Journal: Nature communications

Article Title: Translational control of breast cancer plasticity.

doi: 10.1038/s41467-020-16352-z

Figure Lengend Snippet: Fig. 4 MTOR inhibition induces breast cancer plasticity. a NODAL immunoblot from 0 to 24 h INK 20 nM treated T47D cells (n = 3). b Mean sphere counts from 960 T47D cells pre-exposed to DMSO, INK (20 nM), rhNODAL (100 ng/mL), or INK + SB431542 (10 μM) for 24 h (n = 4) with representative images. Micron bars = 250 μm. c Mean fold change in colony number from T47D cells cultured as in b (n = 3). d Mean percentage of CD44high/CD24low T47D cells treated with DMSO, INK, or INK + SB431542 (n = 6) with representative scatterplots defining CD44 and CD24 subpopulations. e Mean log2-fold change relative to DMSO qRT-PCR of NANOG, SOX2, OCT4, TWIST, ZEB1, SNAIL, VIM transcripts from DMSO or INK (20 nM) for 24 or 24 h INK then 24 h media treated T47D cells (n = 3). f Immunoblot analyses of NANOG, OCT4, and SNAIL in 24 h DMSO or INK (20 nM) treated T47D cells. g Mouse lung colonies 8 weeks following tail vein injection of pretreated SUM149 cells (24 h with DMSO or INK (20 nM)) (n = 10) with representative lung colony images (brown). h Mean PDX401 tumor volumes in mice receiving DMSO or INK (30 mg/kg) every second day for two weeks. Arrow indicates treatment cessation (n = 8). i Mean sphere counts from 96,000 PDX401 cells from endpoint tumors from h (DMSO n = 6, INK n = 7) with representative images. Micron bars = 500 μm. j NODAL and 4E-BP1 immunoblot of T47D cells transfected with 4E-BP1 shRNA, shRNA scrambled (SCR) control, 4E-BP1 ORF, or empty vector. k Mean first and second generation sphere counts from 96 T47D cells described in j (n = 3) with representative images. Micron bars = 250 μm. l Kaplan–Meier plot: 4E-BP1 RNA-Seq expression correlates with survival in 1100 breast cancer patient samples (5.4 years, p = 0.0187). 4E-BP1 RNA-Seq transcript abundance in tumor versus healthy tissue (normal, n = 113; tumor, n = 1062; p = 2.2 × 10−16). Data represents independent experiments. Error bars indicate mean ± SD. Box and whisker plot represents median, IQR, whiskers extend to maximum and minimum. Two-sided t-test for paired samples. The asterisks denote p-values < *0.05, **0.01. Multiple comparisons tested by ANOVA. The same letters indicate relationships with a p ≥0.05. Different letters indicate statistical differences (p < 0.05).

Article Snippet: Zombie aqua was removed and 20 μL of antibody dilution was added to each sample, which was then incubated on ice for 10–15 min. Antibody pairs: CD24 APC (REA832, MiltenyiBiotec, 1:20 dilution), CD44 Vioblue (REA690, Miltenyi Biotec, 1:5 dilution) used in Fig. 1f FITC Mouse Anti-Human CD24 (BD Biosciences; Franklin Lakes, NJ, USA, 1:5 dilution), PE Mouse Anti-Human CD44 (BD Biosciences, 1:5 dilution) used in Fig. 3d and Supplementary Figs. 1b–d and 3f,g Cells were washed with 200 μL FACs buffer (PBS with 1% FBS), pelleted and then resuspended in 100 μL 2% PFA in FACs buffer.

Techniques: Inhibition, Western Blot, Cell Culture, Quantitative RT-PCR, Injection, Transfection, shRNA, Control, Plasmid Preparation, RNA Sequencing, Expressing, Whisker Assay

Journal: European Journal of Medical Research

Article Title: Dickkopf-related protein 3 alters aerobic glycolysis in pancreatic cancer BxPC-3 cells, promoting CD4 + T-cell activation and function

doi: 10.1186/s40001-021-00567-x

Figure Lengend Snippet: Relative mRNA expression levels of A CD3, B CD28, C CD25, D CD69, E CD71 and F HLA-DR in inactivated CD4 + T cells of each group after 72 h of co-culture. DKK3, dickkopf-related protein 3; NC, negative control

Article Snippet: CD4 + T cells were incubated with human CD3 (cat. no. APC-65060; ProteinTech Group, Inc.), CD28 (cat. no. #62-0289-42; eBioscience; Thermo Fisher Scientific, Inc.), CD25, and CD69 (cat. no. FAB1020A and FAB23591P, respectively; R&D Systems, Inc.), and CD71 and HLA-DR (cat. no. ab9179 and ab20181, respectively; Abcam) antibodies (4 ̊C).

Techniques: Expressing, Co-Culture Assay, Negative Control

Journal: European Journal of Medical Research

Article Title: Dickkopf-related protein 3 alters aerobic glycolysis in pancreatic cancer BxPC-3 cells, promoting CD4 + T-cell activation and function

doi: 10.1186/s40001-021-00567-x

Figure Lengend Snippet: Flow cytometric detection of inactivated CD4 + T-cell surface markers. Proportions of A CD3 + , B CD28 + , C CD25 + , D CD69 + , E CD71 + and F HLA-DR + cells. * P < 0.01: compared with the sodium sulphate-pretreated BxPC-3 NC group and BxPC-3-DKK3 group; ** P < 0.01: compared with the BxPC-3-DKK3 group. Data are presented as the mean ± SE of three independent experiments. DKK3, dickkopf-related protein 3; NC, negative control

Article Snippet: CD4 + T cells were incubated with human CD3 (cat. no. APC-65060; ProteinTech Group, Inc.), CD28 (cat. no. #62-0289-42; eBioscience; Thermo Fisher Scientific, Inc.), CD25, and CD69 (cat. no. FAB1020A and FAB23591P, respectively; R&D Systems, Inc.), and CD71 and HLA-DR (cat. no. ab9179 and ab20181, respectively; Abcam) antibodies (4 ̊C).

Techniques: Negative Control

Journal: Frontiers in Pharmacology

Article Title: Platycodon grandiflorum Triggers Antitumor Immunity by Restricting PD-1 Expression of CD8 + T Cells in Local Tumor Microenvironment

doi: 10.3389/fphar.2022.774440

Figure Lengend Snippet: PG enhances antitumor immunity via reducing PD-1 on the surface of CD8 + T. (A) The heat map of Pearson correlation coefficients (PCCs) evaluated by the four methods between the gene expression level of PG targets and immune phenotypes; Benjamini–Hochberg (BH) adjusted p -values of PCCs <0.05 are shown in white. (B) LLC tumor infiltrating CD8 + T cells in PG-treated versus WT mice analyzed by flow cytometry. Flow cytometry analysis of single-cell suspensions obtained from LLC tumors and stained for detecting CD8 + T cells (CD3+CD8+). Representative dot plots with indicated percentages of CD8 + T cells populations within CD45 + cells in LLC tumors from the WT and PG-treated groups, respectively. (C) Protein level of IFN-γ and granzyme B in WT and PG-treated groups by means of western blotting. (D) Transcription levels of IFN-γ and granzyme B in WT and PG-treated groups by means of RT-PCR. (E) Protein level of PD-1 in WT and PG-treated groups. (F) Flow cytometry analysis of single-cell suspensions obtained from LLC tumors and stained for detecting PD-1 + CD8 + T cells. Representative dot plots with indicated percentages of PD-1 + CD8 + T-cell populations within CD8 + T cells in LLC tumors from WT and PG-treated groups, respectively. All plots of flow cytometry analysis were gated on total live cells. Data are from distinct samples and presented as the mean ± SEM. ** p < 0.01, *** p < 0.001 compared with WT by unpaired t-test; n > = 3.

Article Snippet: The untouched and highly purified mouse CD8 + T cells were isolated from single-cell suspensions of splenocytes by immunomagnetic negative selection using the EasySep Mouse CD8 + T-Cell Isolation Kit (19853, STEMCELL), according to the manufacturer’s instructions, and the CD8 + T cells purity (>90%) was identified by flow cytometry Anti-mouse CD3e APC (05122-80-25, Biogems, PeproTech) and anti-mouse CD8a PE (100707, Biolegend).

Techniques: Gene Expression, Flow Cytometry, Staining, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: Platycodon grandiflorum Triggers Antitumor Immunity by Restricting PD-1 Expression of CD8 + T Cells in Local Tumor Microenvironment

doi: 10.3389/fphar.2022.774440

Figure Lengend Snippet: PG decreases PD-1 through VEGF-A–VEGFR-2 axis. (A) Flow cytometer detection of PD-1 expression in CD8 + T cells after treatment with PG. (B) Changes of VEGF-A transcription level in LLC after PG treatment. (C) Western blot analysis of the effect of PG on the expression of VEGF-A protein. (D) The proportion of PD-1 + CD8 + T cells in CD8 + T cells was tested by flow cytometry. Treatment of CD8 + T cells includes different tumor-conditioned media (TCM-C, TCM-1, TCM-5, and TCM-10) groups, and the blank control group and VEGF-A group. The % PD-1 + CD8 + T cells were quantified and are presented in the right panel. (E) The ratio of CD8 + T cells proliferation under different treatments: tumor-conditioned media (TCM-C), TCM-1, TCM-5, TCM-10, blank control group, and VEGF-A group. The % proliferative CD8 + T cells were quantified and are presented in the right panel. (F) Transcription levels of VEGFR-1 and VEGFR-2 were detected by RT-qPCR. Bar chart shows quantification of transcriptional levels compared to GAPDH control in each condition. The results are presented as the mean ± SD of triplicate determination. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The untouched and highly purified mouse CD8 + T cells were isolated from single-cell suspensions of splenocytes by immunomagnetic negative selection using the EasySep Mouse CD8 + T-Cell Isolation Kit (19853, STEMCELL), according to the manufacturer’s instructions, and the CD8 + T cells purity (>90%) was identified by flow cytometry Anti-mouse CD3e APC (05122-80-25, Biogems, PeproTech) and anti-mouse CD8a PE (100707, Biolegend).

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

Journal: Frontiers in Pharmacology

Article Title: Platycodon grandiflorum Triggers Antitumor Immunity by Restricting PD-1 Expression of CD8 + T Cells in Local Tumor Microenvironment

doi: 10.3389/fphar.2022.774440

Figure Lengend Snippet: PG downregulates VEGF-A by p-STAT3 signal. (A) Protein levels of STAT3 and P-STAT3 in WT and PG-treated groups. (B) Protein expressions of STAT3 and P-STAT3 after treated by PG were tested by Western Blot analysis. Bar chart shows quantification of protein levels compared to GAPDH control in each condition. (C) The transcriptional level of the key downstream factors of STAT3: IL-10, IL-6, and VEGF-A in tissues of control and PG-treated groups. (D) Apoptosis-inducing effect of PD on LLC cells. Annexin V/propidium iodide (PI) staining using flow cytometry was performed after LLC cells were treated with various concentrations of PG for 48 h. Taxol at concentration of 250 nM was used as positive control and DMSO (0.05%) without PG was used as negative control. Apoptosis and necrosis were quantified in the right histogram as the percentage of apoptosis. Quantitative analysis of the results of flow cytometry analysis presented the mean ± standard deviation of triplicate. (E) Cell cycle distribution was assayed with PI staining by flow cytometry after 24 h of treatment with or without PG. Percentage of cells in G1, S, or G2 phases of the cell cycle presented in the right bar chart. Data are presented as the mean ± standard deviation of triplicates. (F) Effect of different concentrations of PG on the proliferation in LLC cells was measured by a colony formation assay. (G) The expression of apoptosis-related proteins, cell cycle‐related proteins and cell cycle‐related proteins in LLC cells treated with or without PG and Taxol was estimated by western blot analysis after 48 h (From left to right). All quantitative data are presented as the mean ± standard deviation of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The untouched and highly purified mouse CD8 + T cells were isolated from single-cell suspensions of splenocytes by immunomagnetic negative selection using the EasySep Mouse CD8 + T-Cell Isolation Kit (19853, STEMCELL), according to the manufacturer’s instructions, and the CD8 + T cells purity (>90%) was identified by flow cytometry Anti-mouse CD3e APC (05122-80-25, Biogems, PeproTech) and anti-mouse CD8a PE (100707, Biolegend).

Techniques: Western Blot, Control, Staining, Flow Cytometry, Concentration Assay, Positive Control, Negative Control, Standard Deviation, Colony Assay, Expressing