Journal: Scientific Reports

Article Title: Mechanism of Saikosaponin D in regulating ferroptosis in patient-derived lung adenocarcinoma organoids via upregulation of ATF3/CHOP/CHAC1 signaling

doi: 10.1038/s41598-025-27251-y

Figure Lengend Snippet: SSD induces ferroptosis via downregulation of GPX4 through ER stress. (A) Western blot analysis of GPX4 in PDO and A549 cells treated with 2 µM SSD and control groups, β-Actin was used as a loading control. (B) Statistical analysis of GPX4 expression levels from three independent experiments ( n = 3). (C) Molecular docking of SSD and ATF3 protein. (D) DARTS-Western blot analysis showed the resistance of ATF to pronase E digestion under the treatment of SSD (10 µM), n = 3. (E) RT-qPCR analysis of mRNA expression levels of PDO-related genes ATF3 , CHOP , and CHAC1 treatment with SSD ( n = 3). (F , G) Western blot analysis of protein expression (ATF3, CHOP, CHAC1) in A549 cells and PDOs after treatment with 2 µM SSD( n = 3). (H) Mechanism of SSD acting on lung adenocarcinoma. (Data are presented as mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001). ER, endoplasmic reticulum; PDO, patient-derived organoids; SSD, saikosaponin D. SD, standard deviation.

Article Snippet: Then blocked with 5% BSA for 1 h. Reaction with specific antibodies against the following proteins: β-Actin (A00730, Genscript, China), ATF3 (DF3110, Affinity, USA), GPX4 (ET1706-45, HUABIO, China), CHOP (15204-AF6277, Affinity, USA), and CHAC1 (15207-1-AP, Proteintech, China).

Techniques: Western Blot, Control, Expressing, Quantitative RT-PCR, Derivative Assay, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Aurora A kinase phosphorylates Hec1 to regulate metaphase kinetochore–microtubule dynamics

doi: 10.1083/jcb.201707160

Figure Lengend Snippet: Hec1 S69 is phosphorylated throughout mitosis. (A) Amino acid sequences of the human and PtK1 cell Hec1 N-terminal tail domain. Shown in yellow is the human peptide sequence that was used to generate the S69 phosphospecific antibody. The arrow points to S69 in the human sequence and the corresponding serine residue in the PtK1 sequence. Asterisks indicate all other mapped Aurora B kinase sites in the human Hec1 tail domain ( ; ). (B) Immunofluorescence images of HeLa cells stained with phosphospecific antibodies to Hec1 pS69. Depletion of Hec1 (bottom) results in loss of pS69 staining at kinetochores. Cells are also immunostained with antibody 9G3 (pan-Hec1 antibody) and an anticentromere antibody (ACA) derived from human calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia (CREST) patient serum. (C) Immunofluorescence images of HeLa cells demonstrating kinetochore localization of pS69 during mitosis. Quantification is shown on the right. For each phase shown, ≥400 kinetochores from ≥30 cells were measured. (D) Immunofluorescence images of a HeLa cell stained with antibodies to pS69 and Mad2. For the cell shown, most chromosomes are aligned at the spindle equator, and one chromosome remains near a spindle pole (arrows). A schematic illustrating examples of pole-proximal chromosomes is shown on the left. (E) Immunofluorescence images of HeLa cells depleted of CENP-E to increase the number of pole-proximal chromosomes (arrows) and stained with Hec1 phosphospecific antibodies. Quantification is shown on the right from one representative experiment. n values are as follows: pS69, 20 polar kinetochores and 40 aligned kinetochores; pS55, 17 polar kinetochores and 57 aligned kinetochores; and pS44, 13 polar kinetochores and 29 aligned kinetochores. Error bars indicate SD. Bars: (B, C, and E) 10 µm; (D) 3 µm.

Article Snippet: Two rabbits were immunized with the Mad2 protein (Rockland Immunochemicals Inc), and antisera from the two injected rabbits were affinity purified against full-length Mad2 protein using a HiTrap-NHS column.

Techniques: Sequencing, Residue, Immunofluorescence, Staining, Derivative Assay

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 1: Expression of p33ING1b and p29ING4 in renal cell carcinoma. (a) mRNA expression of p33ING1b and p29ING4 was increased at early and late stages of disease with higher expression of both genes at late stages of disease compared to early stages (Robson stages III/IV vs. I/II) (p33ING1b and p29ING4: stages I/II and III/IV vs. normal tissue p<0.001 and p<0.0001, respectively; p33ING1b and p29ING4: stages III/IV vs. I/II p<0.01 and p<0.001, respectively). (b) Results of the real time RT-PCR were normalized to kidney normal tissue and expressed as x-fold difference (2- ∆∆Ct). Expression of p33ING1b and p29ING4 by renal carcinoma cells was confirmed (Cy3 red; DAPI blue (nuclear counterstaining)). (c) Sections are representative of n=30 patients/group. Representative western blots of p33ING1b and p29ING4 (loading control ß-actin).

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 4: p33ING1b- and p29ING4-specific T cell reactivity. Four peptides resulted in significantly increased expression of IFN-γ compared to controls, independent of tumor stage (p33ING1b (aa109-118) and aa259-268, and p29ING4 (aa149-158) and aa239-248; p<0.001) Stages I/ II (a) and Stages III/IV (b). Elispot analysis was measured as spots/5 x 105 cells/patient.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Enzyme-linked Immunospot

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 5: p33ING1b- and p29ING4-specific T cell reactivity. Two peptides resulted in increased expression of IL-10 compared to controls; peptide p33ING1b (aa109-118) at all stages and p29ING4 (aa239-248) at late stages (p<0.001). Stages I/ II (a) and Stages III/IV (b). Luminex analysis was measured as pg/ml.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 6: p33ING1b- and p29ING4-specific T cell reactivity. Evaluation of IL-2 expression revealed that only a single epitope (p29ING4 (aa149-158)) resulted in high responsiveness of T cells from RCC patients after stimulation with the peptide (stages I/II vs. III/IV p<0.001). PBMCs from healthy volunteers (n=30) had no significant IL-2 expression after stimulation with all peptide epitopes. Luminex analysis for IL-2 expression was measured as pg/ml and presented as percent stimulation index.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Expressing, Luminex

Journal: Journal of Clinical & Cellular Immunology

Article Title: Tumor Suppressor Gene P29ing4 is Overexpressed and Induces a CD8 T Effector Cell Response in Human Renal Cell Carcinoma

doi: 10.4172/2155-9899.1000511

Figure Lengend Snippet: Figure 7: p29ING4 presentation by MHC class I (a) and II (b) molecules. p29ING4 presentation by major histocompatibility complex (MHC) class I and II antigens was shown to be increased in RCC but not in healthy kidneys.

Article Snippet: Analysis of single staining was performed for p33ING1 (Santa Cruz Biotechnology, CA, USA), p29ING4 (Rockland/Biomol, Hamburg, Germany) CD4, CD25, Foxp3, CD8, (Abcam, Cambridge, UK), IFN-γ (BD Pharmingen, Heidelberg, Germany), IL-2, and IL-10 (R&D Systems, Minneapolis, USA).

Techniques: Immunopeptidomics

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: Involvement of LRP1 in Tat-mediated HIV-1 LTR transactivation. a The LRP1 antagonist, receptor-associated protein (RAP), concentration dependently reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Protein expression levels of LRP1 were significantly ( n = 3, p < 0.05) reduced in U87MG cells transfected with LRP1 siRNA (100 nM) compared to cells transfected with control (scrambled) siRNA. c siRNA knockdown of LRP1 reduced significantly ( n = 3; * p < 0.05) Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay, Expressing, Transfection

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE-HDL isoform dependently affected Tat-mediated HIV-1 LTR transactivation. U87MG cells stably transfected with a luciferase gene under the control of HIV-1 Tat responsive LTR promoter were incubated for 48 h with ApoE2-HDL ( a ), ApoE3-HDL ( b ), or ApoE4-HDL ( c ) in the presence of HIV-1 Tat protein (2 μg/ml) and 100 μM chloroquine (CQ). ApoE2-HDL, ApoE3-HDL, and ApoE4-HDL significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001) ( d ). In comparison to ApoE2-HDL and ApoE3-HDL, ApoE4-HDL was less potent and effective at restricting Tat-mediated HIV-1 transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Stable Transfection, Transfection, Luciferase, Incubation, Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: HDL affects the ability of ApoE to restrict Tat-mediated HIV-1 LTR transactivation. a ApoE2 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; * p < 0.05; ** p < 0.01). b ApoE3 significantly and concentration dependently restricted Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3; ** p < 0.01). c ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation in the absence of HDL ( n = 3, ** p < 0.01). d In the presence of HDL, ApoE2 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. e In the presence of HDL, ApoE3 was more potent and effective at restricting Tat-mediated HIV-1 LTR transactivation. f In presence of HDL, ApoE4 concentration dependently restricted Tat-mediated HIV-1 LTR transactivation; however, in the absence of HDL, ApoE4 concentration dependently enhanced Tat-mediated HIV-1 LTR transactivation

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Concentration Assay

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE2 and ApoE3, but not ApoE4, inhibited HIV-1 Tat internalization. Representative images ( a ) and quantified internalized fluorescence intensities ( b ) show that ApoE2 and ApoE3 at 5 and 10 μg/ml inhibited HIV-1 Tat internalization as evidenced by lower levels of punctate staining of internalized FITC-labeled Tat ( n = 30; *** p < 0.001, **** p < 0.0001), whereas ApoE4 did not inhibit HIV-1 Tat internalization

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Fluorescence, Staining, Labeling

Journal: Journal of Neuroinflammation

Article Title: Apolipoprotein E isoform dependently affects Tat-mediated HIV-1 LTR transactivation

doi: 10.1186/s12974-018-1129-1

Figure Lengend Snippet: ApoE mimetic peptide decreased Tat-mediated HIV-1 LTR transactivation. a U87MG cells were treated with an ApoE mimetic peptide in the presence of HIV-1 Tat and chloroquine (CQ) for 48 h. ApoE mimetic peptide significantly reduced Tat-mediated HIV-1 LTR transactivation ( n = 3; * p < 0.05). b Incubation with an ApoE4 structure corrector, that makes the structure and function of ApoE4 more like ApoE3, enhanced the ability of ApoE4 to restrict Tat-mediated LTR transactivation ( n = 3; * p < 0.05)

Article Snippet: Cells plated on glass-bottom 35-mm 2 tissue culture dishes were co-incubated with ApoE2, ApoE3, or ApoE4 (5,10 μg/ml) and FITC-labeled HIV-1 Tat (5 μg/ml; purchased from Rockland) for 90 min at 37 °C in the presence of chloroquine (100 μM).

Techniques: Incubation