Journal: Science Advances

Article Title: PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells

doi: 10.1126/sciadv.ade8222

Figure Lengend Snippet: ( A ) Representative LC3 flow cytometry analysis histograms and quantification of median fluorescent intensity (MFI) of LC3 in WT, δKO littermate control, and TKO HSCs and MPROG after serum and cytokine starvation ( N WT = 3, N δKO = 5, and N TKO = 3). ( B ) Quantification of P62 degradation in serum- and cytokine-starved WT and TKO HSCs with and without chloroquine (CQ) treatment, calculated as the ratio of P62 MFI treated with CQ/P62 MFI without CQ treatment ( N WT = 9 and N TKO = 9). ( C ) Representative confocal images of sorted LT-HSCs stained with 4′,6-diamidino-2-phenylindole (DAPI; blue), anti-LC3 antibody (green), and anti-LAMP1 antibody (red). Colocalization of LC3 with LAMP1 appears yellow (see arrows). ( D ) Quantification of LC3 + events per cell and ( E ) colocalization events of LC3 with LAMP1 assessed by Pearson’s correlation ( N = 20 cells per genotype from three independent samples). ( F ) Representative EM images of autophagic vesicles in sorted WT and TKO HSCs ( N = 20 cells per genotype). ( G ) Quantification of the average area of autophagic vesicles ( N WT = 117 and N TKO = 141) in WT versus TKO HSCs and of the average ratio of autophagic vesicles to cytoplasm per cell in WT and TKO HSCs ( N WT = 23 and N TKO = 25). (A to H) Immunophenotypic populations were defined as follows: HSCs, Lin − cKit + Sca1 + Flk2 − CD48 − ; LT-HSC, Lin − Sca1 + cKit + Flk2 − CD48 − CD150 + ; MPROG, Lin − cKit + Sca1 − ; and LSK, Lin − cKit + Sca1 + . Significance was determined using one-way ANOVA with Tukey’s multiple comparisons test (A) or t test (B, D, and F) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Representative data from individual experiments are shown. Each experiment was performed at least three times.

Article Snippet: For autophagy assessment, an intracellular flow cytometry protocol was adapted and modified from the FlowCellect Autophagy LC3 Antibody-based Assay Kit (Millipore, FCCH100171).

Techniques: Flow Cytometry, Control, Staining

Journal: Science Advances

Article Title: PI3-kinase deletion promotes myelodysplasia by dysregulating autophagy in hematopoietic stem cells

doi: 10.1126/sciadv.ade8222

Figure Lengend Snippet: ( A ) Representative patient sample flow cytometry plots gated on Lin − normal BM (NBM) and MDS BM. ( B and C ) Representative flow cytometry histograms and quantification of the MFI of (B) LC3 and (C) P62 ( N NBM = 3 and N MDS = 6) in patient samples gated on the CD45Ra − CD123 − HSC population. (A to C) The experiment was performed three times with a total of NBM equal to 9 samples and 11 different MDS BM samples. ( D ) Representative electron microscopy images of autophagic vesicles from sorted CD34 + cells of NBM and MDS patient samples. ( E and F ) Quantification of the (E) area of autophagic vesicles ( N NBM = 92 and N MDS = 143) and (F) ratio of total area of autophagic vesicles to area of the cytoplasm per cell in NBM versus MDS CD34 + cells ( N NBM = 15 and N MDS = 18). (D to F) Analyzed images are from three different BM controls and three different patients with MDS. Significance was determined using Welsh’s t test * P ≤ 0.05 and *** P ≤ 0.001. ( G ) Current model: Under low cytokine and serum conditions, a functional PI3K/AKT pathway maintains autophagic degradation and supports functional HSCs. Inactivation of PI3K compromises autophagic degradation, leading to decreased HSC differentiation and MDS initiation. However, pharmacologic autophagy induction can bypass compromised PI3K/AKT activity, improving autophagic degradation and restoring HSC differentiation (image created with Biorender.com ).

Article Snippet: For autophagy assessment, an intracellular flow cytometry protocol was adapted and modified from the FlowCellect Autophagy LC3 Antibody-based Assay Kit (Millipore, FCCH100171).

Techniques: Flow Cytometry, Electron Microscopy, Functional Assay, Activity Assay

Journal: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: Effect of IngC on apoptosis pathway on glioma cell lines. ( A,B ) Panel of 35 proteins related to apoptosis. The data represented by the heat maps show the proteins modulated after 24 and 72 h of IngC treatment (3X IC 50 value (13.09 μM) in glioma cells, GAMG and U373. ( C ) Cells were treated with 3X IC 50 concentrations of IngC (24 and 72 h) for the indicated time periods. GAMG and U373 cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-DR5. The tubulin was used as an internal control to normalize the amount of proteins applied in each lane. This data is representative of three independent experiments. ( D ) After the IngC treatment with 3X IC 50 for 72 h, U373 cells were fixed, stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ( E ) After IngC treatment with 3X IC 50 for 72 h, DNA fragmentation in U373 cell line was measured with the TUNEL assay using flow cytometry. The graphs are representative of at least three independent experiments performed in duplicate. n.s. means non-significant. ** p < 0.005

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Western Blot, Staining, TUNEL Assay, Flow Cytometry

Journal: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: IngC promotes autophagy on glioma cells. ( A ) Cells were treated with the IC 50 value of IngC for the indicated time periods. GAMG cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-LC3. ( A , B ) are representative of three independent experiments with GAMG. Tubulin was used as an internal control to normalize the amount of proteins applied in the treatment without bafilomycin A1 (Baf). ( C ) Development of AVO in IngC-treated cells by detecting green and red fluorescence in acridine orange-stained cells using FACS analysis. U373 cells were treated with IngC (IC 50 value), and Baf (20 nM) for 72 h. The graphs are representative of at least two independent experiments. FITC indicates green color intensity, while PerCP shows red color intensity. ( D ) Effect of baf on GAMG and U373 cell viability of IngC-treated cells. At 3 h after exposure to IngC, baf was added and cultured until 72 h and evaluated by MTS assay. The viability of the untreated cells was considered as 100%. Results shown are the means ± S.D. of three independent experiments. ( E ) Effect of Baf on IngC-induced apoptosis. After, IngC and bafilomicyn treatment for 72 h, GAMG cells were stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ** p < 0.005 and *** p < 0.0001

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Western Blot, Fluorescence, Staining, Cell Culture, MTS Assay

Journal: Frontiers in Immunology

Article Title: Shifting gears: Study of immune system parameters of male habitual marathon runners

doi: 10.3389/fimmu.2022.1009065

Figure Lengend Snippet: The function of Tregs was validated by suppression assays using isolated CD3+CD4+CD25+ Tregs and CD3+CD4+CD25- effector Th cells (Teffs) from PBMCs of marathon runners before and after a race and from control subjects. The suppressive activity of Tregs was assessed by measuring their ability to inhibit the activation and proliferation of activated Teffs (% suppression). Teffs were incubated with CFSE and cultured in the presence of PHA and Tregs at the following ratios of Tregs to Teffs: 0:1, 1:1, 1:2, 1:4, 1:8. Data are expressed as mean (SD). *p<0.05.

Article Snippet: For this purpose, Teffs were incubated with CFSE (CellTraceTM CFSE Cell Proliferation Kit for flow cytometry, ThermoFisher Scientific) according to the manufacturer’s instructions and cultured in the presence of 10μg/ml phytohemagglutinin (PHA, from Sigma Aldrich) and Tregs (10,000 cells per experimental point) at the following Treg:Teff ratios: 0:1, 1:1, 1:2, 1:4, 1:8, for 72h.

Techniques: Isolation, Control, Activity Assay, Activation Assay, Incubation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Enrichment of T-cell proliferation and memory gene signatures of CD79A/CD40 costimulatory domain potentiates CD19CAR-T cell functions

doi: 10.3389/fimmu.2022.1064339

Figure Lengend Snippet: CD19CAR-T cell structure and CD19CAR-T cell response upon CD19 + target cell stimulation in an in vitro assay. (A) Schematic of CD19CAR-T cell structures. Each costimulatory domain either CD28 (CD28z), 4-1BB (BBz), or CD79A/CD40 (CD79A.40z) was fused into anti-CD19 scFv-H-CD28TM followed by CD3ζ and tEGFR. scFv, single chain variable fragment; VL, light-chain variable fragment; VH, heavy chain variable fragment; H, short 12 amino acid of IgG4 Fc-derived spacer of hinge; TM, transmembrane domain; tEGFR, truncated EGFR. (B) Experimental schematic of CD19CAR-T cell generation and functional analysis. EBV-LCL, EBV-transformed lymphoblastoid cell line. (C, D) T-cell proliferation assay. Untransduced or CD19CAR-T cells were stimulated with γ-irradiated CD19-K562 cell line in 1:1 ratio for 10 days and cultured without (C) IL-2 supplementation or with (D) IL-2 supplementation (50 IU/ml). T-cell proliferation were measured by counting viable cells. Arrows mark the day of CD19-K562 cell stimulation. (E) Prolonged co-culture assay. Untransduced or CD19CAR-T cells were co-cultured with Nalm6-FFluc at E:T ratios of 1:1 (left), 1:8 (middle), and 1:16 (right) for a total of nine days without exogenous IL-2. The remaining Nalm6-FF were assessed by flow cytometry at the indicated time points. All data were pooled from three different donors and are presented as mean ± SEM. 2-way ANOVA for (C, D) ; *p < 0.05, **p < 0.01 (CD19.79A.40zCAR- vs. CD19.28z CAR-T cells); One-way ANOVA for (E) .

Article Snippet: CD3 + cells were then cultured in RPMI-1640 medium containing 10% human serum, 0.8mM L-glutamine, 1% penicillin/streptomycin, and 0.5 μM 2-mercaptoethanol (cytotoxic T-cell medium; CTL), which was supplemented with 50 IU/ml of recombinant human interleukin-2 (IL-2).

Techniques: Cell Stimulation, In Vitro, Derivative Assay, Functional Assay, Transformation Assay, Proliferation Assay, Irradiation, Cell Culture, Co-culture Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Enrichment of T-cell proliferation and memory gene signatures of CD79A/CD40 costimulatory domain potentiates CD19CAR-T cell functions

doi: 10.3389/fimmu.2022.1064339

Figure Lengend Snippet: CAR-T cell immunophenotype assays. (A) Experimental schematic of antigen stimulation and CD19CAR-T cell phenotype assays. CD19CAR-T cells were stimulated with γ-irradiated CD19-K562 cell line in a 1:1 ratio for seven days and cultured without IL-2 supplementation. Then, the CD19CAR-T cell proliferation, T-cell differentiation, and exhaustion phenotypes were assessed before and after stimulation on day 0 and day 7, respectively. The remaining CD19CAR-T cells 3–6 x 10 6 cells were harvested at post stimulation on day 7 and extracted for RNA for further RNA sequencing. (B) The percentages of T-cell differentiation subsets were determined by CD62L + CD45RA + naïve T (T N ), CD62L + CD45RA - central memory T (T CM ), CD62L - CD45RA - effector memory T (T EM ), and CD62L - CD45RA + effector memory re-expressing CD45RA T (T EMRA ) cells before and after stimulation. (C) The percentage of T-cell exhaustion was determined by exhausted T-cell markers PD-1 + , TIM-3 + , LAG-3 + , and CTLA-4 + before and after stimulation. Data were pooled from three different donors and are shown as mean ± SEM; One-way ANOVA for (B, C) ; *p < 0.05.

Article Snippet: CD3 + cells were then cultured in RPMI-1640 medium containing 10% human serum, 0.8mM L-glutamine, 1% penicillin/streptomycin, and 0.5 μM 2-mercaptoethanol (cytotoxic T-cell medium; CTL), which was supplemented with 50 IU/ml of recombinant human interleukin-2 (IL-2).

Techniques: Irradiation, Cell Culture, Cell Differentiation, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma

doi: 10.1101/2021.02.23.432584

Figure Lengend Snippet: (A) ELISpot for IFN γ release by CD8 + T cells using parental YUMM1.7 and CCN4-KO YUMM1.7 (KO1) cells as targets and different amount of in vivo activated CD8 + T cells. (B) ELISpot for IFN γ release by in vivo activated CD8 + T cells with CCN4-inducible cells as targets in the presence or absence of 0.5 mg/ml doxycycline. (C) CD8+ T cells isolated from the spleens of C57BL/6 mice that rejected YUMM1.7 tumors were assayed by in vitro ELISpot using variants of the YUMM1.7 cell line as targets (WT YUMM1.7 (Ym1.7) -yellow, CCN4-KO YUMM1.7 (Ym1.7-KO1)-light green, CCN4-KO YUMM1.7 with a blank inducible expression vector (Ym1.7-KO1-IDvector) -dark green and blue, CCN4-KO YUMM1.7 with a CCN4 inducible expression vector (Ym1.7-KO1-IDmCCN4) -purple and red). Variants containing the inducible expression vector were also cultured in the absence (dark green and purple) or presence of 0.5 µ g/ml doxycycline (blue and red). CD8+ T cells expressing IFN γ and TNF α were quantified following 24 hour coculture (bar graph). Results shown as mean ± S.D. for three biological replicates.

Article Snippet: 50 μ l (5 ×10 4 ) of the YUMM1.7-reactive CD8+ T cells were aliquoted into 96-well plates for ELISpot assay using Mouse IFN γ /TNF α Double-Color ELISpot kit (Cellular Technology Limited) following manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, In Vivo, Isolation, In Vitro, Expressing, Plasmid Preparation, Cell Culture

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma

doi: 10.1101/2021.02.23.432584

Figure Lengend Snippet: (A) Average tumor volumes of mice bearing YUMM1.7-WT (squares and triangles) or CCN4-KO (KO1, circles and inverted triangles) tumors (n = 4/group). Groups were treated with either α PD1 (triangles and inverted triangles) or isotype control (squares and circles) antibodies when the tumors reached 100 mm 3 . (B) Expression of H-2K b (top panel) and PD-L1 (bottom panel) were assayed by flow cytometry in WT (red curves) and CCN4-KO (KO1 blue curves) YUMM1.7 cells with (dotted curves) and without (solid curves) preconditioning with IFN γ . Unstained cells were used as a negative control (shaded curve). (C) CD45 − fraction isolated from WT and CCN4-KO YUMM1.7 tumors in a time-matched experiment were assayed for H-2K b and PD-L1 expression by flow cytometry. Contour curves enclose 90% (dotted curve) and 50% (solid curves) of CD45 − events obtained from WT (red) and CCN4-KO (blue) YUMM1.7 tumors. CD8+ T cells expressing PD1 within the tumor (D) and spleen (E) were assayed by flow cytometry in mice bearing WT and CCN4-KO YUMM1.7 tumors (F). Results representative of at least three biological replicates.

Article Snippet: 50 μ l (5 ×10 4 ) of the YUMM1.7-reactive CD8+ T cells were aliquoted into 96-well plates for ELISpot assay using Mouse IFN γ /TNF α Double-Color ELISpot kit (Cellular Technology Limited) following manufacturer’s instructions.

Techniques: Control, Expressing, Flow Cytometry, Negative Control, Isolation