary human brain-derived endothelial cells; MEC, extracts from transformed brain endothelial cell line hCMEC/D3; Astrocyte, extract from brain derived primary astrocytes; iPS-Neu, extracts from Cellular Dynamics iCell human stem cell-derived human neurons; LAN5, extracts from differentiated human neuroblastoma cell line LAN-5 control (Cont) or high-glucose treated (Glu); Brain, extract of human brain samples from AD middle temporal gyrus (MTG).

Article Snippet: TXNIP , TXNIP , Bethyl , Rabbit/Poly , A303-229A , WB , 1:10,000.

Techniques: Comparison, Derivative Assay, Western Blot, Control, Transformation Assay

Journal: NeuroSci

Article Title: Localization of Thioredoxin-Interacting Protein in Aging and Alzheimer’s Disease Brains

doi: 10.3390/neurosci3020013

Figure Lengend Snippet: TXNIP and TRX levels in low-plaque (LP), high-plaque (HP), and Alzheimer’s disease (AD) cases. ( A ) Representatives immunoblot of LP, HP, and AD cases showing TXNIP and TRX bands simultaneously on the same membrane. Membranes were probed with rabbit anti-TXNIP (Bethyl, 1:10,000 dilution) and rabbit anti-TRX (Abcam, 1:5000 dilution). Membranes were subsequently reacted with HRP-labeled anti-β-actin antibody (1:15,000) for normalization of loading. ( B ) Relative levels of TXNIP in LP, HP, and AD middle temporal gyrus brain protein extracts. Relative intensities were normalized for β-actin levels. NS, non-significant differences between groups. ( C ) Relative levels of TRX detected in LP, HP, and AD middle temporal gyrus brain protein extracts. Relative intensities were normalized for β-actin levels. NS, non-significant difference between groups.

Article Snippet: TXNIP , TXNIP , Bethyl , Rabbit/Poly , A303-229A , WB , 1:10,000.

Techniques: Western Blot, Membrane, Labeling