Journal: Oncotarget

Article Title: Amplifications of stemness genes and the capacity of breast tumors for metastasis

doi: 10.18632/oncotarget.27608

Figure Lengend Snippet: ( A ) The experimental design and the CNA genetic landscape of tumors in patients St23784/17 and Ti41749/17, showing that St23784/17 had amplifications at the 3q, 6q, 8q, 9q, and 10q22.1 regions of stemness gene localization (the following genes were amplified: SOX2 , MYC , KLF4 , NOTCH1 , NODAL ), and Ti41749/17 had no stemness gene amplifications. ( B – E ) Analysis of the quantitative and qualitative expression of cells obtained from tumor tissue of breast cancer patients. The cells were analyzed by flow cytometry using a FACSCanto II (Becton Dickinson, USA) and FACS Diva software, using CD326 (PerCP-Cy5.5), CD24 (PE-Cy 7), and CD44 (APC) (BD Biosciences, USA) antibodies. (B) Histogram of the IgG1 PerCP Cy5.5 isotope control; (C) isotype control dot-plot for IgG2a PE-Cy 7/IgG2b APC; (D) phenotypic confirmation and qualitative analysis of CD326 (PerCP-Cy5.5) expression in the histogram; (E) phenotypic confirmation and qualitative analysis of CD326 + CD44 + CD24 -\low expression in the dot-plot. ( F ) The dynamics of the EpCam+ tumor cell mass increase (percentage of the initial number of cells in culture). ( G ) Nonadherent mammospheres of EpCam + cancer cell cultures (St23784/17 patient). Mammosphere formation within 10 days. Scale bar, 200 μm. ( H ) EpCam + cells without mammosphere formation (patient Ti41749/17). ( I ) Thermal map of stemness gene expression in the environment enriched with EpCam+ tumor cells from the patients St23784/17 and Ti41749/17. ( J ) The content of tumor stem cells in the EpCam + cell enriched environment after a cycle of cultivation and IL-6 treatment (% of the initial cell content in each population).

Article Snippet: The EpCam + cell fraction was isolated using the EasySepTM Human EpCam Positive Selection Kit (StemCell Technologies, Catalog #18356).

Techniques: Amplification, Expressing, Flow Cytometry, Software, Control, Gene Expression

Journal: Oncotarget

Article Title: Amplifications of stemness genes and the capacity of breast tumors for metastasis

doi: 10.18632/oncotarget.27608

Figure Lengend Snippet: Stem cell populations (CD326+ fraction) in two breast tumors with (ST23784/17) and without (Ti41749/17) amplification of stemness genes

Article Snippet: The EpCam + cell fraction was isolated using the EasySepTM Human EpCam Positive Selection Kit (StemCell Technologies, Catalog #18356).

Techniques: Amplification, Expressing

Journal: Cell Reports Methods

Article Title: Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

doi: 10.1016/j.crmeth.2022.100350

Figure Lengend Snippet: γδ T cell reactivity accounts for the majority of the mouse extract response and is not mediated by peptides Total MO-specific T cell responses were measured as a percentage of AIM + (CD137 + CD69 + ) CD3 + T cells after stimulation of PBMCs with MO extract, and the individual γδ and αβ T cell reactivity was assessed. (A) Representative FACS plot of total AIM + CD3 + T cells (left) further gated as function of γδ and αβ T cell reactivity (right) (B) Graph shows the relative percentage of γδ and αβ reactivity from the total MO-specific T cell responses across all donors (n = 33). (C) (Left) Representative FACS plots of AIM + (CD137 + CD69 + ) γδ (top row) or αβ (bottom row) T cells after stimulation of PBMCs with MO peptide pools (MPs), MO extract, or control (Neg; media for γδ T cells, or DMSO for αβ T cells). (D) Graphs show the percentage of AIM + reactivity in each condition for γδ or αβ T cells across all donors (n = 20). All graphs show data represented as geometric mean with SD. Each dot represents a unique individual. Pairwise comparisons were performed with the Wilcoxon test, and p values <0.05 were considered statistically significant.

Article Snippet: APCs were separated from PBMCs in the negative flow-through using the human CD3 positive selection kit (EasySepTM Human CD3 Positive Selection Kit II, Stemcell Technologies, Vancouver, BC, Canada).

Techniques: Control

Journal: Cell Reports Methods

Article Title: Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

doi: 10.1016/j.crmeth.2022.100350

Figure Lengend Snippet: γδ T cell reactivity to MO and CR allergen extracts is TCR specific, albeit with different requirements for the presence of APCs (A–C) Allergen-specific γδ T cell responses were measured as percentage of AIM + (CD137 + CD69 + ) γδ T cells after stimulation of PBMCs with (A) MO extract, (B) CR extract, or (C) HDMAPP in the presence (+) or absence (−) of antigen-presenting cells (APCs). (D and E) PBMCs were stimulated with an MO or CR extract, HDMAPP, or α-CD3 and cultured in the absence (−) or presence (+) of a TCR blocking reagent (dasatinib). Graphs show percentage of AIM + γδ (D) or αβ (E) T cells in response to the different stimuli and conditions (MO, n = 8; CR, n = 8; HDM, n = 16; TG, n = 16). Each dot represents a unique individual. Data are represented as geometric mean and SD. Kruskal-Wallis or Wilcoxon paired tests were performed, and p values are indicated. p < 0.05 was considered statistically significant.

Article Snippet: APCs were separated from PBMCs in the negative flow-through using the human CD3 positive selection kit (EasySepTM Human CD3 Positive Selection Kit II, Stemcell Technologies, Vancouver, BC, Canada).

Techniques: Cell Culture, Blocking Assay

Journal: Cell Reports Methods

Article Title: Ex vivo assays show human gamma-delta T cells specific for common allergens are Th1-polarized in allergic donors

doi: 10.1016/j.crmeth.2022.100350

Figure Lengend Snippet:

Article Snippet: APCs were separated from PBMCs in the negative flow-through using the human CD3 positive selection kit (EasySepTM Human CD3 Positive Selection Kit II, Stemcell Technologies, Vancouver, BC, Canada).

Techniques: Recombinant, Software

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs treatment normalizes markers of senescence in MRL/ lpr mice splenic CD4+ T cells in vivo. (A, B) qPCR analysis showing the expression levels of p21, p16 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. (C) Representative western blot showing the expression levels of p21, p16, p53 and acetylation of p53 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. (D, E) Capillary WES and qPCR analysis showing the expression of Sirt1 in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice. GAPDH was used as a protein loading control. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: In Vivo, Expressing, Western Blot, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs increase markers of senescence in splenic CD4+ T cells in vitro . (A-C) Splenic CD4+ T cells from MRL /lpr mice were cultured alone or with hUC-MSCs at ratios of 1:1, 10:1, or 50:1 for 48 h. (D-F) In further experiments, MRL/ lpr splenic CD4+ T cells and hUC-MSCs (10:1) were cultured separated by transwells. Sirt1 (A, D), p21 (B, E) and p16 (C, F) RNA levels of CD4+ T cells were quantitated by qPCR. All experimental data were verified in at least two independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; n.s., not significant.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: In Vitro, Cell Culture

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: Sirt1 is a mediator of hUC-MSCs increasing senescence of splenic CD4+ T cells in MRL/ lpr mice. (A) Western blotting showing the expression of Sirt1, p21, p16, p53 and acetylation of p53 in EX527 and DMSO treated MRL/ lpr splenic CD4+ T cells. (B) MRL/ lpr splenic CD4+ T cells were pretreated with SRT1720 12 h before exposed to hUC-MSCs for 24-48 h, western blotting showing the expression of p21, p16, p53 and acetylation of p53. GAPDH was used as a protein loading control. All experimental data were verified in at least three independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Western Blot, Expressing, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: hUC-MSCs induced miR-199a-5p can increase MRL/ lpr splenic CD4+ T cell senescence. (A) qPCR analysis of the levels of ten potential miRNAs in WT mice, PBS-treated MRL/ lpr mice and hUC-MSCs-treated MRL/ lpr mice splenic CD4+ T cells. (B-D) qPCR and western blotting analysis of the levels of miR-199a-5p, Sirt1, p21, p16 and acetyl-p53 in vehicle and miR-199a-5p mimic-treated MRL/ lpr splenic CD4+ T cells. (E-F) qPCR and western blotting analysis of the levels of Sirt1, p21, p16 and acetyl-p53 in vehicle and miR-199a-5p inhibitor-treated WT splenic CD4+ T cells. (G-I) MRL/ lpr splenic CD4+ T cells and hUC-MSCs were cultured alone or together in the presence or absence of miR-199a inhibitor using a transwell system. MiR-199a-5p, Sirt1, p21, p16 and acetyl-p53 were quantified in hUC-MSCs (the first bar) or splenic CD4+ T cells (the last three bars). GAPDH was used as a protein loading control. All experimental data were verified in at least two independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Western Blot, Cell Culture, Control

Journal: Theranostics

Article Title: Human umbilical cord-derived mesenchymal stem cell therapy ameliorates lupus through increasing CD4+ T cell senescence via MiR-199a-5p/Sirt1/p53 axis

doi: 10.7150/thno.48080

Figure Lengend Snippet: MiR-199a-5p agomir treatment increases senescence of MRL/ lpr splenic CD4+ T cells. (A) Experimental outline. CD4+ T cells were harvested to measures miR-199a-5p (B), Sirt1 (C), p21 and p16 (D). GAPDH was used as a protein loading control. (n = 6 per group). * p < 0.05; ** p < 0.01.

Article Snippet: The splenic CD4+ T cells were purified using immunomagnetic positive selection (EasySepTM mouse CD4+ positive selection Kit, 18952, Stemcell technologies, Canada).

Techniques: Control