Journal: Oncotarget

Article Title: Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

doi: 10.18632/oncotarget.9747

Figure Lengend Snippet: Overview of the patient population in terms of the investigated markers in the different evaluation methods. BP-NEN – bronchopulmonary neuroendocrine neoplasms; TC – typical carcinoids; AC – atypical carcinoids; SCLC – small cell lung cancer; LCNEC – large cell neuroendocrine lung carcinoma; FFPE – formalin-fixed, paraffin-embedded; TOP2A – Topoisomerase 2 alpha; RacGAP1 – Rac GTPase activating protein 1; IHC – immunohistochemistry.

Article Snippet: After dewaxing and microwaving of the sections in 10 mM citrate buffer (pH = 6.0) for 16 minutes at 600 W, the slices were incubated overnight at 4°C with the primary antibodies (Ki-67 (mouse monoclonal, 1:50, Dako Germany GmbH, Hamburg, GER), TOP2A (rabbit monoclonal, 1:100, Abcam, Cambridge, UK), RacGAP1 (rabbit polyclonal, 1:350, Atlas Antibodies, Stockholm, SWE)).

Techniques: Formalin-fixed Paraffin-Embedded, Immunohistochemistry

Journal: Oncotarget

Article Title: Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

doi: 10.18632/oncotarget.9747

Figure Lengend Snippet: The boxplots are depicting the TOP2A and RacGAP1 protein (left) and mRNA (right) levels in the different BP-NEN entities as evaluated by immunohistochemistry and RT-qPCR. To the right the respective Kaplan-Meier-Analyses of each marker in the IHC investigation are shown. Blue indicates low, green moderate and red high TOP2A/RacGAP1 expression levels, according to the cut-off limits of the ROC analysis between TC – AC and AC – SCLC. With both markers, high expression levels imply poor patient survival.

Article Snippet: After dewaxing and microwaving of the sections in 10 mM citrate buffer (pH = 6.0) for 16 minutes at 600 W, the slices were incubated overnight at 4°C with the primary antibodies (Ki-67 (mouse monoclonal, 1:50, Dako Germany GmbH, Hamburg, GER), TOP2A (rabbit monoclonal, 1:100, Abcam, Cambridge, UK), RacGAP1 (rabbit polyclonal, 1:350, Atlas Antibodies, Stockholm, SWE)).

Techniques: Immunohistochemistry, Quantitative RT-PCR, Marker, Expressing

Journal: Oncotarget

Article Title: Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

doi: 10.18632/oncotarget.9747

Figure Lengend Snippet: Spearman's rank correlations of Ki-67, TOP2A and RacGAP1 protein and mRNA levels with clinical data and extent of necrosis

Article Snippet: After dewaxing and microwaving of the sections in 10 mM citrate buffer (pH = 6.0) for 16 minutes at 600 W, the slices were incubated overnight at 4°C with the primary antibodies (Ki-67 (mouse monoclonal, 1:50, Dako Germany GmbH, Hamburg, GER), TOP2A (rabbit monoclonal, 1:100, Abcam, Cambridge, UK), RacGAP1 (rabbit polyclonal, 1:350, Atlas Antibodies, Stockholm, SWE)).

Techniques:

Journal: Oncotarget

Article Title: Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

doi: 10.18632/oncotarget.9747

Figure Lengend Snippet: Below two examples for a strong positivity of RacGAP1 protein expression at the invasive front are shown (magnification 10 × 10). The graph above depicts a Kaplan-Meier-Analysis with a high RacGAP1 protein expression (red line) and a weak/no RacGAP1 expression (blue line) at the invasive front. According to the Log-Rank Test the curves differ highly significantly: High RacGAP1 expression at the invasive front indicates poor patient prognosis.

Article Snippet: After dewaxing and microwaving of the sections in 10 mM citrate buffer (pH = 6.0) for 16 minutes at 600 W, the slices were incubated overnight at 4°C with the primary antibodies (Ki-67 (mouse monoclonal, 1:50, Dako Germany GmbH, Hamburg, GER), TOP2A (rabbit monoclonal, 1:100, Abcam, Cambridge, UK), RacGAP1 (rabbit polyclonal, 1:350, Atlas Antibodies, Stockholm, SWE)).

Techniques: Expressing

Journal: Oncotarget

Article Title: Comparative evaluation of three proliferation markers, Ki-67, TOP2A, and RacGAP1, in bronchopulmonary neuroendocrine neoplasms: Issues and prospects

doi: 10.18632/oncotarget.9747

Figure Lengend Snippet: Immunohistochemical staining of Ki-67, TOP2A and RacGAP1 at the same location of each one TC, AC, SCLC and LCNEC sample (magnification 40 × 10).

Article Snippet: After dewaxing and microwaving of the sections in 10 mM citrate buffer (pH = 6.0) for 16 minutes at 600 W, the slices were incubated overnight at 4°C with the primary antibodies (Ki-67 (mouse monoclonal, 1:50, Dako Germany GmbH, Hamburg, GER), TOP2A (rabbit monoclonal, 1:100, Abcam, Cambridge, UK), RacGAP1 (rabbit polyclonal, 1:350, Atlas Antibodies, Stockholm, SWE)).

Techniques: Immunohistochemical staining, Staining

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Gal‐9 expression in the brains of AD patients and APP/PS1 mice. (a) Representative images of Gal‐9 expression in the hippocampus of AD patients and age‐matched control subjects. (b) Quantification of the percentage of Gal‐9‐positive cells. Scale bars, 20 μm. Bars represent means ± SEM. Unpaired Student's t ‐test. n = 10 AD patients and 10 control subjects (one slide from each subject). *** p < 0.001. (c) Western blot analysis of Gal‐9 in the hippocampus of AD patients and age‐matched controls. n = 4 AD patients and 3 controls. Bars represent means ± SEM. Unpaired Student's t ‐test. * p < 0.05. (d) Gal‐9 concentrations in the CSF of AD patients ( n = 11) and healthy controls ( n = 16). Bars represent means ± SEM. Unpaired Student's t ‐test, ** p < 0.01. (e) Co‐immunostaining of Gal‐9 and Aβ in brain sections from APP/PS1 mice. Scale bars, 20 μm. (f) Western blot analysis of Gal‐9 levels in the brains of APP/PS1 and WT mice at different ages. Bars represent means ± SEM. Unpaired Student's t ‐test. ** p < 0.01, *** p < 0.001. (g) Co‐immunostainings of Gal‐9, Aβ plaques, and microglia in the brains of AD patients (upper panel) and APP/PS1 mice (lower panel). Scale bars, 50 μm. (h) RT‐PCR analysis of Gal‐9 mRNA levels in BV2 cells exposed to Aβ fibrils. (i) ELISA analysis of Gal‐9 levels in the supernatant of BV2 cells exposed to Aβ fibrils. Bars represent means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. ** p < 0.01, *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Expressing, Control, Western Blot, Immunostaining, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Gal‐9‐Aβ fibrils induce cognitive impairment and synaptic degeneration in APP/PS1 mice. Three‐month‐old male mice were injected with PBS, Gal‐9, Aβ, or Gal‐9‐Aβ. The mice were sacrificed 6 months after the operation. Spatial memory was assessed in the Morris water maze test (a–d) and Y maze test (e, f). (a) The latency to the platform in the Morris water maze test. (b) Integrated time traveled in the Morris water maze test. AUC, area under the curve. (c) The average swim speed of mice. (d) Time spent in the target quadrant in the probe trial. (e, f) Y‐maze test results. Shown are the time spent in the new arm (e) and the number of new arm entries (f). n = 7 mice in the PBS group, n = 6 mice in the Gal‐9 group, n = 6 mice in the Aβ group, n = 8 mice in the Gal‐9‐Aβ group. (g) Electron microscopy of synapses (top) and magnified images (below). Scale bar, 1 μm. (h) Width of synaptic clefts. (i) The number of synaptic clefts. (j) Postsynaptic density. (k) Length of the active zone. n = 14 slices from 4 different mice per group. (l) Golgi staining of dendritic spines of hippocampal slices. Scale bar, 15 μm. n = 10 slices from 4 different mice per group. (m, n) Representative immunoblots (m) and quantification (n) of synapsin I, synaptophysin, and PSD‐95 in the hippocampus of mice. n = 4 mice per group. Data are presented as means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Injection, Electron Microscopy, Staining, Western Blot

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Gal‐9‐Aβ fibrils accelerate Aβ deposition and induce neuroinflammation in APP/PS1 mice. Three‐month‐old male mice were injected with PBS, Gal‐9, Aβ, or Gal‐9‐Aβ. The mice were sacrificed 6 months after the operation. (a) Representative images of Aβ deposits in the hippocampus of APP/PS1 mice. Scale bar, 200 μm. (b, c) Quantification of the number (b) and area (c) of Aβ deposits. n = 12 slices from 4 mice per group. ELISA of Aβ concentrations in the hippocampus (d) and cortex (e) in APP/PS1 mice. n = 6 samples from 3 mice per group. (f, g) Representative immunoblots and quantification of APP, α‐ and β‐C‐terminal fragments (α‐CTF and β‐CTF), IBA‐1, and GFAP in the hippocampal lysates. n = 4 mice per group. Data are presented as means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (h, i) Co‐immunostaining and quantification of Aβ and the microglial markers IBA1 in the cortex of APP/PS1 mice. Scale bar, 20 μm. n = 10 slices from 4 mice per group. Data are presented as means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, * p < 0.05, *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Immunostaining

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Gal‐9 deficiency rescues cognitive decline in APP/PS1 mice. WT, Gal‐9 KO, APP/PS1 and APP/PS1;Gal‐9 KO mice were sacrificed at 3, 6, 9, or 12 months old. (a) Representative images of Aβ deposits in the mouse hippocampus. (b, c) Quantification of the area (b) and number (c) of Aβ deposits in the hippocampus of APP/PS1 and APP/PS1;Gal‐9 KO mice. n = 12 mice. (d, e) ELISA analysis of Aβ 42 concentrations in the cortex (d) and hippocampus (e) in APP/PS1 and APP/PS1;Gal‐9 KO mice. n = 6 mice per group. (f, g) Brain lysates were immunoblotted (f) and quantified (g) for APP, α‐ and β‐C‐terminal fragments (α‐CTF and β‐CTF), IBA‐1, and GFAP in the hippocampus of mice. n = 4 mice per group. Data are presented as means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. (h–k) Immunostaining and quantification of Aβ and the microglial markers IBA1 and in the cortex (h, j) and hippocampus (i, k) of 10‐month‐old APP/PS1 and APP/PS1;Gal‐9 KO mice in (a). Scale bar, 20 μm in the cortex; 100 μm in the hippocampus. n = 10 slices from 4 mice per group. Data are presented as means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Gal‐9 deficiency rescues memory deficits and synaptic dysfunction in APP/PS1 mice. The spatial memory of WT, Gal‐9 KO, APP/PS1 and APP/PS1;Gal‐9 KO mice was assessed in the Morris water maze test and Y maze test. (a) The latency traveled to the platform. (b) Integrated time traveled in the Morris water maze test. AUC, area under the curve. (c) Average swim speed of mice. (d) Probe trial results. (e, f) Time spent in the new arm (e) and number of new arm entries (f) in the Y‐maze test. n = 9 mice in the WT group, n = 11 mice in the Gal‐9 KO group, n = 8 mice in the APP/PS1 group, n = 12 mice in the APP/PS1; Gal‐9 KO group. (g) Long‐term potentiation (LTP) of fEPSPs was induced by 3 × TBS (4 pulses at 100 Hz, repeated three times with a 200 ms interval). The magnitude of LTP and synaptic transmission was assessed by input/output (I/O). (h) Golgi staining and quantification of dendritic spines in hippocampal slices. Scale bar, 15 μm. n = 10 slices per group. (i) Electron microscopy of synapses (top) and magnification (bottom). Scale bar, 1 μm. (j) The number of synaptic clefts, length of active zone, width of synaptic clefts, and postsynaptic density of synapses. n = 14 slices from 4 mice per group. (k, l) Hippocampal lysates were immunoblotted for synapsin I, synaptophysin, and PSD95. n = 4. Data are presented as the means ± SEM. One‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Transmission Assay, Staining, Electron Microscopy

Journal: Aging Cell

Article Title: Microglia‐derived Galectin‐9 drives amyloid‐β pathology in Alzheimer's disease

doi: 10.1111/acel.14396

Figure Lengend Snippet: Blockade of Gal‐9 halts the progression of Aβ pathology. (a–d) Three‐month‐old APP/PS1 mice received unilateral intra‐hippocampal injections of: (1) brain homogenates derived from APP/PS1 mice, (2) brain homogenates derived from APP/PS1;Gal‐9 KO mice, (3) APP/PS1 mouse brain homogenates pre‐treated with anti‐Gal‐9 antibody, or (4) APP/PS1 mouse brain homogenates pre‐treated with iso‐IgG. (a) Schematic for brain lysate and antibody injection experiments. (b) Immunohistochemistry using 6E10 antibody showing Aβ deposits in the hippocampus. (c) Total Aβ‐positive area and number of Aβ plaques. n = 12 samples from 4 mice per group. (d) ELISA analysis of Aβ 42 in the cortex and hippocampus. (e, f) APP/PS1 mice or APP/PS1; Gal‐9 KO mice received a unilateral injection of brain homogenates from APP/PS1 and WT mice, respectively. (e) Schematic for brain lysate injection experiments. (f) Representative immunohistochemistry images showing Aβ deposits in the hippocampus. (g) Quantification of the number and area of Aβ‐positive deposits in the hippocampus. (h) ELISA analysis of Aβ 42 in the cortex and hippocampus. Scale bar, 200 μm. n = 12 slices from 4 mice per group. Data are presented as means ± SEM. Two‐way ANOVA followed by Tukey's post hoc test. n.s, not significant, ** p < 0.01, *** p < 0.001.

Article Snippet: Human APP swe and PSEN1 dE9 transgenic mice (APP/PS1 mice) were obtained from the Jackson Laboratory (stock number 005864).

Techniques: Derivative Assay, Injection, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Allele frequencies of ATXN1 with different CAG repeat lengths in the probands of NIMH AD families and healthy individuals of CEPH families. (B) Western blot analysis of APP processing in the brains of 3 month-old WT (+/+), Ataxin-1 hetero-KO (+/−) and KO (−/−) mice. RIPA buffer-soluble lysates were used to detect Ataxin-1 (ATXN1), APP, APP-CTF, ADAM10, BACE1. TBS-soluble lysates for sAPPα and sAPPβ. Actin is a loading control. Arrow head, proADAM10; *, non-specific band. (C) Densitometric quantification of western blot results. WT, relative value of 100. Values are mean ± SEM. n = 5. *p < 0.05, **p < 0.01, t-test. (D) Immunohistochemical (left) and immunofluorescence (right) staining for BACE1. Ctx, cortex; Hip, hippocampus; Cbl, cerebellum; Str, striatum; BSt, brain stem. Bar = 500 μm. (E) Ataxin-1, BACE1, and APP levels in dissected brain regions. OB, olfactory bulb. (F) Densitometry of BACE1 levels. n = 3. n.s.: not significant. ***p < 0.001. (G) RT-qPCR analysis of Atxn1 and Bace1 mRNA expression in the whole brain. n = 4. **p < 0.01, ***p < 0.001 versus WT Atxn1 mRNA; ##p < 0.01 versus WT Bace1 mRNA. (H) Bace1 mRNA levels in dissected cortex and cerebellum. n = 4. (I) Representative real-time qPCR amplification graphs for cortical cDNA. RFU, relative fluorescence unit. See also Figure S1.

Article Snippet: Other antibodies used for western blotting were Ataxin-1 (76/8, NeuroMab), ADAM10 (422751, Millipore; ab124695, Abcam), mouse sAPPα (826801, Biolegend), sAPPβ (18957, IBL), sAPPβ-swe (10321, IBL), total sAPP (22C11, Millipore), BACE1 (D10E5, Cell Signaling; ab2077, Abcam), 6E10 (detects human sAPPα, 803015, Biolegend), CIC (A301-204A, Bethyl Laboratories), ETV4 (10684-1-AP, ProteinTech), ETV5 (13011-1-AP, ProteinTech), PS1 (D39D1, Cell Signaling), pan-actin (MS-1295-P0, Fisher Scientific) and GAPDH (MAB374, EMD Millipore).

Techniques: Western Blot, Control, Immunohistochemical staining, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing, Amplification, Fluorescence

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Actinomycin D or vehicle (DMSO) was treated for 48 hrs in acute brain slice cultures. Results are from two independent brain slice cultures (2 mice each for WT and Ataxin-1 KO). n (brain slices) = 3–4. *p < 0.05, **p < 0.01, ***p < 0.001, t-test. (B) Left: extracellular field potential records of multiple unit activity in CA1 areas of brain slices 48 hrs after incubation. Right: histograms representing spike amplitude distribution (bin size 10 mV) and inter-spike interval (bin size 2 ms). (C) Representative real-time qPCR amplification graphs of steady-state (total) and nascent Bace1 mRNA. Brain slices were incubated for 16 hrs in ACSF containing 5-ethynyl uridine. n (brain slices) = 4. (D) Locations of predicted CIC and PEA3 (ETV1/4/5) binding sites in Etv1/4/5 and Bace1 promoter regions, respectively. The CIC binding site in Etv1 promoter is relative distal (−1486) than those in Etv4/5. (E) CIC expression in medial cortex (left) and hippocampal CA1 (right). Bar = 50 μm. (F) mRNA levels of Etv1, Etv4, and Etv5 in the cortex and cerebellum. Gapdh was used as an internal control for RT-qPCR.All levels were presented as relative to Etv1-WT (100). Compared to cortex, the relative levels of Etv4 and Etv5 versus Etv1 mRNA are very low in cerebellum. n= 6 (cortex), 4 (cerebellum). (G) Western blot analysis for cortical levels of Ataxin-1, CIC, ETV4, ETV5, BACE1, sAPPβ, and sAPPα. (H) Cerebellar levels of ETV4 and ETV5. Vertical lines indicate that different parts within same blot were placed together for better comparison. (I) Densitometric quantification. n = 5, except ETV4 and ETV5 (n = 7). (J) Luciferase activity in HEK293T cells measured 24 hr after transfection with pGL3 plasmids harboring BACE1 promoter and vectors expressing either ETV4 or ETV5. Results from 3 independent experiments of sextuplicate transfection were normalized and combined. n = 18. See also Figure S2.

Article Snippet: Other antibodies used for western blotting were Ataxin-1 (76/8, NeuroMab), ADAM10 (422751, Millipore; ab124695, Abcam), mouse sAPPα (826801, Biolegend), sAPPβ (18957, IBL), sAPPβ-swe (10321, IBL), total sAPP (22C11, Millipore), BACE1 (D10E5, Cell Signaling; ab2077, Abcam), 6E10 (detects human sAPPα, 803015, Biolegend), CIC (A301-204A, Bethyl Laboratories), ETV4 (10684-1-AP, ProteinTech), ETV5 (13011-1-AP, ProteinTech), PS1 (D39D1, Cell Signaling), pan-actin (MS-1295-P0, Fisher Scientific) and GAPDH (MAB374, EMD Millipore).

Techniques: Slice Preparation, Activity Assay, Incubation, Amplification, Binding Assay, Expressing, Control, Quantitative RT-PCR, Western Blot, Comparison, Luciferase, Transfection

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: (A) Analysis of Ataxin-1, BACE1, ADAM10, sAPPα, sAPPβ, and sAPP levels in AD mouse brains. sAPPα and sAPPβ derived from either transgenic APPswe or endogenous APP were detected by different antibodies. (B) Densitometric quantification. Number of mice analyzed, n = 11–12. ***p < 0.001, t-test. (C) Ratio of transgenic sAPPα and sAPPβ. **p < 0.01. (D-E) Levels of APP-CTFα and - CTFβ in brains. Number in parenthesis denotes the number of mice analyzed. *p < 0.05. (F) TBS-insoluble Aβ40 and Aβ42 amounts in the brains of 5-month-old mice. The amounts of TBS-insoluble Aβ at this age were ~ 20 times higher than those of TBS-soluble Aβ. (G) Western blot analysis of detergent-soluble brain lysates with 6E10 antibody targeting N-terminal region of human Aβ. *, non-specific bands. (H) Densitometric quantification of Aβ levels. See also Figure S3.

Article Snippet: Other antibodies used for western blotting were Ataxin-1 (76/8, NeuroMab), ADAM10 (422751, Millipore; ab124695, Abcam), mouse sAPPα (826801, Biolegend), sAPPβ (18957, IBL), sAPPβ-swe (10321, IBL), total sAPP (22C11, Millipore), BACE1 (D10E5, Cell Signaling; ab2077, Abcam), 6E10 (detects human sAPPα, 803015, Biolegend), CIC (A301-204A, Bethyl Laboratories), ETV4 (10684-1-AP, ProteinTech), ETV5 (13011-1-AP, ProteinTech), PS1 (D39D1, Cell Signaling), pan-actin (MS-1295-P0, Fisher Scientific) and GAPDH (MAB374, EMD Millipore).

Techniques: Derivative Assay, Transgenic Assay, Western Blot

Journal: Cell

Article Title: Loss of Ataxin-1 Potentiates Alzheimer's Pathogenesis by Elevating Cerebral BACE1 Transcription

doi: 10.1016/j.cell.2019.07.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Other antibodies used for western blotting were Ataxin-1 (76/8, NeuroMab), ADAM10 (422751, Millipore; ab124695, Abcam), mouse sAPPα (826801, Biolegend), sAPPβ (18957, IBL), sAPPβ-swe (10321, IBL), total sAPP (22C11, Millipore), BACE1 (D10E5, Cell Signaling; ab2077, Abcam), 6E10 (detects human sAPPα, 803015, Biolegend), CIC (A301-204A, Bethyl Laboratories), ETV4 (10684-1-AP, ProteinTech), ETV5 (13011-1-AP, ProteinTech), PS1 (D39D1, Cell Signaling), pan-actin (MS-1295-P0, Fisher Scientific) and GAPDH (MAB374, EMD Millipore).

Techniques: Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Reporter Assay, Staining, Expressing, Knock-In, SYBR Green Assay, Cloning, Mutagenesis, Software, Microscopy, Real-time Polymerase Chain Reaction, Transfection

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Systematically profiling the expression of TRIP13 in glioma. ( A ) The expression profiles of TRIP13 mRNA in glioblastoma and normal brain tissues based on TCGA are represented. Quantification data shows the expression levels of TRIP13 in gliomas from the TCGA, GSE16011 datasets, and CGGA stratified by the IDH status ( B – D ) and WHO grade ( E – G ). ( H ) The Human Protein Atlas database shows that TRIP13 was colocalized with nucleoplasm or microtubules in the cytoplasm of A549 and U-2 OS cells. ( I , J ) Validation of TRIP13 mRNA expression and protein production in U87MG, LN229, GBM8401, U118MG, and LNZ308 glioma cell lines in comparison with normal brain tissues. ( K ) TRIP13 expression was detected in the different subcellular location of GBM in the IVY GBM dataset. * p < 0.05, *** p < 0.001, and **** p < 0.0001 compared to the normal brain tissue group. n.s: not significant.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing, Biomarker Discovery, Comparison

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: TRIP13 mRNA expression was related to clinical outcome in lower-grade glioma (LGG) and glioblastoma (GBM). ( A – C ) Kaplan–Meier estimates of overall survival for ( A ) all grade, ( B ) LGG and ( C ) GBM patients in the GSE16011 data. ( D – F ) Kaplan–Meier estimates of overall survival for ( D ) all grade, ( E ) LGG and ( F ) GBM patients in the CGGA. ( G – I ) Kaplan–Meier estimates of overall survival for ( G ) all grade, ( H ) LGG and ( I ) GBM patients in the TCGA. ( J – L ) Kaplan–Meier estimates of progression-free survival for ( J ) all grade, ( K ) LGG and ( L ) GBM patients in the TCGA. ( M – O ) TRIP13 expression individual in two distinct clinical status including disease free and recurred in ( M ) all grade, ( N ) LGG and ( O ) GBM patients (TCGA, Firehose Legacy). The p -values were calculated using the log-rank test. OS, overall survival. PFS, progression-free survival.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: TRIP13 is preferentially expressed in recurrent glioblastoma and stem-like cell lines. ( A ) The expression profiles of TRIP13 mRNA in patients with or without radiotherapy based on TCGA lower-grade glioma (LGG) and glioblastoma (GBM) are represented. ( B ) Comparison of TRIP13 mRNA expression levels in primary and recurrent GBM in the TCGA dataset. ( C ) Comparison of TRIP13 mRNA expression levels in primary tumors ( n = 12), GS neurospheres ( n = 17) and glioblastoma stem-like cell lines ( n = 27) in the GDS3885 dataset. Correlation analysis between TRIP13 and ( D ) MKI67, ( E ) PAF, and ( F ) PROM1 mRNA expression levels in primary tumors, neurospheres and glioblastoma stem-like cell lines in the GDS3885 dataset. r, Pearson correlation coefficient. The p -value indicates the significance of the correlation.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing, Comparison

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Functional enrichment analysis associated with TRIP13 expression level in human gliomas based on three datasets (TCGA, CGGA301 and GSE16011). ( A ) Heatmaps of clinicopathological parameters, TRIP13 co-expressed genes (R > 0.6) expression based on TRIP13 expression in glioma from TCGA, CGGA301 and GSE16011 array data. ( B ) Venn analysis of TRIP13 co-expressed genes. There were 121 overlapping genes positively associated (R > 0.6) with TRIP13 in TCGA, CGGA301 and GSE16011 array data. ( C ) GO and ( D ) KEGG pathway analysis of 121 overlapping genes in TCGA, CGGA301 and GSE16011 array data. CGGA, Chinese Glioma Genome Atlas; TCGA, The Cancer Genome Atlas; GO, Gene Ontology; Mut, mutant; WT, wild type.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Functional Assay, Expressing, Mutagenesis

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Identification of TRIP13 co-expressed genes functions and canonical pathway using IPA. ( A ) Ingenuity pathways analysis (IPA) Diseases and Functions Annotation. Bars with positive z-scores indicate that functional activity is increased (red color), whereas negative z-scores represent decreased activity (blue color). (|z-score| > 1.4, p -value < 0.0001). ( B ) Mitotic roles of polo-like kinases. TRIP13 co-expressed genes (in red) are highly expressed in all mitotic pathways involving polo-like kinases (Plks). ( C ) IPA shows canonical pathways most significantly ( p < 0.05) associated with TRIP13. The p -value for each pathway is representative of a blue bar and reported as the negative log of the p -value.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Functional Assay, Activity Assay

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Network of interactions between the TRIP13 co-expressed genes and potential upstream regulators; confirmed relationships between TRIP13 and these regulators in TCGA-GBM database by correlation analysis. ( A ) The network was generated by the Path Designer tool of IPA. TP53, FOXM1, TCF1, E2F1 and E2F3 were the top regulators (different colors distinguish between them, with regulator–target gene connections by arrows/lines in the same colors), with high z-scores for activation. The circles represent proteins; squares indicate mRNA or genes. All mRNA for genes are displayed as expression values (low to high expression, light to dark red) from the TCGA database. ( B ) The scatter plot shows Pearson correlation (r) of TRIP13 expression with these transcription regulators.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Generated, Activation Assay, Expressing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: GeneMANIA network of TRIP13 and its co-expressed genes. The red node is the TRIP13 gene, the yellow nodes are its co-expressed genes and the gray nodes are predicted interactors. The between-nodes edges indicate relationship types, including co-expression, co-localization, physical interaction, pathway, genetic interaction and predicted interaction, which are colored according to the legend. The thickness of edges represents interaction weight (i.e., strength of pairing relationships).

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Associations among TRIP13 expression and DNA methylation and clinical characteristics in lower-grade glioma (LGG) and glioblastoma (GBM). ( A ) Visualization of the correlation between TRIP13 mRNA expression and DNA methylation in ( a ) TCGA-LGG and ( b ) TCGA-GBM cohort using the MEXPRESS program. The Pearson correlation coefficients (r) and p -value are indicated in the bottom panel. TSS, transcription start site; UTR, untranslated region. ( B ) The methylation status of TRIP13 expression in low-grade glioma ( n = 473) and GBM ( n = 106) samples in TCGA. ( C ) Correlation analysis between methylation and gene expression levels of TRIP13 of low-grade and high-grade glioma samples in TCGA. r, Pearson correlation coefficient of methylation and gene expression. P, p -value indicates the significance of the correlation. ( D , E ) Volcano plot of the distribution of all differentially expressed genes significantly associated with TRIP13 methylation of LGG and GBM samples in TCGA. The red nodes represent upregulated differentially expressed genes; the blue nodes represent downregulated differentially expressed genes. The orange nodes represent the downregulated TRIP13 co-expressed genes (LGG, n = 118; GBM, n = 14). The gray dashed line indicates the −Log 10 ( p -value) cutoff value of less than 1.30 (means p -value < 0.05). ( F ) KEGG pathway analysis in TRIP13 methylation using GSEA incorporated with LGG and GBM samples. Normalized enrichment scores and p -value corrected by FDR were calculated by GSEA. Only significantly enriched pathways (FDR adj. p -value < 0.05) are shown. Color represents the enrichment analysis results (blue, negative; red, positive). ( G , H ) Kaplan–Meier estimates of survival for LGG and GBM patients with hyper- or hypo-methylated TRIP13 in TCGA. TCGA, The Cancer Genome Atlas; GBM, glioblastoma; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; FDR, false discovery rate.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing, DNA Methylation Assay, Methylation, Gene Expression

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: The miRNAs predicted to regulate TRIP13 based on lower-grade glioma (LGG) miRNA- sequence analysis. ( A ) Venn analysis of selected miRNAs predicted interactive with TRIP13. There were 4 overlapping miRNAs significantly negatively associated (r > −0.2) with TRIP13 in LinkedOmics, miRanda and PITA datasets. ( B ) Heatmaps of miRNAs negatively correlated with TRIP13 mRNA expression in the TCGA-LGG dataset (LinkedOmics). The miR-29 family (29a, 29b, 29c) are shown significantly inversed with TRIP13 based on 512 lower-grade glioma samples. ( C ) The starBase program demonstrated that TRIP13 mRNA expression was negatively correlated with the expression of miR-29 family (522 lower-grade glioma samples). ( D ) The expression profiles of miR-29 family (29a, 29b, 29c) mRNA were validated in LGG and GBM samples, respectively, in the GSE112009 dataset.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Sequencing, Expressing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: TRIP13 correlates with tumor aneuploidy and reduced immune expression signatures. ( A ) OncoPrint depicting somatic copy number alterations, aneuploidy score (reflecting the number of altered chromosome arms) ( a ) and overall survival time ( b , c ) using the cBioportal. The bottom-right “Kaplan–Meier curve” shows the overall survival time in TCGA-LGG ( n = 443) and GBM ( n = 458) cohorts stratified on the basis of high versus low aneuploidy score. ( B ) The plot shows the percentage of tumor samples in TCGA-LGG and GBM cohorts ( n = 507 and n = 566, respectively) versus corresponding aneuploidy score which is determined by ABSOLUTE algorithm. ( C , D ) TRIP13 expression in low and high aneuploidy levels in TCGA LGG ( n = 507) and GBM ( n = 153) samples. ( E ) The correlation analysis between the transcriptional levels of TRIP13 and aneuploidy score for GBM ( n = 153) and LGG ( n = 514) tumor samples. r, Pearson correlation coefficient. The p -value indicates the significance of the correlation. ( F ) Volcano plot of the distribution of all differentially expressed genes significantly associated with TRIP13 of ( a ) LGG and ( b ) GBM samples in TCGA. The red nodes indicate upregulated differentially expressed genes; the blue nodes indicate downregulated differentially expressed genes. Among them, the 948 genes in LGG and 402 genes in GBM are significantly negative correlated (r < −0.3) and highlighted in deep blue, and several immune-related genes (e.g., c5orf53, MAP3K5, SELL, CYLD, MAPK3 and PSTPIP1 ) are labeled on the plot. The gray dashed line indicates the –Log10 ( p -value) cutoff value of less than 1.3 (means p -value < 0.05). ( G ) The Reactome pathway analysis reveals significant enrichment of negatively correlated genes with TRIP13 involved in major immune pathways in ( a ) LGG and ( b ) GBM. The top significant term is “immune system”. ( H ) Heat map demonstrating expression (z-score in RNA-seq by Expectation-Maximization) of the top 20 genes in the “immune system” term of ( F ) based on the Pearson correlation coefficient r in ( a ) LGG and ( b ) GBM. TCGA LGG and GBM samples are ordered by TRIP13 expression level. ( I ) The expression of genes specific for distinct types of immune cells (see ) were evaluated in TCGA LGG and GBM with low or high TRIP13 levels. The legend illustrating gene symbols and expression pattern is obtained from the RNA-seq analysis (% dec., percent decrease). ( J ) The network of TRIP13 and immune-related genes based on ( I ). The blue node is TRIP13 gene, the other color nodes are immune-related genes, and the gray nodes are predicted interactors. The between-nodes edges indicate relationship types, including co-expression, co-localization, physical interaction and pathway which are colored according to the legend. The thickness of edges represents interaction weight (i.e., strength of pairing relationships).

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing, Labeling, RNA Sequencing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: Immune genes negatively correlated with TRIP13 expression in TCGA GBM and LGG samples.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing

Journal: Cancers

Article Title: Clinical Significance and Systematic Expression Analysis of the Thyroid Receptor Interacting Protein 13 (TRIP13) as Human Gliomas Biomarker

doi: 10.3390/cancers13102338

Figure Lengend Snippet: The ratio of immune cell type associated with TRIP13 expression and the corresponding Kaplan–Meier curves in lower-grade glioma (LGG) and glioblastoma (GBM) form TCGA. The boxplots displaying the gene expression ratio (RSEM normalization values) in LGG and GBM based on ( A , B ) CD8 + T cell-specific to Treg-specific genes, ( C , D ) anti-tumor to pro-tumor modulators specific genes and ( E , F ) M1 macrophage-specific genes to M2 macrophage-specific genes. Kaplan–Meier estimates of survival for high/low ratio of patients in TCGA LGG and GBM data based on ( G , H ) CD8 + T cell-specific to Treg-specific genes, ( I , J ) anti-tumor to pro-tumor modulators specific genes and ( K , L ) M1 macrophage-specific genes to M2 macrophage-specific genes.

Article Snippet: The primary antibodies for TRIP13 (Atlas Antibodies, STO, SWE) and GAPDH (Santa-Cruz, Dallas, TX, USA) were used.

Techniques: Expressing, Gene Expression