Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: The mRNA transcription levels of the WNT gene family in LUAD and LUSC. (A) The mRNA transcription levels of WNT gene family in lung cancer (ONCOMINE). Red for overexpression, blue for downregulated expression. (B) Differential expression of WNT gene family in different tumor tissues and normal tissues (TIMER). Statistical significance of differential expression was assessed using the Wilcoxon test ( p value significant codes: 0 ≤ *** < 0.001 ≤ ** < 0.01 ≤ * < 0.05 ≤. < 0.1). (C) The expression matrix plots for WNT gene family in LUAD and LUSC. Indicates the relative expression level among WNT genes.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Over Expression, Expressing, Quantitative Proteomics

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Compared with normal tissues, the WNT gene family’s mRNA expression and methylation levels in LUAD and LUSC (UALCAN). (A) The mRNA transcription levels of the WNT gene family in LUAD and LUSC. (B) The methylation level of the WNT gene family in LUAD and LUSC. p < 0.05 was considered statistically significant.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Expressing, Methylation

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: The mRNA expression of WNT gene family in different pathological stages of LUAD and LUSC (GEPIA). (A) Correlation between WNT gene family and pathological stage in patients with LUAD. (B) Correlation between WNT gene family and pathological stage in patients with LUSC.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: The overall survival (OS) curves of WNTs in LUAD and LUSC. (A) Kaplan-Meier survival analysis of WNTs in LUAD. (B) Kaplan-Meier survival analysis of WNTs in LUSC.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques:

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of WNT gene expression and clinical characteristics and the nomogram predicting 1-y, 3-y, and 5-y survival in patients with LUAD and LUSC. (A, B, E, F) Hazard ratio and p value of constituents involved in univariate and multivariate Cox regression in LUAD (A, B) and LUSC (E, F) . (C, G) Nomogram to predict the 1-y, 3-y and 5-y overall survival of LUAD (C) and LUSC (G) patients. (D, H) Calibration curve for the overall survival nomogram model in the discovery group. The dashed diagonal line represents the ideal nomogram, and the red, orange and blue represent the 1-y, 3-y and 5-y observed nomograms. (D) for LUAD. (H) for LUSC.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Gene Expression

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Genetic alterations and association analysis of the WNT gene family in NSCLC patients. (A) Genetic alterations of the WNT Gene Family in LUAD (cBioPortal). (B) Genetic alterations of the WNT Gene Family in LUSC (cBioPortal). (C, D) Correlation analysis heatmap between different WNT genes in LUAD (C) and LUSC (D) . (E) Correlation analysis between different WNT genes (GEPIA).

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques:

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Correlation of WNT genes with immune cell infiltration in LUAD and LUSC (TIMER). (A) WNT1. (B) WNT2. (C) WNT2B. (D) WNT3. (E) WNT3A. (F) WNT4. (G) WNT5A. (H) WNT5B. (I) WNT6. (J) WNT7A. (K) WNT7B. (L) WNT8A. (M) WNT8B. (N) WNT9A. (O) WNT9B. (P) WNT10A. (Q) WNT10B. (R) WNT11. (S) WNT16.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques:

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: The association of WNT2B and WNT7A with immune cell infiltration (TISIDB). (A, C) The landscape of relationship between WNT2B (A) and WNT7A (C) expression and TILs in different types of cancer (red is positive correlated and blue is negative correlated). (B) Relationship between WNT2B and immune infiltration levels of B cell, CD4+ T cell, macrophage, neutrophil, dendritic cell in LUAD, and CD8+ T cell, neutrophil, dendritic cell in LUSC. (D) Relationship between WNT7A and immune infiltration levels of CD4+ T cells, macrophage, neutrophil, dendritic cell in LUAD, and B cell, CD4+ T cell, macrophage in LUSC.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Comparison of tumor infiltration levels among tumors with different SCNA for WNTs in LUAD and LUSC samples ( p value significant codes: 0 ≤ *** < 0.001 ≤ ** < 0.01 ≤ * < 0.05 ≤. < 0.1). (A) WNT1. (B) WNT2. (C) WNT2B. (D) WNT3. (E) WNT3A. (F) WNT4. (G) WNT5A. (H) WNT5B. (I) WNT6. (J) WNT7A. (K) WNT7B. (L) WNT8A. (M) WNT8B. (N) WNT9A. (O) WNT9B. (P) WNT10A. (Q) WNT10B. (R) WNT11. (S) WNT16.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Comparison

Journal: Frontiers in Oncology

Article Title: Validation and analysis of expression, prognosis and immune infiltration of WNT gene family in non-small cell lung cancer

doi: 10.3389/fonc.2022.911316

Figure Lengend Snippet: Validation of mRNA and protein expression levels of WNT2B and WNT7A in NSCLC. (A) Comparison of mRNA expression levels of WNTs in 20 paired NSCLC tissues and adjacent normal tissue samples by qRT-PCR. (B) Comparison of mRNA expression levels of WNTs in LUAD cell line (PC9) and LUSC cell line (NCI-H520) with human normal lung epithelial cell line (BEAS-2B). (C-F) The western blot analyses. (G–J) The immunohistochemical staining in 20 NSCLC tissues and adjacent normal tissues.

Article Snippet: Human normal lung epithelial cell line (BEAS-2B), LUAD cell line (PC9) and LUSC cell line (NCI-H520) were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Biomarker Discovery, Expressing, Comparison, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Sciences

Article Title: Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression

doi: 10.3390/ijms22157782

Figure Lengend Snippet: PML is required for FANCD2 mono-ubiquitination and foci formation in response to genotoxic agents inducing interstrand DNA crosslinks. ( A , C ) U2OS and HeLa cells were transfected with siControl, si PML , or siATR and treated with 1 μM mitomycin C (MMC) for 24 h. FANCD2 protein expression was determined though Western blotting of cell lysates from U2OS and HeLa. Long form (L) of FANCD2 represents mono-ubiquitination and short form (S) is non-ubiquitinated FANCD2 . ( B , D ) FANCD2 foci formation in U2OS and HeLa cells treated with siControl or si PML under 1 μM MMC exposure for one day. Representative immunofluorescence images and their quantification. Over 100 nuclei were counted for statistics. Data represent the mean ± SD from three independent experiments (*** p < 0.001). ( E , F ) Proliferation level of PML - or ATR-deficient U2OS and HeLa cells treated with indicated doses of MMC for four or five days.

Article Snippet: After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C.

Techniques: Ubiquitin Proteomics, Transfection, Expressing, Western Blot, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression

doi: 10.3390/ijms22157782

Figure Lengend Snippet: PML NBs control gene expression of Fanconi anemia core complex. ( A ) After depletion of PML , U2OS cells were treated with 1 μM MMC or 10 μM MG132 for 24 h. Expression of FANCD2 and FA core proteins including FANCA , FANCG , and FANCL was analyzed by immunoblotting assay. The asterisk indicates a cross-reactive artifact. ( B ) Relative mRNA expression of FANCD2 , FANCA , FANCG , FANCL , Chk1 , and PML was determined by real-time quantitative PCR in the U2OS cells depleted with PML . Data represent the mean ± SD from three independent experiments (*** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant ( p > 0.05)).

Article Snippet: After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C.

Techniques: Control, Gene Expression, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression

doi: 10.3390/ijms22157782

Figure Lengend Snippet: Depletion of PML impairs phosphorylation of CHK1 in response to DNA damage. ( A ) Phosphorylation of CHK1 in U2OS and HeLa cells treated with si PML in the absence or presence of 1 μM MMC. ( B ) U2OS cells treated with CHK1 siRNA were immunostained by anti- FANCD2 or anti- FANCA antibody in the absence or presence of 1 μM MMC exposure. Representative immunofluorescence images and their quantification are shown. Over 37 nuclei were counted for statistics. Data represent the mean ± SD from three independent experiments (** p < 0.01; * p < 0.05).

Article Snippet: After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C.

Techniques: Phospho-proteomics, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Promyelocytic Leukemia Proteins Regulate Fanconi Anemia Gene Expression

doi: 10.3390/ijms22157782

Figure Lengend Snippet: CHK1 is required for the transcriptional regulation of Fanconi anemia core protein and FANCD2 . ( A ) U2OS cells were transfected with siControl or siCHK1 and treated with 1 μM mitomycin C (MMC) or 10 μM MG132 for 24 h. Protein expressions were determined from whole cell lysates using antibodies against FANCD2 , FANCA , FANCG , FANCL , and CHK1. The asterisk indicates a cross-reactive artifact. ( B ) After depletion of CHK1, lysates prepared from GM6914/ FANCA cell lines were subjected to immunoblot analysis with HA, FANCA , and CHK1 antibodies. ( C ) After depletion of CHK1, relative mRNA levels of core FA genes, FANCD2 , and CHK1 were assessed through RT-qPCR assay in U2OS cells. Data represent the mean ± SD from three independent experiments (*** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant ( p > 0.05)).

Article Snippet: After they were washed, cells were blocked with blocking buffer (0.2% gelatin and 0.5% BSA in PBS) for 1~2 h at room temperature and then incubated with primary antibodies, such as FANCA (Merck Millipore: MABC557), FANCD2 (Novus Biologicals: NB100-182), PML (Santa Cruz Biotechnology: sc-966), or γH2AX (Cell Signaling Technology: #2595) overnight at 4 °C.

Techniques: Transfection, Western Blot, Quantitative RT-PCR

Journal: Oncology Reports

Article Title: SNAI2 enhances HPV‑negative cervical cancer cell dormancy by modulating u‑PAR expression and the activity of the ERK/p38 signaling pathway in vitro

doi: 10.3892/or.2024.8763

Figure Lengend Snippet: SNAI2 regulates the expression of proliferation-related genes by suppressing the ERK/p38 pathway ratio. (A) The proliferated-related gene expression was analyzed by reverse transcription-quantitative PCR. (B and C) p-p38 and p-ERK were detected by WB. (D) The ratio of ERK/p38 was calculated by densitometric analysis of WB. *P<0.05 and **P<0.01. SNAI2, snail family transcriptional repressor 2; p-, phosphorylated; WB, western blotting; CDK, cyclin-dependent kinases; u-PAR, urokinase plasminogen activator receptor.

Article Snippet: The PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibodies [SNAI2 (1:1,000; cat. no. sc-166476; Santa Cruz Biotechnology, Inc.), ERK1/2 (1:1,000; cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), phosphorylated (p-)ERK1/2 (1:2,000; cat. no. sc-7976; Santa Cruz Biotechnology, Inc.), p38 (1:1,000; cat. no. 9212; Cell Signaling Technology, Inc.), p-p38 (1:1,000; cat. no. 9211; Cell Signaling Technology, Inc.) and β-actin (1:2,000; cat. no. 4967; Cell Signaling Technology, Inc.)] at 4°C overnight.

Techniques: Expressing, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncology Reports

Article Title: SNAI2 enhances HPV‑negative cervical cancer cell dormancy by modulating u‑PAR expression and the activity of the ERK/p38 signaling pathway in vitro

doi: 10.3892/or.2024.8763

Figure Lengend Snippet: Schematic diagram. SNAI2 enhances HPV-negative cervical cancer cell dormancy, which is characterized by G0/G1 arrest, by downregulating u-PAR expression, and the activity of the ERK1/2 and p38MAPK signaling pathways, further regulating the expression of proliferation-associated genes (downregulation of cyclin D1 and CDK1, upregulation of p21 expression). SNAI2, snail family transcriptional repressor 2; HPV, human papillomavirus; u-PAR, urokinase plasminogen activator receptor.

Article Snippet: The PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibodies [SNAI2 (1:1,000; cat. no. sc-166476; Santa Cruz Biotechnology, Inc.), ERK1/2 (1:1,000; cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), phosphorylated (p-)ERK1/2 (1:2,000; cat. no. sc-7976; Santa Cruz Biotechnology, Inc.), p38 (1:1,000; cat. no. 9212; Cell Signaling Technology, Inc.), p-p38 (1:1,000; cat. no. 9211; Cell Signaling Technology, Inc.) and β-actin (1:2,000; cat. no. 4967; Cell Signaling Technology, Inc.)] at 4°C overnight.

Techniques: Expressing, Activity Assay, Protein-Protein interactions

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition decreases cell proliferation and survival in prostate cancer cells. ( a ) Western blots from DU145, PC3, VCaP, LNCaP, V16D and MR49F cells showing AR expression. ( b ) Indicated cell lines were treated with 10 µM SB203580 (SB) under normoxia (21% O 2 ) (left) or hypoxia (0.2% O 2 ) (right) for 150 h. Confluency was measured with the IncuCyte Live Cell Imaging system after confirming proportionality to cell numbers. Each data point represents an independent experiment, bars represent the mean value ± S.E.M. ( c ) V16D cells were transfected with siRNA targeting MAPK11, MAPK14, or non-targeting negative control (NTC). Expression of target genes was assessed by qPCR (left) or western blotting (right). ( d ) Cells as in (c) were placed in normoxia (21% O 2 ) or hypoxia (0.2% O 2 ) for 72 h and confluency was measured with the IncuCyte Live Cell Imaging system. ( e ) V16D cells were treated with various doses of SB203580 (5, 10, 20 µM) for 72 h under normoxia (21% O 2 ) or hypoxia (0.2% O 2 ). Single cells were seeded for clonogenic survival in triplicate and surviving fraction calculated from colony formation 14 days later. Data points represent independent experiments and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition, Western Blot, Expressing, Live Cell Imaging, Transfection, Negative Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition decreases Hsp27 phosphorylation, AR activity and expression of AR target genes under normoxia and hypoxia. ( a ) Prostate cancer cell lines (DU145, PC3, VCaP, LNCaP, V16D, MR49F) were treated with 10 µM SB203580 for one hour and subjected to western blotting (left). V16D cells were treated with 25 µM of anisomycin for one hour as a positive control for p38 MAPK activation. Densitometry analysis was performed to calculate fold change of p-Hsp27 (Ser82) relative to Hsp27 for each cell line. ( b ) V16D cells were transfected with a plasmid encoding luciferase driven by the AR promoter. 24 h later, cells were treated with 1 nM DHT, 10 µM SB203580, or 10 µM enzalutamide (Enza) in 10% charcoal treated (CT)-FBS media (androgen depleted) and incubated under normoxia (21% O 2 ) or hypoxia (0.2% O 2 ) for 48 h. Relative light units (RLU) from luciferase was measured after addition of luciferin. ( c–e ) V16D cells were treated with 10 µM SB203580, or 10 µM enzalutamide for 48 h and relative mRNA expression of KLK3 ( c ), NKx3.1 ( d ), and FKBP5 ( e ) was measured by RT-qPCR normalized to the average of HPRT1 and GUSB gene expression. Fold change was calculated relative to the 10% CT-FBS negative normoxia control. Data points represent an independent experiment and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition, Phospho-proteomics, Activity Assay, Expressing, Western Blot, Positive Control, Activation Assay, Transfection, Plasmid Preparation, Luciferase, Incubation, Quantitative RT-PCR, Gene Expression, Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: Androgen and hypoxia activate p38 MAPK and Hsp27. ( a ) V16D cells were exposed to 0 nM, 1 nM, or 10 nM DHT in charcoal-stripped media for 6 h in normoxia (21% O 2 ) or hypoxia (0.2% O 2 ). 25 µM of anisomycin for one hour was used as a positive control for p38 MAPK activation. Total cell lysates were subjected to western blotting. Densitometry analysis of protein bands was performed to calculate fold change of p-p38 (Tyr182) ( b ) and p-Hsp27 (Ser82) ( c ) relative to normoxia control with 0 nM DHT and normalized to eIF4E. Data points represent independent experiments and bars represent the mean value ± S.E.M. (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001).

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Positive Control, Activation Assay, Western Blot, Control

Journal: Cancers

Article Title: p38 MAPK Inhibition Mitigates Hypoxia-Induced AR Signaling in Castration-Resistant Prostate Cancer

doi: 10.3390/cancers13040831

Figure Lengend Snippet: p38 MAPK inhibition prolongs survival of mice bearing CRPC xenografts. Subcutaneous V16D xenografts were established and treatment with vehicle or SB203580 (10 mg/kg) started when tumors reached 200 mm 3 , in a 5 days on and 2 days off schedule until endpoint. Kaplan-Meier survival curves are shown for the time to reach tumor volume endpoint (1000 mm 3 ) from treatment start. Tumor growth curves from individual mice are shown in .

Article Snippet: The membrane was incubated with antibodies diluted in Odyssey blocking buffer (LI-COR Biosciences) overnight at 4 °C to detect the following proteins: p-p38 MAPK (Tyr182; E-1; 1:500; Santa Cruz Biotechnology, Dallas, TX, USA), p38 MAPK (1:1000), p-Hsp27 (Ser82; 1:1000), Hsp27 (G31; 1:1000), AR (D6F11; 1:1000), HIF-1ɑ (1:1000; BD Biosciences, San Jose, CA, USA), eIF4E (1:1000; BD Biosciences), and beta-tubulin (1:10,000; Abcam, Cambridge, UK).

Techniques: Inhibition

Journal: The FASEB Journal

Article Title: Fructose induces inflammatory activation in macrophages and microglia through the nutrient‐sensing ghrelin receptor

doi: 10.1096/fj.202402531R

Figure Lengend Snippet: Ghsr Inhibition suppresses fructose‐induced CREB–AKT, NF‐κB p65, and p38 MAPK signaling cascades in macrophages and microglia. WT & Ghsr ‐mutant ( Ghsr mutant ) RAW 264.7 macrophages and IMG microglia were treated with fructose (+Fru) for 24 h. (A, B) Representative immunoblot images and quantification results of p‐CREB (Ser133), CREB, p‐AKT (Ser473), p‐AKT (Thr308), AKT, p‐NF‐κB p65 (Ser536), and NF‐κB p65 in RAW 264.7 (A) and IMG (B). (C, D) Representative immunoblot images and quantification results of p‐p38 MAPK (Thr180/Tyr182) and p38 MAPK in RAW 264.7 (C) or IMG (D). Data were presented as mean ± SEM, # p < .05; ## p < .01; ### p < .001, +Fru vs. −Fru; * p < .05; ** p < .01; *** p < .001, Ghsr mutant vs. WT. (E) The schematic diagram illustrates the effect of GHSR on fructose‐induced inflammatory pathways of RAW 264.7 macrophages and IMG microglia. The dashed arrow from GHSR to p38 indicates that the regulation of p38 by GHSR is likely to involve multiple steps.

Article Snippet: Primary antibodies for AKT (Cat# 4691), AMPKα (Cat# 2603), β‐Actin (Cat# 4970), CREB (Cat# 9197), NF‐κB p65 (Cat# 8242), p38 MAPK (Cat# 9212), phospho‐AKT‐Ser473 (Cat# 9271), phospho‐AKT‐Thr308 (Cat# 4056), phospho‐AMPKα‐Thr172 (Cat# 2535), phospho‐CREB‐Ser133 (Cat# 9198), phospho‐NF‐κB p65‐Ser536 (Cat# 3033), and phospho‐p38 MAPK‐Thr180/Tyr182 (Cat# 4511) were purchased from Cell Signaling Technology.

Techniques: Inhibition, Mutagenesis, Western Blot

Journal: The FASEB Journal

Article Title: Fructose induces inflammatory activation in macrophages and microglia through the nutrient‐sensing ghrelin receptor

doi: 10.1096/fj.202402531R

Figure Lengend Snippet: The proposed model illustrating the role of GHSR in fructose‐induced pro‐inflammatory activation in macrophages and microglia. In this study, we demonstrated that fructose exposure promotes inflammatory activation in macrophages and microglia. GHSR is involved in three aspects of the regulation: (1) GHSR stimulates the CREB–AKT and NF‐κB pro‐inflammatory signaling pathways, which are essential for fructose‐induced inflammation (noted as number 1 in the diagram); (2) GHSR expression directly or indirectly regulates fructose uptake and metabolism by promoting GLUT5 and KHK. This regulation alters the intracellular energy balance and AMP/ATP ratio, and the upregulation of AMP can subsequently promote inflammation via the AMPK–AKT and p38 pathways (noted as number 2 in the diagram); (3) Fructose increases Ghsr gene expression in macrophages and microglia potentially through CREB, resulting in a positive feedback loop that further exacerbates the inflammatory state (noted as number 3 in the diagram).

Article Snippet: Primary antibodies for AKT (Cat# 4691), AMPKα (Cat# 2603), β‐Actin (Cat# 4970), CREB (Cat# 9197), NF‐κB p65 (Cat# 8242), p38 MAPK (Cat# 9212), phospho‐AKT‐Ser473 (Cat# 9271), phospho‐AKT‐Thr308 (Cat# 4056), phospho‐AMPKα‐Thr172 (Cat# 2535), phospho‐CREB‐Ser133 (Cat# 9198), phospho‐NF‐κB p65‐Ser536 (Cat# 3033), and phospho‐p38 MAPK‐Thr180/Tyr182 (Cat# 4511) were purchased from Cell Signaling Technology.

Techniques: Activation Assay, Protein-Protein interactions, Expressing, Gene Expression

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Cyclic stretch induced the nuclear translocation of p38. a , b Nuclear translocation of total or phosphorylated p38 (p-p38) induced by cyclic stretch (CS). a AECs were treated with or without CS for 10 min, and immunostaining was performed with p38/p-p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). b Quantification of the nucleo-cytoplasmic distribution of p38/p-p38. c Cell fractionation analyses of CS-induced nuclear translocation of p38. AECs were treated with or without CS for 10 min, followed by nuclear and cytoplasmic extraction. Western blotting was performed with p-p38, p38, p-ATF2, β-actin, and Lamin B antibodies. d , e Nuclear translocation of p38 induced by high tidal volume ventilation in a mouse model of ventilator-induced lung injury (VILI). d The animals were ventilated for 4 h with a tidal volume of 15 ml/kg and a respiratory rate of 100 breaths/min, and immunostaining was performed with p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). e Quantification of the nucleo-cytoplasmic distribution of p38. f The time course of nuclear translocation of p38 induced by CS. AECs were treated with CS for the indicated time. Immunostaining was performed with p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). g The time course of nuclear translocation of p38 induced by CS. AECs were treated with CS for the indicated time. Western blotting was performed with p-p38, p38, and β-actin antibodies. h , i The effect of p38 phosphorylation on its nuclear translocation. AECs were preincubated with p38 specific inhibitor SB203580 (5 µM), MKK3/6 inhibitor gossypetin (60 µM), and treated with or without CS for 10 min. h Immunostaining was performed with p-p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). i Quantification of the nucleo-cytoplasmic distribution of p-p38. Three independent experiments were analyzed. Significance: * P < 0.05, *** P < 0.001, NS no significance; two-tailed Student’s t tests. (color figure online)

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Translocation Assay, Immunostaining, Cell Fractionation, Extraction, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Microtubule and dynein are required for the cyclic stretch (CS) induced nuclear translocation of p38. a AECs were preincubated with or without nocodazole (Noc, 1 µM), paclitaxel (Pac, 100 nM), cytochalasin D (Cyt D, 1 µM), or phalloidin (Pha, 10 µM), and treated with CS for 10 min, then Western blotting was performed with p-p38, p38, p-ATF2, and β-actin antibodies. b , c AECs were preincubated with paclitaxel (Pac, 100 nM) or nocodazole (Noc, 1 µM), and treated with CS for 10 min. b Immunostaining was performed with p-p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). c Quantification of the nucleo-cytoplasmic distribution of p-p38. d , e AECs were preincubated with or without ciliobrevin D (Cil D, 20 µM), ML-7 (10 µM), p150Glued siRNA (sip150), MLCK siRNA (siMLCK), and treated with CS for 10 min. d Immunostaining was performed with p-p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). e Quantification of the nucleo-cytoplasmic distribution of p-p38. f AECs were preincubated with or without the chemicals described above, and treated with CS for 10 min, and Western blotting was performed with p-p38, p38, p-ATF2, and β-actin antibodies. Cells that were not subjected to CS served as a negative control (static). Three independent experiments were analyzed. Significance: *** P < 0.001, NS no significance; two-tailed Student’s t tests. (color figure online)

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Translocation Assay, Western Blot, Immunostaining, Negative Control, Two Tailed Test

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Cyclic stretch-induced interaction between p38 and Imp7. a AECs were treated with or without CS, lysed, and incubated with a Sepharose bead-conjugated p-p38 antibody. The proteins bound to the beads were subjected to SDS-PAGE. The arrows indicate notable bands that co-immunoprecipitated in CS-induced AECs. b GST-tagged Imp7 and GST were purified and used for an in vitro binding assay with His-tagged p-p38 bound Ni 2+ -NTA Resin. c AECs were either transfected with HA-Imp7 alone, or co-transfected with FLAG-p38 and HA-Imp7 and treated with or without CS. Western blotting of whole cell extracts and immunoprecipitates were performed using anti-FLAG or anti-HA antibodies. d AECs were treated with or without CS, then immunoprecipitation with Imp7 antibody was performed. Western blotting of whole cell extracts and immunoprecipitates were performed using anti-Imp7, anti-p-p38 or anti-p38 antibodies. e , f AECs were treated with or without CS and double-stained with primary antibodies against p38 (red) or Imp7 (green), followed by incubation with Cy3/Cy5-conjugated secondary antibodies, respectively. The nuclei of cells were stained with DAPI (blue). f Quantification of the nucleo-cytoplasmic distribution of p38. g , h In situ PLA detection of the association between endogenous p38 and Imp7 in AECs. Red dots represent PLA events and indicate the close proximity between p38 and Imp7 proteins. Cell nuclei are stained in blue with DAPI. h Quantification of the PLA signal. Three independent experiments were analyzed. Significance: *** P < 0.001; two-tailed Student’s t tests. (color figure online)

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Incubation, SDS Page, Immunoprecipitation, Purification, In Vitro, Binding Assay, Transfection, Western Blot, Staining, In Situ, Two Tailed Test

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Imp7 is required for CS-induced nuclear translocation of p38. a Knockdown efficiency for NTR siRNA verified by Western blotting. b , c AECs were transfected with siRNA specific to Imp7 (siImp7), Imp-α1 (siImpα1), Imp-α5 (siImpα5), Imp-β1 (siImpβ1), Imp3 (siImp3), Imp9 (siImp9), or control (siCtrl), respectively, and treated with CS for 10 min. b Immunostaining was performed with p-p38 (red), F-actin (green) antibodies, and counterstained with DAPI to detect nuclei (blue). Cells that were not subjected to CS served as a negative control (static). c Quantification of the nucleo-cytoplasmic distribution of p-p38. d AECs were preincubated with siImp7 or siCtrl, and treated with CS for 10 min, then Western blotting was performed with Imp7, p-p38, p38, and β-actin antibodies. e , f Immunofluorescence of p-p38 localization in AECs silenced with siImp7. An empty vector or HA-Imp7 was overexpressed. Cell nuclei are stained in blue with DAPI. f Quantification of the nucleo-cytoplasmic distribution of p-p38. g Quantification of the effect on CS-induced p38 nucleo-cytoplasmic distribution upon silencing of the indicated importins. Three independent experiments were analyzed. Significance: *** P < 0.001, NS no significance; two-tailed Student’s t tests. (color figure online)

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Translocation Assay, Knockdown, Western Blot, Transfection, Control, Immunostaining, Negative Control, Immunofluorescence, Plasmid Preparation, Staining, Two Tailed Test

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Imp7 is necessary for p38-dependent gene expression. a AECs were transfected with siRNA specific to Imp7 (siImp7), Imp-α1 (siImpα1), Imp-α5 (siImpα5), Imp-β1 (siImpβ1), Imp3 (siImp3), Imp9 (siImp9), or control (siCtrl), respectively, and treated with CS for 10 min, then Western blotting was performed with p-p38, p38, p-ATF2, p-MK2, p-Elk1, and β-actin antibodies. Cells that were not subjected to CS served as a negative control (static). b AECs were transfected with siImp7 or siCtrl, and an empty vector or HA-Imp7 were overexpressed. The cells were treated with CS for 10 min, then Western blotting was performed with Imp7, p-p38, p38, p-ATF2, p-MK2, and β-actin antibodies. c AECs were transfected with the siImp7 or siCtrl, and an empty vector or a HA-Imp7 were overexpressed. The cells were treated with or without CS for 10 min, and qRT-PCR was performed to check TNF-α, IL-1β, and IL-6 mRNA expression. d AECs were transfected with siImp7 or siCtrl, and an empty vector or HA-Imp7 were overexpressed. The cells were treated with or without CS for 10 min, and then ELISA was performed to check the levels of TNF-α, IL-1β, and IL-6 in the cell culture supernatant. Three independent experiments were analyzed. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001, NS no significance; two-tailed Student’s t tests

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Gene Expression, Transfection, Control, Western Blot, Negative Control, Plasmid Preparation, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Model depicting the mechanism by which targeting the nuclear translocation of p38 by Imp7 inhibition attenuates inflammatory response in response to mechanical stretch

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Translocation Assay, Inhibition

Journal: Inflammation Research

Article Title: Inhibition of importin-7 attenuates ventilator-induced lung injury by targeting nuclear translocation of p38

doi: 10.1007/s00011-023-01727-x

Figure Lengend Snippet: Administration of Imp7 siRNA-loaded nanoparticles attenuated VILI. a Representative IVIS images show the fluorescence signal was mainly concentrated in the lung in the DiR-labeled Imp7 siRNA (siImp7) nanoparticles (NP) administered group compared with the control group. b The mice were administered siImp7 or control siRNA (siCtrl) NP intratracheally (500 µL), and immunostaining was performed with Imp7 (green), F-actin (white) antibodies, and counterstained with DAPI to detect nuclei (blue). c The mice were administered siImp7 NP intratracheally (500 µL), and Western blotting was performed at the indicated time point with Imp7 and β-actin antibodies. d – g Mice were administered siImp7 or siCtrl NP in PBS (500 µL) intratracheally, and were treated with or without mechanical ventilation (MV) for 4 h with a tidal volume of 15 ml/kg and a respiratory rate of 100 breaths/min. d Mice lungs were sectioned and stained with H&E, and tissue damage induced by MV was assessed by light microscopy. Immunostaining was performed with p-p38 (line3, red), p-MK2 (line4, red), p-Elk1 (line5, red), F-actin (white) antibodies, and counterstained with DAPI to detect nuclei (blue). e The wet/dry weight ratio was calculated as the ratio of the wet weight to the dry weight. f MPO activity in lung tissue lysates was determined by ELISA. g The levels of TNF-α, IL-1β, and IL-6 in BALF were determined by ELISA. Three independent experiments were analyzed. Significance: * P < 0.05, ** P < 0.01, *** P < 0.001, NS no significance; two-tailed Student’s t tests. (color figure online)

Article Snippet: For Western blotting, equal amounts of proteins were separated by SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes followed by immunostaining with primary antibody specific for p38 (1:1000), phosphorylated p38 (p-p38) (1:1000), ATF-2 (1:1000), p-ATF-2 (1:1000), p-MK2 (1:1000), p-Elk1 (1:1000) (Cell Signaling Technology, Danvers, MA, USA), and Imp7 (1:1000, Abcam, Cambridge, MA, USA).

Techniques: Fluorescence, Labeling, Control, Immunostaining, Western Blot, Staining, Light Microscopy, Activity Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Journal of cellular physiology

Article Title: Loss of NLRP6 increases the severity of kidney fibrosis.

doi: 10.1002/jcp.31347

Figure Lengend Snippet: FIGURE 6 ERK1/2 and p38 phosphorylation in UUO. (a) Increased kidney ERK1/2 phosphorylation and (b) increased kidney p38 phosphorylation in obstructed kidneys from Nlrp6 deficient mice as compared to obstructed kidneys from WT mice 14 days after obstruction during experimental CKD. Western Blot of whole kidney protein. Representative image of the Western blot and quantification of the p‐ERK1/2/ ERK1/2 and p‐p38/p38 ratios. *p < 0.05, ***p < 0.0005 versus WT mice. Mean ± SEM of five mice per group at 14 days following UUO. UUO, unilateral ureteral obstruction; WT, wild‐type.

Article Snippet: Additional primary antibodies were mouse monoclonal anti‐p‐ERK (1:500), rabbit polyclonal anti‐ERK1/2 (1:2000), goat polyclonal anti‐p38 (1:2500; all from Santa Cruz) and rabbit anti‐p‐SMAD3 (1:1000, Cell Signaling).

Techniques: Phospho-proteomics, Western Blot

Journal: Journal of cellular physiology

Article Title: Loss of NLRP6 increases the severity of kidney fibrosis.

doi: 10.1002/jcp.31347

Figure Lengend Snippet: FIGURE 7 Increased fibrogenic cytokine expression in Nlrp6 deficient mice with UUO and in Nlrp6 deficient tubular cells: role of p38 MAPK. (a, b) Kidney fibrogenic cytokine mRNA expression. Both (a) TGF‐β1 and (b) CTGF mRNA expression were higher in Nlrp6−/−

Article Snippet: Additional primary antibodies were mouse monoclonal anti‐p‐ERK (1:500), rabbit polyclonal anti‐ERK1/2 (1:2000), goat polyclonal anti‐p38 (1:2500; all from Santa Cruz) and rabbit anti‐p‐SMAD3 (1:1000, Cell Signaling).

Techniques: Expressing

Journal: Journal of cellular physiology

Article Title: Loss of NLRP6 increases the severity of kidney fibrosis.

doi: 10.1002/jcp.31347

Figure Lengend Snippet: FIGURE 8 Current working hypothesis Nlrp6 and maladaptive proximal tubular cell phenotypes. (a) We postulate that tubular cell Nlrp6 functions as a gatekeeper under stress conditions, keeping in check proinflammatory and profibrotic responses. (b) However, fibrogenic and inflammatory cytokines downregulate tubular cell Nlrp6 expression, facilitating inflammatory (as shown in reference Valiño‐Rivas et al., 2020) and fibrogenic (as shown in the present study) responses, including decreased Klotho expression, increased kidney chemokine expression and macrophage infiltration, and increased fibrogenic cytokines, extracellular matrix deposition and myofibroblast numbers. (c) Integrating the present findings with the recent literature on proximal tubular cell phenotypes and their relation to kidney fibrosis, during kidney injury, fibrogenic and inflammatory cytokines may decrease Nrlp6 expression in proximal tubular cells. Decreased Nrlp6 expression has been observed in a novel proximal tubular cell cluster (New‐PT1) that represented injured cells in transition towards repair (New‐PT2, not shown) or towards a maladaptive phenotype (New‐PT3). New‐PT3 is characterized by more severe Nrlp6 downregulation, Klotho downregulation and upregulation of CTGF (CCN2) and MCP‐1 (CCL2) expression, among other profibrotic and proinflammatory changes in gene expression. Klotho, CTGF and MCP‐1 were the most responsive genes to Nrlp6 siRNA targeting in cultured proximal tubular cells, a response that was dependent on p38 MAPK activity. The overall result is a positive feed‐back loop that magnifies tissue injury when Nlrp6 is downregulated. Thus, NLPRP6 appears to play a key role in the system of checks and balances that modulates tubular cell homeostasis and kidney fibrosis. TGF‐β1, transforming growth factor beta 1.

Article Snippet: Additional primary antibodies were mouse monoclonal anti‐p‐ERK (1:500), rabbit polyclonal anti‐ERK1/2 (1:2000), goat polyclonal anti‐p38 (1:2500; all from Santa Cruz) and rabbit anti‐p‐SMAD3 (1:1000, Cell Signaling).

Techniques: Expressing, Gene Expression, Cell Culture, Activity Assay