Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: IL‐36 γ promotes and IL‐36Ra inhibits NSCLC progression of the KL mouse model. A) A scheme of tumor induction of Kras LSL‐G12D/+ Lkb1 fl/fl (KL) and Kras LSL‐G12D/+ Lkb1 fl/fl Il1f9 −/‐ (KL9) mice (left). Survival of KL ( n = 21) and KL9 ( n = 22) mice that were intranasally injected with Ad‐Cre (2 × 10 6 pfu) (right). B) Images of HE staining of tumor‐burdened lungs of KL and KL9 mice that were intranasally injected with Ad‐Cre for 8, 10, or 12 weeks, respectively. C) Tumor burden in the lungs of KL and KL9 mice that were injected with Ad‐Cre for 8 weeks ( n = 7 and 10 mice for KL and KL9, respectively), 10 weeks ( n = 9 and 6 mice for KL and KL9, respectively), or 12 weeks ( n = 9 and 9 mice for KL and KL9, respectively). D) Box plot of individual tumor size in the lungs of KL and KL9 mice that were intranasally injected with Ad‐Cre for 8, 10, or 12 weeks, respectively. E) Images (left) and quantification analysis (right) of Ki67 staining in individual lung tumors from KL ( n = 28) and KL9 ( n = 32) mice that were intranasally injected with Ad‐Cre for 10 weeks. F) A scheme of tumor induction of KL and Kras LSL‐G12D/+ Lkb1 fl/fl Il1f5 −/− (KL5) mice (left). Survival of KL ( n = 25) and KL5 ( n = 31) mice that were intranasally injected with Ad‐Cre (2 × 10 6 pfu) (right). G) Images of HE staining of tumor‐burdened lungs of KL and KL5 mice that were intranasally injected with Ad‐Cre for 6, 8, or 10 weeks, respectively. (H) Tumor burden in the lungs of KL and KL5 mice that were intranasally injected with Ad‐Cre for 6 weeks ( n = 5 and 8 mice for KL and KL5, respectively), 8 weeks ( n = 6 and 8 mice for KL and KL5, respectively) or 10 weeks ( n = 20 and 12 mice for KL and KL5, respectively). I) Box plot of individual tumor size in the lungs of KL and KL5 that were intranasally injected with Ad‐Cre for 6, 8, or 10 weeks, respectively. J) Images (left) and quantification analysis (right) of Ki67 staining in individual lung tumors from KL ( n = 16) and KL5 ( n = 19) mice that were intranasally injected with Ad‐Cre for 8 weeks. Graphs show mean ± SEM (C–E, H–J). Two‐tailed student's t ‐test (C–E, H–J) or Log‐Rank analysis (A,F). Scale bars represent 5 mm (B,G) or 50 µm (E,J), respectively. Data are combined results of three independent experiments (A,F) or representative results of two independent experiments (B–E,G–J).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Injection, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: IL‐36 γ promotes and IL‐36Ra inhibits NSCLC progression of the KP mouse model. A) A scheme of tumor induction of Kras LSL‐G12D/+ Tp53 fl/fl (KP) and Kras LSL‐G12D/+ Tp53 fl/fl Il1f9 −/‐ (KP9) mice (left). Survival of KP ( n = 21) and KP9 ( n = 20) mice that were intranasally injected with Ad‐Cre (2 × 10 6 pfu) (right). B) Images of HE staining of tumor‐burdened lungs of KP and KP9 mice after intranasal injection with Ad‐Cre for 8, 10, or 12 weeks, respectively. C) Tumor burden in the lungs of KP and KP9 mice that were intranasally injected with Ad‐Cre for 8 weeks ( n = 6 and 5 mice for KP and KP9, respectively), 10 weeks ( n = 9 and 9 mice for KP and KP9, respectively), or 12 weeks ( n = 10 and 9 mice for KP and KP9, respectively). D) Box plot of individual tumor size in the lungs of KP and KP9 mice that were intranasally injected with Ad‐Cre for 8, 10, or 12 weeks, respectively. E) Images (left) and quantification analysis (right) of Ki67 staining in individual lung tumors from KP ( n = 53) and KP9 ( n = 33) mice that were intranasally injected with Ad‐Cre for 10 weeks. F) A scheme of tumor induction of KP and Kras LSL‐G12D/+ Tp53 fl/fl Il1f5 −/− (KP5) mice (left). Survival of KP ( n = 23) and KP5 ( n = 23) mice that were intranasally injected with Ad‐Cre (2 × 10 6 pfu) (right). G) Images of HE staining of tumor‐burdened lungs of KP and KP5 mice after intranasal injection with Ad‐Cre for 6, 8, or 10 weeks, respectively. H) Tumor burden in the lungs of KP and KP5 mice that were intranasally injected with Ad‐Cre for 6 weeks ( n = 6 and 7 mice for KP and KP5, respectively), 8 weeks ( n = 7 and 10 mice for KP and KP5, respectively), or 10 weeks ( n = 13 and 15 mice for KP and KP5, respectively). I) Box plot of individual tumor size in the lungs of KP and KP5 that were intranasally injected with Ad‐Cre for 6, 8, or 10 weeks, respectively. J) Images (left) and quantification analysis (right) of Ki67 staining in individual lung tumors from KP ( n = 27) or KP5 ( n = 30) mice that were intranasally injected with Ad‐Cre for 8 weeks. Graphs show mean ± SEM (C–E,H–J).Two‐tailed student's t ‐test (C–E,H–J) or Log‐Rank analysis (A,F). Scale bars represent 5 mm (B,G) or 50 µm (E,J), respectively. Data are combined results of three independent experiments (A,F) or representative results of two independent experiments (B–E,G–J).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Injection, Staining, Two Tailed Test

Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: IL‐36 γ and IL‐36Ra reciprocally regulate glutathione homeostasis and ROS levels during NSCLC progression. A–C) GSEA plot of the glutathione metabolism pathway (A) and z‐score heatmap of the indicated genes from the transcriptome data of lung tumors from KL ( n = 2) and KL9 ( n = 2) (B) or KL ( n = 2) and KL5 ( n = 2) (C) mice that were intranasally injected with Ad‐Cre for 10 weeks or 8 weeks, respectively. D–E) Quantitative real‐time PCR (qRT‐PCR) analysis of the signature genes for GSH homeostasis in lung tumors isolated from KL ( n = 6) and KL9 ( n = 6) mice that were intranasally injected with Ad‐Cre for 10 weeks (D) or from KL ( n = 6) and KL5 ( n = 6) mice that were injected with Ad‐Cre for 8 weeks (E). F–H) GSEA plot of the glutathione metabolism pathway (F) and z‐score heatmap of the indicated genes from the transcriptome analysis of lung tumors from KP ( n = 2) and KP9 ( n = 3) (G) or KP ( n = 2) and KP5 ( n = 3) (H) mice that were intranasally injected with Ad‐Cre for 10 weeks or 8 weeks, respectively. I–J) qRT‐PCR analysis of the signature genes for GSH metabolism in the lung tumors of KP ( n = 6) and KP9 ( n = 6) mice that were that were intranasally injected with Ad‐Cre for 10 weeks (I) or from KP ( n = 6) and KP5 ( n = 6) mice that were intranasally injected with Ad‐Cre for 8 weeks (J). K) Immunoblot analysis of the signature proteins for GSH homeostasis in lung tumors from KL ( n = 2), KL5 ( n = 2), and KL9 ( n = 2) (left), or KP ( n = 2), KP5 ( n = 2), and KP9 ( n = 2) (right) mice that were intranasally injected with Ad‐Cre for 10 weeks. L–M) GSH levels in the lung tumors from KL ( n = 6), KL5 ( n = 4), and KL9 ( n = 4) mice (L) or KP ( n = 7), KP5 ( n = 7), and KP9 ( n = 4) mice (M) that were were intranasally injected with Ad‐Cre for 10 weeks. M–O) Images (left) and quantification analysis (right) of 8‐oxo‐dGuo staining in the lung tumors from KL ( n = 7), KL5 ( n = 6), and KL9 ( n = 4) mice (N) or KP ( n = 6), KP5 ( n = 5), and KP9 ( n = 5) mice (O) that were intranasally injected with Ad‐Cre for 10 weeks. P) Flow cytometry (left) and quantification analysis (right) of H 2 DCFDA staining of single‐cell suspensions of lung tumors from KL ( n = 7), KL5 ( n = 6), and KL9 ( n = 4) mice that were intranasally injected with Ad‐Cre for 10 weeks. Q) Flow cytometry (left) and quantification analysis (right) of SYTOX Green staining of single‐cell suspensions of lung tumors from KL ( n = 5), KL5 ( n = 5), and KL9 ( n = 7) mice that were intranasally injected with Ad‐Cre for 10 weeks. R) Kaplan–Meier survival curves pf NSCLC patients with IL1F9 high IL1F5 low ( n = 23) and IL1F9 low IL1F5 high low ( n = 23) expression pattern (TCGA AgilentG4502A_07). Graphs show mean ± SEM (D,E,I,J,L,M,P,Q). Two‐tailed student's t ‐test (D,E,I,J,L–Q) or Log‐Rank analysis ( R ). Scale bars represent 50 µm (N,O). Data are representative results of two independent experiments (D,E,L–Q).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Injection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Western Blot, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: IL‐36 γ promotes GSH biogenesis and protects against oxidative stress‐induced cell death. A) qRT‐PCR analysis of Gclm , Gpx2 , Gpx4 , and G6pd in primary mouse lung epithelial cells ( n = 4 technical replicates) that were unstimulated or stimulated with IL‐36 γ (20 ng mL −1 ) in the presence or absence of IL‐36Ra (20 ng mL −1 ) for 6 h. B) qRT‐PCR analysis of Gclm , Gpx2 , Gpx4 , and G6pd in lungs from C57BL/6 mice that were intranasally injected with PBS ( n = 8, 50 µL), IL‐36 γ ( n = 8, 0.5 µg in 50 µL PBS), or IL‐36 γ plus IL‐36Ra ( n = 8, 0.5 µg IL‐36 γ , and 0.5 µg IL‐36Ra in 50 µL PBS) for 24 h. C,D) qRT‐PCR analysis of Gclm , Gpx2 , Gpx4 , and G6pd in A549 cells (C) ( n = 4 technical replicates) or in human lung organoid (D) ( n = 4 technical replicates) that were unstimulated or stimulated with IL‐36 γ (20 ng mL −1 ) in the presence or absence of IL‐36Ra (20 ng mL −1 ) for 6 h. E) Immunoblot analysis of Gclm , Gpx4 , and Gsr in A549 cells (left) or HEK293‐mIL‐36R cells (right) that were unstimulated or stimulated with human or mouse IL‐36 γ (20 ng mL −1 ) in the presence or absence of human or mouse IL‐36Ra (20 ng mL −1 ) for 8 h. F) GSH levels in in A549 cells (left), mouse lung epithelial cells (middle), or human lung organoids (right) ( n = 4 technical replicates) that were unstimulated or stimulated with h/mIL‐36 γ (20 ng mL −1 ) in the presence or absence of h/mIL‐36Ra (20 ng mL −1 ) for 8 h. G) Flow cytometry and quantification analysis of H 2 DCFDA staining in A549 cells that were treated with cisplatin (Cis, 20 µ m ) for 12 h followed by stimulation with IL‐36 γ (20 ng mL −1 ), IL‐36 γ plus IL‐36Ra (20 ng mL −1 ), or IL‐36 γ plus NAC (5 m m ) for 8 h (left), cultured in glucose‐free DMEM (G − ) for 16 h followed by stimulation with IL‐36 γ (20 ng mL −1 ), IL‐36 γ plus IL‐36Ra (20 ng mL −1 ), or IL‐36 γ plus NAC (5 m m ) for 8 h (middle), or stimulated with IL‐36 γ (20 ng mL −1 ), IL‐36 γ plus IL‐36Ra (20 ng mL −1 ), or IL‐36 γ plus NAC (5 m m ) for 7 h followed by H 2 O 2 (16 m m ) treatment for 1 h (right) ( n = 3 technical replicates). H) Images (left) and flow cytometry and quantification analysis of SYTOX Green dead cell (right) of A549 cells treated as in (G) ( n = 3 technical replicates). Graphs show mean ± SEM (A–D,F–H). Two‐tailed student's t ‐test (A–D,F–H). Scale bars represent 100 µm (H). Data are representative results of two independent experiments (A–H).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Quantitative RT-PCR, Injection, Western Blot, Flow Cytometry, Staining, Cell Culture, Two Tailed Test

Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: Inhibition of IL‐36 γ maturation alleviates NSCLC progression. A) Schematic illustration of tumor induction and API treatment (upper) and the survival of mice (lower). KL or KL9 mice that were intranassally injected with Ad‐Cre for 4 weeks followed by intraperitoneal injection of PBS (200 µL, n = 22, and 11 mice for KL and KL9, respectively) or API (100 µg in 200 µL per mouse, n = 21 and 11 mice for KL and KL9, respectively) every other day for 6 weeks. B) Images (left) and quantification analysis (right) of HE staining of tumor‐burdened lungs of KL ( n = 9 and 9 mice for PBS and API, respectively) and KL9 ( n = 6 and 6 mice for PBS and API, respectively) mice treated as in (A). C) Box plot of individual tumor size in the tumor‐burdened lungs of KL ( n = 9 and 9 mice for PBS and API, respectively) and KL9 ( n = 6 and 6 mice for PBS and API, respectively) mice treated as in (A). D) Images (left) and quantification analysis (right) of Ki67 staining in individual lung tumors of KL ( n = 13 and 12 mice for PBS and API, respectively) and KL9 ( n = 15 and 13 mice for PBS and API, respectively) mice treated as in (A). E) qRT‐PCR analysis of the indicated genes or proteins in lung tumors from KL ( n = 6 and 6 mice for PBS and API, respectively) and KL9 ( n = 6 and 6 mice for PBS and API, respectively) treated as in (A). F) Immunoblot analysis of Gclm , Gpx4 , and Gsr in lung tumors from KL ( n = 2 and 2 mice for PBS and API, respectively) and KL9 ( n = 2 and 2 mice for PBS and API, respectively) treated as in (A). G) GSH levels in lung tumors from KL ( n = 5 and 5 mice for PBS and API, respectively) and KL9 ( n = 4 and 4 mice for PBS and API, respectively) mice treated as in (A). H) Images (left) and quantification analysis (right) of 8‐oxo‐dGuo + staining in lung tumors from KL ( n = 9 and 9 mice for PBS and API, respectively) and KL9 ( n = 6 and 6 mice for PBS and API, respectively) mice treated as in (A). I) Flow cytometry (left) and quantification analysis (right) of H 2 DCFDA staining of single‐cell suspensions of lung tumors from KL ( n = 5 and 5 mice for PBS and API, respectively) and KL9 ( n = 6 and 5 mice for PBS and API, respectively) mice treated as in (A). Graphs show mean ± SEM (B–E,G,I). Two‐tailed student's t ‐test (B–E,G,I) or Log‐Rank analysis (A). Scale bars represent 5 mm (B) or 50 µm (D,H), respectively. Data are combined results of three independent experiments (A) or representative results of two independent experiments (B–I).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Inhibition, Injection, Staining, Quantitative RT-PCR, Western Blot, Flow Cytometry, Two Tailed Test

Journal: Advanced Science

Article Title: IL‐36 γ and IL‐36Ra Reciprocally Regulate NSCLC Progression by Modulating GSH Homeostasis and Oxidative Stress‐Induced Cell Death

doi: 10.1002/advs.202101501

Figure Lengend Snippet: Neutralization of IL‐36 γ inhibits NSCLC progression. A) Schematic illustration of tumor induction and α IL‐36 γ treatment. KL/KP mice were intranasally injected with Ad‐Cre for 4 weeks followed by intraperitoneal injection of control IgG or α IL‐36 γ (100 µg in 200 µL PBS per mouse) every other day for 6 weeks. B) Kaplan–Meier survival curves for KL (left, n = 14 and 14 mice for IgG or α IL‐36 γ , respectively) or KP (left, n = 16 and 16 mice for IgG or α IL‐36 γ , respectively) mice treated as in (A). C,D) Images of HE staining (left), tumor area (middle) and tumor size (right) of tumor‐burdened lungs of KL (C, n = 7 and 6 mice for IgG and α IL‐36 γ , respectively) or KP (D, n = 7 and 7 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). E,F) Images (left) and quantification analysis (right) of Ki67 staining in lung tumors from KL (E, n = 30 and 32 mice for IgG and α IL‐36 γ , respectively) or KP (F, n = 29 and 20 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). G,H) qRT‐PCR analysis of the indicated genes for GSH metabolism in lung tumors from KL (G, n = 6 and 6 mice for IgG and α IL‐36 γ , respectively) or KP (H, n = 6 and 6 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). I) Immunoblot analysis of GCLM, GPX4 and GSR in lung tumors from KL (left, n = 3 and 3 mice for IgG and α IL‐36 γ , respectively) or KP (right, n = 3 and 3 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). J) GSH levels in the lung tumors of KL ( n = 8 and 10 mice for IgG or α IL‐36 γ , respectively) mice treated as in (A). K,L) Images (K) and quantification analysis (L) of 8‐oxo‐dGuo staining in lung tumors of KL ( n = 7 and 6 mice for IgG and α IL‐36 γ , respectively) and KP ( n = 7 and 7 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). M–N) Flow cytometry (left) and quantification analysis (right) of H 2 DCFDA staining in single‐cell suspensions of lung tumors from KL (M, n = 4 and 5 mice for IgG and α IL‐36 γ , respectively) or KP (N, n = 4 and 5 mice for IgG and α IL‐36 γ , respectively) mice treated as in (A). O) Flow cytometry (left) and quantification analysis (right) of SYTOX Green staining in KP ( n = 5 and 5 mice for IgG and α IL‐36 γ , respectively) or KL ( n = 5 and 5 mice for IgG and α IL‐36 γ , respectively) treated as in (A). Graphs show mean ± SEM (C–H,J,L–O). Two‐tailed student's t ‐test (C–H,J,L–O) or Log‐Rank analysis (B). Scale bars represent 5 mm (C,D, left), 2 mm (C,D, right), or 50 µm (E,F,K), respectively. Data are combined results of two independent experiments (B) or representative results of two independent experiments (C–O).

Article Snippet: For CHX and TNF α ‐induced apoptosis, the cells were cultured in 12‐well plate in DMEM containing 10% fetal bovine serum and 1% streptomycin and penicillin for 12 h. The medium were supplemented with CHX (100 mg mL −1 , 1:3000 in use, Enzo Biochem, ALX‐380‐269) and TNF α (10 ng per well, SRP2102‐10UG, Sigma‐Aldrich) with IL‐36 γ or Z‐VAD‐FMK (50 μ m , HY‐16658B, MedChemExpress).

Techniques: Neutralization, Injection, Control, Staining, Quantitative RT-PCR, Western Blot, Flow Cytometry, Two Tailed Test