Journal: bioRxiv

Article Title: SMALL MOLECULE CLPP AGONISTS INDUCE SENESCENCE AND ALTER TRAIL-MEDIATED APOPTOTIC RESPONSE OF TRIPLE-NEGATIVE BREAST CANCER CELLS

doi: 10.1101/2022.07.11.499620

Figure Lengend Snippet: A. Immunoblots showing the effect of TR-57 (150 nM) or abemaciclib (500 nM) treatments for 48 hrs on Wee1 levels in SUM159 WT and SUM159 ClpP null cells. B . Measurement of caspase 3 activity in SUM159. Cells were treated with Adavosertib for 24 hrs, STS (100 nM) for 24 hrs, TRAIL (100 ng/ml) for 6 hrs, or Adavosertib for 48 hrs in combination with TRAIL (100 ng/ml) for 6 hrs. C. Proposed model on how ClpP agonists lead to senescence and TRAIL sensitivity. Data shown in this figure is representative of 3 independent experiments.

Article Snippet: TRAIL (Cat. No. HY-P7306) and Adavosertib (Cat. No. HY-10993) were obtained from MedChemExpress.

Techniques: Western Blot, Activity Assay

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 5 IL-18/poly I:C-primed NK cells kill hepatic stellate cells in a TRAIL- involved degranulation manner. IL-18 and poly I:C-primed NK cells were treated by anti-TRAIL or isotype antibody before coculture. HSC death was measured (A), and NK cell degranulation was evaluated by CD107a expression (C). Soluble TRAIL was added to cell coculture, the cell death of HSCs (B) and CD107a expression of NK cells (D) was shown. Results were shown as mean ± SEM of 6 independent experiments performed with 6 different donor NK cells. *P < 0.05, **P < 0.01, ***P < 0.001, paired t-test. The kinetics of the activated NK cell-mediated LX2 cell apoptosis were observed by using live-cell imaging (E).

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Expressing, Live Cell Imaging

Journal: Journal of leukocyte biology

Article Title: Activated NK cells kill hepatic stellate cells via p38/PI3K signaling in a TRAIL-involved degranulation manner.

doi: 10.1002/JLB.2A0118-031RR

Figure Lengend Snippet: FIGURE 6 IL-18/poly I:C-primed hepatic NK cells kill HSCs in a TRAIL-involved degranulation manner. Liver NK cells were purified from speci- men, then primed by IL-18 and/or poly I:C before cocultured with primary HSCs as above. CD107a expression of liver NK cells (A) and PI/AnnexinV level of primary HSCs (B) were detected. Anti-TRAIL or isotype antibody were used to treat IL-18/poly I:C-primed liver NK cells before cocultured with primary HSCs, then CD107a expression of liver NK cells (C) and the cell death of primary HSCs (D) was measured. Results were shown as mean ± SEM of 5 independent experiments performed with 5 different donor NK cells. *P < 0.05, **P < 0.01, paired t-test.

Article Snippet: Soluble TRAIL (R&D System; Catalog number: 375-TL-010) was added to HSCs half hour earlier than NK cells in coculture.

Techniques: Purification, Expressing

Journal: JCI insight

Article Title: Balanced engagement of activating and inhibitory receptors mitigates human NK cell exhaustion.

doi: 10.1172/jci.insight.150079

Figure Lengend Snippet: Figure 6. Engagement of NKG2A during exhaustion rescues defects in cytotoxicity and cytokine production. (A) NK cells were incubated with both positive (anti-NKp46 plus MICA/B) and inhibitory (anti-NKG2A) stimuli or with positive stimuli plus isotype IgG. NK cells plated on isotype-bound cells served as a control for stimulation through the low-affinity Fc receptor CD16. NK cells were incubated with K-562 target cells (E/T 2:1), and live cell killing assays were performed using the IncuCyte platform as previously described. (B) Percentage of live targets was quantified for each group of cells at multiple time points (n = 6). One-way ANOVA with Dunnett’s multiple comparisons was used for statistical analysis (with each time point compared independently). (C and D) Expression of granzyme B (C) (n = 5) and TRAIL (D) (n = 4) was assessed via flow cytometry. One-way ANOVA with multiple comparisons was used for statistical analysis. (E) Irradiated P815 cells were coated with either anti-NKp30 and anti-NKG2A or anti-NKp30 and isotype IgG. P815 coated in isotype alone served as a negative control. NK cells were incubated with P815 target cells (E/T 2:1) for 5 days and subsequently stimulated with PMA/ionomycin for 4 hours. Following stimulation, cytokine production was assessed via flow cytometry (n = 5). One-way ANOVA with multiple comparisons was used for statistical analysis (with IFN-γ and TNF-α compared independently). *P ≤ 0.05; **P < 0.01.

Article Snippet: Fluorochrome-conjugated antibodies were purchased from BioLegend (CD3 [catalog 317330], CD56 [catalog 362510], NKG2D [catalog 320806], CD96 [catalog 338418], granzyme B [catalog 515408], perforin [catalog 308112], IFN-γ [catalog 502538], TNF-α [catalog 502932], CD107a [catalog 328606], TIM-3 [catalog 345018], LAG-3 [catalog 369320], Ki67 [catalog 350515], human CD45 [catalog 304041], mouse CD45 [catalog 103149]); BD Biosciences (anti-CD3 [catalog 562406], CD16 [catalog 560717]); R&D Systems (TIGIT [catalog FAB7898A], TRAIL [catalog FAB687G]); Invitrogen (Fas-L [catalog 12-9919-42]; and Miltenyi Biotec (PD-1 [catalog 130-117-384], isotype IgG2b-PE [catalog 130-092-215], NKG2A [catalog 130-113-563]).

Techniques: Incubation, Control, Expressing, Flow Cytometry, Irradiation, Negative Control