Journal: British Journal of Cancer

Article Title: Adenovirus-mediated interferon α gene transfer induces regional direct cytotoxicity and possible systemic immunity against pancreatic cancer

doi: 10.1038/sj.bjc.6602713

Figure Lengend Snippet: Expression of NKG2D ligands on pancreatic cancer cell lines. ( A ) RT–PCR analyses of NKG2D ligands. Total RNA was extracted from cell lines and after reverse transcription the mRNA of MICA, MICB, ULBP-1, ULBP-2 and ULBP-3 was detected by PCR. ( B ) Flow cytometric analysis of MICA and MICB. Cells were incubated with anti-human MICA/B monoclonal antibody (filled curve) or isotype control antibody (open curve), and flow cytometry was carried out using a FACScan system.

Article Snippet: Cells (5 × 10 5 ) were incubated at 4°C for 30 min with phycoerythrin-conjugated mouse anti-human MICA/B monoclonal antibody (6D4; IgG, BD Biosciences Pharmingen, San Jose, CA, USA) or isotype control antibody (IgG), and then washed twice with PBS containing 2% BSA and 0.05% NaN 3 and fixed with 2% paraformaldehyde solution.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Flow Cytometry

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: High expression of NKG2D ligands contributes to longer survival in AML patients. (A) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B on primary AML cell surface from patients was detected by flow cytometry (BD FACSCalibur). Mouse IgG-APC was used as isotype control. (B) The MFI (Median Fluorescence Intensity) value was quantified from (A) . (C–G) Kaplan-Meier analysis of overall survival of AML patients stratified by the expression levels of PAPR1. Numbers of patients, log-rank p-value and Hazard Ratio (HR) value were shown.

Article Snippet: Primary antibodies against human NKG2D ligands (MICA/B, ULBP1, ULBP2/5/6, and ULBP3) using in flow cytometry were purchased from R&D System, with Cat. No. as follows: FAB13001A, FAB1380A, FAB1298A, and FAB1517A, respectively; mouse anti-NKG2D antibody (Cat#: MAB139) used in functional blockade for NKG2D receptor and isotype control Goat anti-mouse IgG2A-APC (Cat#: IC003A) were obtained from R&D System.

Techniques: Expressing, Flow Cytometry, Control, Fluorescence

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: LSCs is out of response to the cytotoxicity of NK cells. (A) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B on the cell surface of KG-1α, Kasumi-1, HL60, and LSCs (enriched from KG-1α) were detected by flow cytometry (BD FACSCalibur). Mouse IgG-APC was used as isotype control. (B) LSCs, KG-1α and HL60 were co-cultured with NK92 MI cells pretreated with IgG or anti-NKG2D antibody at E:T ratio of 2.5:1, 5:1, and 10:1, respectively for 4 h The cytotoxic activity of NK92 MI cells were measured by Non-Radioactive Cytotoxicity kit according to the released LDH from target cells. Results were representative of three different experiments. (C) Quantification of the cytotoxicity of NK92 cells from (B) at E:T ratio of 10:1.

Article Snippet: Primary antibodies against human NKG2D ligands (MICA/B, ULBP1, ULBP2/5/6, and ULBP3) using in flow cytometry were purchased from R&D System, with Cat. No. as follows: FAB13001A, FAB1380A, FAB1298A, and FAB1517A, respectively; mouse anti-NKG2D antibody (Cat#: MAB139) used in functional blockade for NKG2D receptor and isotype control Goat anti-mouse IgG2A-APC (Cat#: IC003A) were obtained from R&D System.

Techniques: Expressing, Flow Cytometry, Control, Cell Culture, Activity Assay

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: PARP1 is negatively correlated with NKG2D ligands expression in AML patients. (A) LSCs were treated with the indicated compounds for 24 hours, and then cells were collected to extract total RNA. The mRNA transcriptional level of ULBP1, ULBP2, ULBP3, MICA and MICB were detected by quantitative real-time RT-PCR. Results were representative of three different experiments. (B) Primary LSCs isolated from patient 3, 5, 8 and 11 were treated the PARPi at a concentration of 1 μM for 24 hours, and then the quantitative real-time RT-PCR was performed as (A) to measure the transcriptional level of ULBP1, ULBP2, ULBP3, MICA and MICB. Results were representative of three different experiments. (C) LSCs were treated with PARPi at a concentration of 1 μM for 24 hours, and then co-cultured with primary NK cells pretreated with IgG or anti-NKG2D antibody at E:T ratio of 2.5:1, 5:1, and 10:1, respectively for 4 hours. The cytotoxic activity of primary NK cells was measured by Non-Radioactive Cytotoxicity kit according to the released LDH from target cells. Results were representative of three different experiments. (D) The correlation between PARP1 and NKG2D ligands (ULBP1, ULBP2, ULBP3, MICA and MICB) in NKG2D ligands-negative (NKG2DLneg) AML patients from microarray data set GSE127200 were analyzed by R cor.test. The Pearson Correlation Coefficient also known as R value was shown. (E) The mRNA transcriptional level of ULBP1, ULBP2, ULBP3, MICA and MICB in AML patients stratified by the expression levels of PAPR1 (Low (n=4), Medium (n=5), and High (n=10)) were detected by quantitative real-time RT-PCR. Data was shown as box plot. The statistical significance was determined by one-way ANOVA analysis (* p < 0.05, ** 0.001 < p < 0.01, *** p < 0.001). All experiments were repeated three times. (F) The expression level of PARP1 in PBMCs from healthy donors (n=2) and primary AML cells from AML patients (n=3) were detected by WB. GAPDH was employed as an internal loading control. (G) Quantification of the WB shown in (E) . The Integrated Optical Density of bands was measured by the software ImageJ. Data was presented as mean ± SD from three experiments. (H) Kaplan-Meier analysis of overall survival of AML patients stratified by the expression levels of PARP1. Numbers of patients and log-rank p-value are shown.

Article Snippet: Primary antibodies against human NKG2D ligands (MICA/B, ULBP1, ULBP2/5/6, and ULBP3) using in flow cytometry were purchased from R&D System, with Cat. No. as follows: FAB13001A, FAB1380A, FAB1298A, and FAB1517A, respectively; mouse anti-NKG2D antibody (Cat#: MAB139) used in functional blockade for NKG2D receptor and isotype control Goat anti-mouse IgG2A-APC (Cat#: IC003A) were obtained from R&D System.

Techniques: Expressing, Quantitative RT-PCR, Isolation, Concentration Assay, Cell Culture, Activity Assay, Microarray, Control, Software

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: PARP1 is involved in the regulation of NKG2D ligand expression in LSCs. (A) WB was used to detect the knockdown efficiency of PRAP1 in LSCs. GAPDH was employed as an internal loading control. (B) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B in PARP1-stable knockdown or -scramble control LSCs were detected by flow cytometry (BD FACSCalibur). Mouse IgG-APC was used as isotype control. (C) Formation of γH2AX foci mediated by the pretreatment of 1 μM PARP1 inhibitor (PARPi) for 6 hours in LSCs was detected by immunofluorescence and observed in a fluorescence microscope (Zeiss). DMSO was used as vehicle control. Scale bars, 10 μm. (D) The protein expression level of γH2AX, H2AX, phosphorylated-ATM (Ser1981), ATM, and PARP1 in LSCs pretreated with 1μM PARPi or Vehicle for 6 hours were detected by WB. GAPDH was employed as an internal loading control. (E) Quantification of the WB shown in (D) . The Integrated Optical Density of bands was measured by the software ImageJ. Data was presented as mean ± SD from three experiments. (F) The expression level of ULBP1, ULBP2, ULBP3 and MICA/B on the cell surface of LSCs treated with 1 μM PARPi or co-treated with 1 μM PARPi and 1 μM ATM inhibitor (ATMi) for 6 hours were measured by flow cytometry (BD FACSCalibur). DMSO was used as Vehicle control. Mouse IgG-APC was used as isotype control. ***p < 0.001. ns, no significance.

Article Snippet: Primary antibodies against human NKG2D ligands (MICA/B, ULBP1, ULBP2/5/6, and ULBP3) using in flow cytometry were purchased from R&D System, with Cat. No. as follows: FAB13001A, FAB1380A, FAB1298A, and FAB1517A, respectively; mouse anti-NKG2D antibody (Cat#: MAB139) used in functional blockade for NKG2D receptor and isotype control Goat anti-mouse IgG2A-APC (Cat#: IC003A) were obtained from R&D System.

Techniques: Expressing, Knockdown, Control, Flow Cytometry, Immunofluorescence, Fluorescence, Microscopy, Software

Journal: Frontiers in Oncology

Article Title: cGAS/STING-mediated upregulation of NKG2D ligands in LSCs contributes to enhanced sensitivity to NK cells

doi: 10.3389/fonc.2026.1764427

Figure Lengend Snippet: The cGAS-STING signaling is involved in the regulation of NKG2D ligands in LSCs. (A) WB was used to detect the protein level of cGAS, STING, phosphorylated-STING, TBK1, phosphorylated-TBK1, IRF3, and phosphorylated-IRF3 in LSCs pretreated with 1 μM PARPi or Vehicle for 6 hours. GAPDH was employed as an internal loading control. (B) Quantification of the WB shown in (A) . The Integrated Optical Density of bands was measured by the software ImageJ. Data was presented as mean ± SD from three experiments. (C) Primary LSCs isolated from patient 3, 5, 8 and 11 were treated with 1 μM PARPi as (A) , and then the indicated protein levels were detected by WB. α-Tubulin was employed as an internal loading control. (D) WB was used to detect the knockdown effect of STING and IRF3 (E) in LSCs. α-Tubulin and GAPDH were employed as internal loading control, respectively. (F) The expression level of NKG2D ligands on the cell surface of STING-knockdown or IRF3-knockdown (G) LSCs pretreated with 1 μM PARPi for 6 hours were detected by flow cytometry (BD FACSCalibur). Scramble was used as shRNA control. Mouse IgG-APC was used as isotype control. (H) HEK293T cells were co-transfected with ULBP1-, ULBP3-, MICA- or MICB-promoter reporter plasmid and IRF3/5D or empty vector for 24 hours, Renilla luciferase reporter plasmid pRL-TK was used as an internal control. DLR assay was performed to determine the reporter activity. Data was shown as means ± standard deviations from three independent experiments.

Article Snippet: Primary antibodies against human NKG2D ligands (MICA/B, ULBP1, ULBP2/5/6, and ULBP3) using in flow cytometry were purchased from R&D System, with Cat. No. as follows: FAB13001A, FAB1380A, FAB1298A, and FAB1517A, respectively; mouse anti-NKG2D antibody (Cat#: MAB139) used in functional blockade for NKG2D receptor and isotype control Goat anti-mouse IgG2A-APC (Cat#: IC003A) were obtained from R&D System.

Techniques: Control, Software, Isolation, Knockdown, Expressing, Flow Cytometry, shRNA, Transfection, Plasmid Preparation, Luciferase, Activity Assay