Journal: Journal for Immunotherapy of Cancer

Article Title: LAL deficiency induced myeloid-derived suppressor cells as targets and biomarkers for lung cancer

doi: 10.1136/jitc-2022-006272

Figure Lengend Snippet: Metabolic reprogramming in Lal -/- Ly6G + cells. (A) The tSNE plot of genes involved in glycolysis in Lal -/- versus Lal +/+ Ly6G + cells. (B) The tSNE plot of genes involved in the citrate cycle in Lal -/- versus Lal +/+ Ly6G + cells. (C) The tSNE plot of genes responding to ROS in Lal -/- versus Lal +/+ Ly6G + cells. In (A), (B) and (C), green dots represent cluster 123, red dots represent cluster 0468, and blue dots represent other clusters. (D) Expressions of metabolic enzymes in Lal -/- versus Lal +/+ Ly6G + cells by western blot analysis. (E) Levels of glucose, pyruvate, and α-ketoglutarate in Lal -/- versus Lal +/+ Ly6G + cells. Data are expressed as mean±SD; experiments were independently repeated, n=4. *p<0.05, **p<0.01. G6PD, glucose-6-phosphate dehydrogenase; GLUD, glutamate dehydrogenase; HK, hexokinase; KO, knockout; LDHA, lactate dehydrogenase A; LDHB, lactate dehydrogenase B; MDSC, myeloid-derived suppressor cell; ROS, reactive oxygen species; tSNE, T-stochastic neighbor embedding; WT, wild type.

Article Snippet: Western blot analysis was performed as previously described using antibodies against hexokinase 1 (HK1), HK2, HK3, glucose-6-phosphate dehydrogenase (G6PD), PDH, lactate dehydrogenase A (LDHA), LDHB, and glutamate dehydrogenase (GLUD) (rabbit monoclonal antibodies, 1:1000; Cell Signaling).

Techniques: Western Blot, Knock-Out, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: LAL deficiency induced myeloid-derived suppressor cells as targets and biomarkers for lung cancer

doi: 10.1136/jitc-2022-006272

Figure Lengend Snippet: Expressions of metabolic enzymes in NSCLC versus healthy subjects. (A) A representative gating strategy of PDH + , G6PD + , LDH + , and GLUD + cells in the whole blood cells. (B) MFI and percentages of PDH + , G6PD + , LDH + , and GLUD + cells in the leucocytes of patients with NSCLC versus healthy individuals. (C) A representative gating strategy of PDH + , G6PD + , LDH + , and GLUD + cells in blood CD11b + HLA-DR - cells. (D) MFI and percentages of PDH + , G6PD + , LDH + , and GLUD + cells in blood CD11b + HLA-DR - cells of patients with NSCLC versus healthy individuals. (E) MFI and percentages of PDH + , G6PD + , LDH + , and GLUD + cells in blood CD11b + CD13 + HLA-DR - cells of patients with NSCLC versus healthy individuals. Data are expressed as mean±SD; experiments were independently repeated, n=9–13. *p<0.05, **p<0.01. G6PD, glucose-6-phosphate dehydrogenase; GLUD, glutamate dehydrogenase; LDH, lactate dehydrogenase; NSCLC, non-small cell lung cancer; PDH, pyruvate dehydrogenase.

Article Snippet: Western blot analysis was performed as previously described using antibodies against hexokinase 1 (HK1), HK2, HK3, glucose-6-phosphate dehydrogenase (G6PD), PDH, lactate dehydrogenase A (LDHA), LDHB, and glutamate dehydrogenase (GLUD) (rabbit monoclonal antibodies, 1:1000; Cell Signaling).

Techniques:

Journal: Journal for Immunotherapy of Cancer

Article Title: LAL deficiency induced myeloid-derived suppressor cells as targets and biomarkers for lung cancer

doi: 10.1136/jitc-2022-006272

Figure Lengend Snippet: Checkpoint inhibitor treatment. (A) Statistical analysis of percentages of PD-L1 + cells in leucocytes of patients with NSCLC before versus after treatment. (B) Statistical analysis of percentages of CD11b + HLA-DR - , CD13 + , CD14 + , CD15 + , and CD33 + cells in the leucocytes of patients with NSCLC before versus after treatment. (C) Statistical analysis of percentages of CD13 + , CD14 + , CD15 + , and CD33 + cells in CD11b + HLA-DR - cells of patients with NSCLC before versus after treatment. (D) MFI of PDH + , G6PD + , LDH + , and GLUD + cells in the leucocytes of patients with NSCLC before versus after treatment. (E) MFI of PDH + , G6PD + , LDH + , and GLUD + cells in blood CD11b + HLA-DR - cells of patients with NSCLC before versus after treatment. (F) MFI of PDH + , G6PD + , LDH + , and GLUD + cells in blood CD11b + CD13 + HLA-DR - cells of patients with NSCLC before versus after treatment. Data are expressed as mean±SD; experiments were independently repeated, n=5–9. *p<0.05, **p<0.01. G6PD, glucose-6-phosphate dehydrogenase; GLUD, glutamate dehydrogenase; LDH, lactate dehydrogenase; NSCLC, non-small cell lung cancer; PD-L1, programmed death ligand-1; PDH, pyruvate dehydrogenase.

Article Snippet: Western blot analysis was performed as previously described using antibodies against hexokinase 1 (HK1), HK2, HK3, glucose-6-phosphate dehydrogenase (G6PD), PDH, lactate dehydrogenase A (LDHA), LDHB, and glutamate dehydrogenase (GLUD) (rabbit monoclonal antibodies, 1:1000; Cell Signaling).

Techniques:

Journal: Redox Biology

Article Title: Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

doi: 10.1016/j.redox.2023.102646

Figure Lengend Snippet: Dampened expression of glucose-6-phosphate dehydrogenase (G6PD) impairs spontaneous recovery of glutathione oxidation and NADPH following ISOPOOH exposure of HAEC. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of G6PD KD HAEC expressing Grx1-roGFP2 (A) or iNAP1 (B). G6PD KD HAEC were pre incubated in glucose deficient medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 5–6 independent experiments where the responses from 10 individual cells were averaged for each experiment.

Article Snippet: Electrophoresed proteins were electrotransferred onto nitrocellulose, and the blots were blocked with 5% BSA in 1X tris-buffered saline containing 0.1% tween-20 (TBST) at 4 °C for 1 h. Blots were incubated overnight at 4 °C with an anti-G6PD (Cell Signaling Technology; Danvers, MA, USA; cat# D5D2) monoclonal antibody (1:1000) in TBST containing 5% BSA.

Techniques: Expressing, Confocal Microscopy, Incubation, Fluorescence

Journal: Redox Biology

Article Title: Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

doi: 10.1016/j.redox.2023.102646

Figure Lengend Snippet: Linear regression analysis of ISOPOOH-induced and glucose-mediated changes in Grx1-roGFP2 and iNAP1 signal in glucose deprived G6PD KD HAEC as shown in Fig. 5 A and B, respectively.

Article Snippet: Electrophoresed proteins were electrotransferred onto nitrocellulose, and the blots were blocked with 5% BSA in 1X tris-buffered saline containing 0.1% tween-20 (TBST) at 4 °C for 1 h. Blots were incubated overnight at 4 °C with an anti-G6PD (Cell Signaling Technology; Danvers, MA, USA; cat# D5D2) monoclonal antibody (1:1000) in TBST containing 5% BSA.

Techniques:

Journal: Redox Biology

Article Title: Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

doi: 10.1016/j.redox.2023.102646

Figure Lengend Snippet: A knockout of glucose-6-phosphate dehydrogenase (G6PD) in a BALB/c cell model impairs glucose-mediated recovery of glutathione oxidation following ISOPOOH exposure. The ratio of oxidized glutathione to reduced glutathione (GSSG:GSH) and NADPH flux was monitored using live cell confocal microscopy of G6PD knockout (A, C) or wild-type BALB/c cells (B, D), respectively. BALB/c cells expressing Grx1-roGFP2 (A, B) or iNAP1 (C, D) were pre incubated in glucose deficient medium for 2 h prior to exposure to 1–9 μM ISOPOOH. Following the acquisition of a baseline signal, vehicle, 1–9 μM ISOPOOH, and 1 mM glucose were added at the indicated times. Emitted fluorescence intensity values are shown normalized to their respective baselines and expressed as the ratio of signal induced by 405/488 nm excitation. Data are expressed as MEAN ± SEM for n = 3–5 independent experiments where the responses from 10 individual cells were averaged for each experiment.

Article Snippet: Electrophoresed proteins were electrotransferred onto nitrocellulose, and the blots were blocked with 5% BSA in 1X tris-buffered saline containing 0.1% tween-20 (TBST) at 4 °C for 1 h. Blots were incubated overnight at 4 °C with an anti-G6PD (Cell Signaling Technology; Danvers, MA, USA; cat# D5D2) monoclonal antibody (1:1000) in TBST containing 5% BSA.

Techniques: Knock-Out, Confocal Microscopy, Expressing, Incubation, Fluorescence

Journal: Redox Biology

Article Title: Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

doi: 10.1016/j.redox.2023.102646

Figure Lengend Snippet: Linear regression analysis of ISOPOOH-induced and glucose-mediated changes in Grx1-roGFP2 signal in glucose deprived G6PD KO BALB/c and WT BALB/c cells as shown in Fig. 6 A and B, respectively.

Article Snippet: Electrophoresed proteins were electrotransferred onto nitrocellulose, and the blots were blocked with 5% BSA in 1X tris-buffered saline containing 0.1% tween-20 (TBST) at 4 °C for 1 h. Blots were incubated overnight at 4 °C with an anti-G6PD (Cell Signaling Technology; Danvers, MA, USA; cat# D5D2) monoclonal antibody (1:1000) in TBST containing 5% BSA.

Techniques:

Journal: Redox Biology

Article Title: Real-time redox adaptations in human airway epithelial cells exposed to isoprene hydroxy hydroperoxide

doi: 10.1016/j.redox.2023.102646

Figure Lengend Snippet: Linear regression analysis of ISOPOOH-induced and glucose-mediated changes in iNAP1 signal in glucose deprived G6PD KO BALB/c and WT BALB/c cells as shown in Fig. 6 C and D, respectively.

Article Snippet: Electrophoresed proteins were electrotransferred onto nitrocellulose, and the blots were blocked with 5% BSA in 1X tris-buffered saline containing 0.1% tween-20 (TBST) at 4 °C for 1 h. Blots were incubated overnight at 4 °C with an anti-G6PD (Cell Signaling Technology; Danvers, MA, USA; cat# D5D2) monoclonal antibody (1:1000) in TBST containing 5% BSA.

Techniques:

Journal: Journal of Food and Drug Analysis

Article Title: A novel NRF2/ARE inhibitor gossypol induces cytotoxicity and sensitizes chemotherapy responses in chemo-refractory cancer cells

doi: 10.38212/2224-6614.3376

Figure Lengend Snippet: Antibody list

Article Snippet: G6PD , Immunoblot , rabbit , 8866S (Cell Signaling Technology).

Techniques: Western Blot

Journal: Journal of Food and Drug Analysis

Article Title: A novel NRF2/ARE inhibitor gossypol induces cytotoxicity and sensitizes chemotherapy responses in chemo-refractory cancer cells

doi: 10.38212/2224-6614.3376

Figure Lengend Snippet: Gossypol enhances cisplatin sensitivity through inhibition of NRF2/G6PD axis in cisplatin resistant HNSCC cells. (A) The box plot of G6PD expression in different cancer cell lines. The expression levels of G6PD in cisplatin-resistant (left plot) and cisplatin-sensitive cancer cells (right plot) were retrieved from the Oncomine database. (B) Dose-dependent effect of gossypol on protein levels of NRF2, G6PD and NQO1. After treatment with 0.625, 1.25, 2.5, and 5 μM of gossypol for 24 h, the expression levels of NRF2, G6PD and NQO1 proteins were detected by Western blot analysis. β-Actin was used as the internal control. (C) The IC 50 values of cisplatin in parental HONE-1 cell and the two resistant sub-lines, cis6 and cis15. The Resistance Index was calculated by dividing the IC 50 value of cisplatin in resistant sub-line by the IC 50 value of cisplatin in parental HONE-1 cell. (D) Total and phospho-NRF2 levels in parental and cisplatin-resistant HONE-1 cells. β-Actin was used as the internal control. (E ~ G) Cytotoxicity effects of gossypol combined with cisplatin in HONE-1-derieved cells. The cells were co-treated with non-toxic concentration of gossypol (0.5 μM) with cisplatin for 72 h, and cell viability was determined by methylene blue assays (E: parental HONE-1 cells; F: cisplatin-resistant cis6 cells; G: cisplatin-resistant cis15 cells).

Article Snippet: G6PD , Immunoblot , rabbit , 8866S (Cell Signaling Technology).

Techniques: Inhibition, Expressing, Western Blot, Concentration Assay

Journal: Journal of Food and Drug Analysis

Article Title: A novel NRF2/ARE inhibitor gossypol induces cytotoxicity and sensitizes chemotherapy responses in chemo-refractory cancer cells

doi: 10.38212/2224-6614.3376

Figure Lengend Snippet: Antibody list

Article Snippet: G6PD , Immunoblot , rabbit , 8866S (Cell Signaling Technology).

Techniques: Western Blot