Journal: Translational Psychiatry

Article Title: Astrocytic APOE3-Christchurch expression ameliorates brain amyloid-β pathology in 5xFAD mice

doi: 10.1038/s41398-026-04002-9

Figure Lengend Snippet: ( A - C ) Aβ40 and Aβ42 levels in the soluble TBS fractions ( A ), detergent soluble (TBS-X) fractions ( B ), and insoluble neutralized formic acid (FA) fractions ( C ) of cortical brain homogenates in the 5xFAD mice were measured by ELISA at 8 months of age, normalized to protein concentrations of each fraction. ( D ) Soluble oligomeric Aβ (oAβ) levels in the TBS-X fractions of cortical brain homogenates in the 5xFAD mice were measured by ELISA at 8 months of age, normalized to protein concentrations. ( E, F ) Amounts of full-length APP in the TBS-X fractions of mouse cortical lysates were measured by western blot at 8 months of age, normalized to those of β-actin (ACTB). Data are expressed as means ± SEM (GFP: N = 12; APOE3: N = 18; APOE3Ch: N = 13; Open circles: females, closed circles: males). Injection group differences were analyzed using two-way ANOVA, with Tukey’s multiple comparisons test, adjusting for sex. *, P < 0.05; **, P < 0.01.

Article Snippet: Recombinant Aβ42 monomer (1 mg/ml) (Stressmarq, #SPR-485) was incubated at room temperature for 10 min. To dissolve the peptide, 7.5 μl of cold DMSO (Sigma Aldrich) was added, followed by 92.5 μl of cold PBS (PH 7.4) to reach a final concentration of 220 μM monomeric Aβ42.

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

Journal: Translational Psychiatry

Article Title: Astrocytic APOE3-Christchurch expression ameliorates brain amyloid-β pathology in 5xFAD mice

doi: 10.1038/s41398-026-04002-9

Figure Lengend Snippet: ( A ) Isogenic iPSC-derived astrocytes (iPSC-ACs) with homozygous APOE3 (left) and APOE3Ch (right) were immunostained for GFAP (green) and S100b (red). DAPI (blue) stains nuclei. Scale bars: 100 µm. ( B ) APOE mRNA levels in the iPSC-ACs were measured by RT-qPCR, normalized to those of β-actin ( ACTB ). ( C, D ) Amounts of APOE in the conditioned medium (CM) from iPSC-ACs were measured by western blot, normalized to protein amounts of cell lysates. ( E ) Effects of the CM from iPSC-ACs on Aβ42 aggregation was assessed by western blot using 6E10 antibody. ( F - G ) Populations of Aβ fibrils (F; > 150 kDa), oligomers (G; 37–150 kDa), and monomers ( H ; < 10 kDa) were quantified. Data expressed as means ± SEM (n = 3–5 independent differentiation batches). Group differences were analyzed using two-tailed student t-test or one-way ANOVA with Tukey’s multiple comparisons test. *, P < 0.05.

Article Snippet: Recombinant Aβ42 monomer (1 mg/ml) (Stressmarq, #SPR-485) was incubated at room temperature for 10 min. To dissolve the peptide, 7.5 μl of cold DMSO (Sigma Aldrich) was added, followed by 92.5 μl of cold PBS (PH 7.4) to reach a final concentration of 220 μM monomeric Aβ42.

Techniques: Derivative Assay, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 6. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against the HuCCT-1 cell line. A): peripheral NK cell degranulation, evaluated as frequency of CD107a+ NK cells, in iCCA patients (n = 13) and HC (n = 16) in the presence of 7C6 mAb or IgG1-Fc. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56+ CD107a+ NK cells in a HC and a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): the proportion of circulating IFNγ+ NK cells in patients (n = 10) and HC (n = 11) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, the parametric t test and the non-parametric Wilcoxon t test were used. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 7. Anti-MICA/B 7C6 mAb boosts anti-tumor cytotoxic function of peripheral NK cells from iCCA patients against patient-derived iCCA cell lines. A): peripheral NK cell degranulation, evaluated as CD107a+NK frequency, in iCCA patients (n = 12) and HC (n = 8) in the presence of 7C6 mAb or IgG1-Fc using patient- derived primary tumor cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): dot plots showing the frequency of CD3-CD56 + CD107a+ NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc. C): proportion of circulating IFNγ+NK cells in patients (n = 10) and in HC (n = 8) in the presence of 7C6 mAb compared with IgG1-Fc. To compare paired data, we used the parametric t test and the non-parametric Wilcoxon t test. To compare unpaired data, the parametric t test and the non-parametric Mann-Whitney U test were used. D): representative dot plots showing the frequency of CD3-CD56+ IFNγ +NK cells in a HC and in a patient (iCCA) in the presence of 7C6 mAb or IgG1-Fc.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay, MANN-WHITNEY

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 8. 7C6 mAb enhances the anti-tumor effect of liver- and tumor-infiltrating NK cells in iCCA patients. A): Frequency of degranulating CD107a+NK cells in LIL (n = 13) and TIL (n = 10) of iCCA patients in the presence of anti-MICA/B 7C6 mAb or IgG1-Fc using autologous tumor-derived cell lines as targets. Parametric paired and unpaired t tests were used to compare data. B): representative dot plots showing the frequency of CD3-CD56+ CD107a+ LIL- and TIL-NK cells in the presence of 7C6 mAb or IgG1-Fc. C): proportion of IFNγ+ NK cells in LIL (n = 10) and TIL (n = 8) of iCCA patients in the presence of 7C6 mAb compared with IgG1-Fc using autologous tumor-derived cell lines as targets. The parametric t test and non-parametric Wilcoxon t test were used to compare paired data. The parametric t test was used to compare unpaired data.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Derivative Assay

Journal: OncoImmunology

Article Title: MICA/B-targeted antibody promotes NK cell–driven tumor immunity in patients with intrahepatic cholangiocarcinoma

doi: 10.1080/2162402x.2022.2035919

Figure Lengend Snippet: Figure 9. Cytotoxicity assay of HC PBMC, patient PBMC, LIL and TIL cells. A, B): Frequency of CFSE+LIVE/DEAD (LD)+ HuCCT-1 cell line targets when HC PBMC (n = 5), patient PBMC (n = 10), LIL (n = 8) and TIL (n = 5) were used as effector cells in the presence of 7C6 mAb and isotype control (IgG1). The parametric paired t tests were used to compare data. C, D): Frequency of CFSE+LD+ patient-derived cell line targets when HC PBMC (n = 5), patient PBMC (n = 8), LIL (n = 8) and TIL (n = 4) were used as effectors in the presence of 7C6 and isotype control. The parametric paired t test was used to compare data in panel C. The non-parametric Wilcoxon t test was used to compare paired data in panel D. Target cell death was determined as frequency of CFSE+LD+ cells.

Article Snippet: The recombinant human IgG1 Fc (BioXcell, Lebanon, NH, USA) and the humanized anti-MICA/B 7C6-IgG1 mAb were added at a final concentration of 10 μg/ml.

Techniques: Cytotoxicity Assay, Control, Derivative Assay

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 3. Induction of autophagy in response to VK2 in HL-60neo/HL-60bcl-2 cells. Involvement of LC3B in VK2-induced autophagy: After exposure to 10 μM VK2 for 48 and 72 hr, the cells were processed for fluorescent immunocytostaining with anti-LC3B Ab and counterstaining with DAPI for nucleus as described in Materials ant Methods. (A) HL-60neo/bcl-2 cells (original magnification x 1,000). (B) Quantification of the cells showing the punctuated pattern of LC3B staining, which marks cells with autophagosome forma- tion. One hundred cells were assessed and ratios for the cells showing the punctuated pattern of LC3B staining were expressed. Results shown are the means ± SD from the results of three independent experi- ments. *p < 0.001. Development of acidic vesicular organelles (AVOs) after VK2 treatment: (C) After staining the cells with acridine orange, AVOs were quantified using flowcytometer in HL-60neo (upper) and HL-60bcl-2 (lower) treated with or without VK2 (5 μM, 10 μM or 20 μM) for 72 hr. X-axis, green color intensity; Y-axis, red color intensity. Expression of isoforms of LC3B and caspase-3: (D) Cellular pro- teins were lysed at the indicated time after incubation with or without 10 μM of VK2. Proteins were separated by either 11.25% or 15% SDS-PAGE. Aliquots of 40 μg of protein extracts were used for immunoblot- ting using anti-caspase-3 and anti-LC3B Abs, respectively. The anti-β-actin mAb was used for protein-loading equivalence. Expression of isoforms of LC3B in the pres- ence and absence of protease inhibitors: (E) After treatment with 10 μM of VK2 for 44 hrs in HL-60neo and for 92 hrs in HL-60bcl-2 cells, cells were further cultured with/without protease inhibitors, E-64-d (10 μg/ml) and pepstatin A (10 μg/ml) in the presence of VK2 for 4 hrs. Cellular proteins were lysed and immunoblotted with anti-LC3B Ab.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Staining, Expressing, Incubation, SDS Page, Western Blot, Cell Culture

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 4. Effects of a caspase-3 inhibitor on VK2-induced autophagy in HL-60neo cells. (A) HL- 60neo cells were cultured with Z-DEVD-FMK at various concentrations with/without 10 μM of VK2 for 96 hr. The number of cells was assessed with the WST cell counting kit as described in Materials and Methods. *p < 0.001. (B) HL-60neo cells were treated with 10 μM VK2 with/without 100 μM of Z-DEVD-FMK for 48 hr and 96 hr, respectively. Cellular proteins were lysed and separated by either 11.25% or 15% SDS-PAGE. Aliquots of 40 μg of protein extracts were used for immunoblotting using anti-cleaved caspase-3 Ab, anti-LC3B Ab, and anti-β-actin mAb, respectively.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Cell Culture, Cell Counting, SDS Page, Western Blot

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 5. Capsulated fragmented nuclei in HL-60bcl-2 cells after treatment with VK2. (A) Morphological features of HL-60neo after 72 hr-treatment with VK2 (10 μM) with/without Z-DEVD-FMK (100 μM) and of HL-60bcl-2 cells after 96 hr-treatment with VK2. May-Grünwald-Giemsa staining, original magnification x 1,000. (B) Percentages of HL-60 cells with apoptotic bodies containing either capsulated nuclear fragments or uncapsulated nuclear. After treatment HL- 60neo/bcl-2 cells with 10 μM of VK2 for various length of time, morphologic changes were assessed in 100 cells after May-Grünwald Giemsa stain as well as Figure 4A. (This is one of representative result from 3 separate experiments.) (C) Electron microscopy of HL-60bcl-2 cells after treatment with 10 μM VK2 for 96 hr. (D) Fluorescent immunocytostaining with anti-LC3B Ab in HL-60bcl-2 cells after 96 hr-treatment with 10 μM VK2. Fluorescent immunocytostaining with anti-LC3B Ab (original magnification x 1,000) was performed as described in Figure 3A and C, respectively. The slides stained with anti-LC3B Ab were monitored using Zeiss LSM510 confocal microscope (Original magnification x 600).

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Staining, Giemsa Stain, Electron Microscopy, Microscopy

Journal: Autophagy

Article Title: Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells.

doi: 10.4161/auto.5941

Figure Lengend Snippet: Figure 7. Effects of inhibition of VK2-inducing autophagy in a Atg5-/- mouse embryonic fibroblast (MEF) cell line with the Atg5 Tet-off system. Tet-off Atg5 MEF cells were incubated with 10 ng/ml doxycycline hydrochloride (Dox) for 120 hrs, and thereafter cultured in the presence or absence of 1 to 50 μM of VK2 for 72 hrs. (A) Cell growth inhibition in response to VK2 was assessed using WST assay kit. (B) MEF cells treated with or without Dox were cultured in the presence or absence of 10 μM of VK2 for 72 hr. Then, cellular proteins were separated by 11.25% SDS-PAGE and immunoblotted with either anti-Atg5, anti-LC3B, or anti-β-actin Abs.

Article Snippet: The membranes were probed with antibodies (Abs) such as anti-human Bcl-2 monoclonal (m) Ab (BD Biosciences Pharmingen, San Jose, CA), anti-microtubule-associated protein 1 light chain 3B (LC3B) Ab, anti-caspase-3 Ab (Cell Signaling Technology, Danvers, MA), anti-cleaved caspase-3 Ab (Cell Signaling Technology), antiAtg5 Ab (Abgent, San Diego, CA), anti-Atg7 Ab (ProSci, Poway, CA), anti-human catalase rabbit mAb (Epitomics Inc., Burlingame, CA), and anti-β-actin Ab (Sigma-Aldrich), respectively.

Techniques: Inhibition, Incubation, Cell Culture, WST Assay, SDS Page

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 1. NR4A1 regulates β1-integrin expression in breast cancer cells and tumors. (A) Breast 598

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 3. Role of NR4A1/p300/Sp1 in regulation of β1- and β3-integrin. (A) Analysis of polII, 620

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: NR4A1 Antagonists Inhibit β1-Integrin-Dependent Breast Cancer Cell Migration

doi: 10.1128/mcb.00912-15

Figure Lengend Snippet: Fig. 5. Role of NR4A1 on TGFβ-induced migration of MDA-MB-231 cells. (A) MDA-MB-231 647

Article Snippet: NR4A1 antibody was purchased from Novus Biologicals (Littleton, CO).

Techniques: Migration