Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: Investigation of the association of ATP1A1 genetic variation with CaOx stone through integration analysis of the gene expression profiles of GSE73680 and the WES data. (A) WGCNA analysis for 52 samples of P group (n = 27) and N group (n = 25) from GSE73680. The salmon module was identified to be related to calcium stone ( p = 0.01). (B) ATP1A1 was identified by Venn diagram analysis for the genes obtained from the salmon module (434 genes) and the candidate genes from the WES data (67 genes). (C) SNPrs11540947 (NM_000701: c.-78C > T) was genotyped with HRM. Each sample was tested at least thrice, independently. The results were determined by the aligned melt curves (the left panel) and the difference plots (the right panel). Three genotypes were identified (CC: green; CT: blue; TT: red). (D) Sanger sequencing was performed for rs11540947 in ATP1A1 , which is indicated with black arrows. (E–F) The allele difference of rs11540947 in the promoter activity of ATP1A1 was detected by the dual-luciferase reporting system in HEK293 cells (E) and HK2 cells (F). The relative luciferase activity was calculated as the fold change relative to the control group. Three independent experiments were performed, and the values are expressed as the mean ± SD (n = 3). **** p < 0.0001 vs. pGL3-basic; ### p < 0.001 vs. the wild-type (CC). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Gene Expression, Sequencing, Activity Assay, Luciferase, Control

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: List of 67 candidate genes and the corresponding 76 SNPs identified from the WES data of 28 patients with CaOx stone.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques:

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: Whole exome sequencing analysis for rs11540947 (C > T) in ATP1A1 .

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Sequencing, Control

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: COM exposure to HK2 cells activated the ATP1A1/Src/ROS/MAPKs/NF-κB signaling pathway. HK2 cells were exposed to 100 μg/mL COM for 0, 3, 6, 12, 24, and 48 h. Each experiment was independently repeated at least thrice. (A) The activity of NKA in HK2 cells decreased after COM stimulation. The data were expressed as the mean ± SD (n = 5). ** p < 0.01 vs. levels at 0 h. (B) COM exposure to HK2 cells inhibited the mRNA level of ATP1A1 . The data were expressed as the mean ± SEM (n = 3). ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. levels at 0 h. (C) Western blotting analysis for the expressions of ATP1A1, p65, p50, Nrf2, and the phosphorylation of Src, p38, and JNK after COM exposure. The relative levels of proteins were calculated as a fold change relative to the level at 0 h. The data were expressed as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; **** p < 0.0001 vs. the level at 0 h. (D–E) HK2 cells were exposed to 100 μg/mL COM for 24 h. Intracellular ROS was detected with a green fluorescent probe DCFHDA. The nuclei were stained with DAPI (blue). Scale bar = 50 μm. The relative fluorescence intensity was calculated as the fold change relative to the control group. The data were expressed as the mean ± SD (n = 3). *** p < 0.001 vs. the control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Activity Assay, Western Blot, Phospho-proteomics, Staining, Fluorescence, Control

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: ATP1A1 overexpression inhibited the activation of the ATP1A1/Src/ROS/MAPKs/NF-κB signaling pathway induced by COM exposure. HK2 cells were infected with Ad-hATP1A1 and HBAD-mCherry at 20 MOI for 48 h and then exposed to 100 μg/mL COM for 0, 3, 6, 12, and 24 h. Each experiment was repeated thrice, independently. (A) Western blotting analyzed the effects of ATP1A1 overexpression on the activation of Src, p38, JNK, p65, p50, and Nrf2. The protein levels were calculated as fold changes relative to the level at 0 h in the HBAD-mCherry group. The values were expressed as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. levels in the HBAD-mCherry group. (B–C) HK2 cells infected with Ad-ATP1A1 and HBAD-mCherrry were exposed to 100 μg/mL COM for 24 h. Intracellular ROS was then measured with DCFHDA. Scale bar = 50 μm. The relative fluorescence intensity was calculated as fold changes relative to the level in the HBAD-mCherry group without COM treatment. The values were expressed as the mean ± SD (n = 3). **** p < 0.0001; *** p < 0.001 vs. COM (−). ### p < 0.001 vs. the HBAD-mCherry group.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Over Expression, Activation Assay, Infection, Western Blot, Fluorescence

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: pNaKtide attenuated ROS accumulation and inflammation induced by COM by inhibiting the ATP1A1/Src signaling pathway. HK2 cells were pretreated with 1 μM tat-pNaktide and tat-scramble, and then exposed to100 μg/mL COM for 0, 3, 6, 12, 24, and 48 h. Each experiment was independently repeated thrice. (A) Western blotting results revealed pNaKtide inhibited the activation of Src, p38, JNK, p65, and p50 while promoting the Nrf2 expression. The levels of these proteins were expressed as fold changes relative to the level at 0 h in the tat-scramble group. The values were expressed as the mean ± SD (n = 3). *** p < 0.001; **** p < 0.0001 vs. levels in the tat-scramble group. (B–C) After 24 h of COM exposure, pNaKtide decreased the intracellular ROS levels. Scale bar = 50 μm. The level of intracellular ROS was calculated as fold changes relative to the level in the tat-scramble group without COM treatment. The values were expressed as the mean ± SD (n = 3). ** p < 0.01; **** p < 0.0001 vs. COM (−). ### p < 0.001 vs. the tat-scramble group.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Western Blot, Activation Assay, Expressing

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: ATP1A1 overexpression and pNaKtide treatment suppressed COM-induced apoptosis and attenuated crystal-cell adhesion. Each experiment was tested thrice, independently. (A–B) Apoptosis of Ad-hATP1A1 infected and pNaKtide-treated cells measured after 24 h of exposure to COM. Scale bar = 50 μM. The apoptosis rate was calculated as the percentage of apoptotic cells to the total adherent cells. The values were expressed as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001 vs. COM (−). # p < 0.05 vs. the HBAD-mCherry group or the tat-scramble group. (C–D). Cleaved caspase 3 in Ad-hATP1A1 infected and pNaKtide-treated cells as measured by Western blotting after 24 h of exposure to COM. The values were expressed as the mean ± SD (n = 3). *** p < 0.001; **** p < 0.0001 vs. the HBAD-mCherry group or the tat-scramble group. (E–F) Crystal deposition on cells was investigated after infection with Ad-hATP1A1 or treatment with pNaKtide. (−) represents no adenovirus and no COM exposure. Control represents only COM exposure. The values were expressed as the mean ± SD (n = 3). **** p < 0.0001 vs. the (−) group. ### p < 0.001 vs. the HBAD-mCherry group or the scramble group.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Over Expression, Infection, Western Blot, Control

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: DNA methylation participated in the downregulation of ATP1A1 induced by COM. (A) After exposing 100 μg/mL COM to HK2 cells, the mRNA levels of DNMT1 , DNMT3a , and DNMT3b , were measured by qRT-PCR. The values were expressed as the mean ± SEM (n = 4). * p < 0.05; ** p < 0.01; *** p < 0.001; vs. the levels at 0h. (B) The protein levels of DNMT1, DNMT3a, and DNMT3b were determined by Western blotting. The relative expression level was expressed as a fold change relative to the level at 0 h. Each experiment was repeated thrice, independently. The values were expressed as the mean ± SEM (n = 3). * p < 0.05; *** p < 0.001; **** p < 0.0001 vs. the levels at 0 h. (C) The ATP1A1 mRNA level was detected after pretreatment with 5Aza-2dc, a specific inhibitor of DNA methyltransferase, at the concentration of 0.1–10 μM. The values were expressed as the mean ± SEM (n = 3). * p < 0.05 vs. COM (−); ### p < 0.001; #### p < 0.0001 vs. only COM (+). (D) The protein level of ATP1A1 was examined by Western blotting after pretreatment with 10 μM 5Aza-2dc. Three independent experiments were performed and the values were expressed as the mean ± SD (n = 3). ** p < 0.01 vs. COM (−). ## p < 0.01 vs. only COM (+).

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: DNA Methylation Assay, Quantitative RT-PCR, Western Blot, Expressing, Concentration Assay

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: The ATP1A1/Src/MAPKs/NF-κB signaling pathway was activated in the CaOx stone rat model. 0 w represents the time before inducing the CaOx stone model. The rats in the 0 w group received natural water and were used as controls (n = 3). 2 w and 4 w represent the CaOx stone rats who received water containing 20 g/L HYP and 2 g/L CaCl 2 for 2 and 4 weeks, respectively (n = 5 per group). (A) The mRNA level of ATP1A1 in the rat kidneys was measured by qRT-PCR. The values were expressed as the mean ± SD (n = 3 at 0 w, n = 5 at 2 w and 4 w). *** p < 0.001; **** p < 0.0001 vs. the levels at 0 w. (B–C) The protein level of ATP1A1 in the rat kidneys was analyzed by Western blotting and was calculated as the fold change relative to the level at 0 w. The values were expressed as the mean ± SD (n = 3). ** p < 0.01; *** p < 0.001 vs. the level at 0 w. (D) Immunohistochemical staining for ATP1A1 in the rat kidneys. Scale bar = 25 μM. Relative mean optical density (MOD) was calculated as the fold change relative to the value at 0 w (n = 3 at 0 w, n = 5 at 2 w and 4 w). (E–F) Western blotting analysis for the activation of Src, p38, JNK, p65, p50, and Nrf2 in the rat kidneys. The control represents the CaOx stone rats treated with 0.9% saline. Scramble and pNaKtide represent the CaOx stone rats treated with 17.5 mg/kg tat-scramble and tat-pNaKtide, respectively. The protein levels were expressed as the fold change relative to the level at 0 w. The values were expressed as the mean ± SD (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. the control group. # p < 0.05; ## p < 0.01; ### p < 0.001; #### p < 0.0001 vs. the tat-scramble group. (G–H) The ATP1A1 protein level in rat kidneys was reversed by treatment with 1 mg/kg 5Aza-2dc. The controls represent the CaOx stone rats treated with 0.9% saline. Three independent experiments were performed. The values were expressed as the mean ± SD (n = 3). ** p < 0.01; **** p < 0.0001 vs. the level at 0 w. ## p < 0.01; #### p < 0.0001 vs. the level in the control group.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining, Activation Assay, Control, Saline

Journal: Redox Biology

Article Title: Meta-data analysis of kidney stone disease highlights ATP1A1 involvement in renal crystal formation

doi: 10.1016/j.redox.2023.102648

Figure Lengend Snippet: A schematic diagram of the mechanism of ATP1A1 genetic variation and ATP1A1/Src/ROS signaling pathway involved in kidney stone formation. In the resting condition, the α1 subunit encoded by ATP1A1 gene was coupled with Src to form a receptor complex, thereby retaining the Src in an inactivated state. ① The T-allele of rs11540947 in the 5′UTR region of ATP1A1 decreased the promoter activity. ② Crystal adhesion induced ATP1A1 methylation by upregulating methyltransferases. ③ DNA methylation or genetic variation in ATP1A1 decreased the expression of the α1 subunit. ④ The decreased ATP1A1 (α1, in light black box) released the Src kinase domain, which led to the phosphorylation of Src. ⑤ The activation of Src resulted in the generation of ROS. ⑥ ROS could reactivate the ATP1A1/Src signaling pathway as a ligand of the α1 subunit to form the Na/K-ATPase/ROS amplification loop. ⑦Excessive accumulation of ROS induced inflammation and apoptosis by activating the stress-related signaling pathway, including p38-MAPK and JNK. ⑧ Damaged cells further promoted crystal adhesion to the cell surface. ⑨ pNaKtide, a specific inhibitor of the ATP1A1/Src signal complex, attenuated the generation of ROS and reduced cell apoptosis and crystal-cell adhesion, thereby preventing kidney stone formation.

Article Snippet: After dewaxing, antigen repair, treatment with 3% hydrogen peroxide and blocking with goat serum, the rat kidney slice was incubated with ATP1A1 rabbit polyclonal antibody (Proteintech, China), followed by treatment with HRP goat anti-rabbit IgG (Abclonal, China).

Techniques: Activity Assay, Methylation, DNA Methylation Assay, Expressing, Phospho-proteomics, Activation Assay, Amplification

Journal: Cancer Cell International

Article Title: Prognosis-related molecular subtyping in head and neck squamous cell carcinoma patients based on glycolytic/cholesterogenic gene data

doi: 10.1186/s12935-023-02880-3

Figure Lengend Snippet: ENO1, PFKFB3, NSDHL and SQLE expression in HNSCC. A The typical staining and IHC score of ENO1 in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). B The typical staining and IHC score of PFKFB3 in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). C The typical staining and IHC score of NSDHL in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). D The typical staining and IHC score of SQLE in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). Data were shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Slides were incubated overnight at 4 °C with the following primary antibodies: anti-ENO1 rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-PFKFBS rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-SQLE rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-NSDHL rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Prognosis-related molecular subtyping in head and neck squamous cell carcinoma patients based on glycolytic/cholesterogenic gene data

doi: 10.1186/s12935-023-02880-3

Figure Lengend Snippet: ENO1, PFKFB3, NSDHL and SQLE expression in HNSCC. A The typical staining and IHC score of ENO1 in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). B The typical staining and IHC score of PFKFB3 in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). C The typical staining and IHC score of NSDHL in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). D The typical staining and IHC score of SQLE in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). Data were shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Slides were incubated overnight at 4 °C with the following primary antibodies: anti-ENO1 rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-PFKFBS rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-SQLE rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-NSDHL rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China).

Techniques: Expressing, Staining

Journal: Cancer Cell International

Article Title: Prognosis-related molecular subtyping in head and neck squamous cell carcinoma patients based on glycolytic/cholesterogenic gene data

doi: 10.1186/s12935-023-02880-3

Figure Lengend Snippet: ENO1, PFKFB3, NSDHL and SQLE expression in HNSCC. A The typical staining and IHC score of ENO1 in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). B The typical staining and IHC score of PFKFB3 in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). C The typical staining and IHC score of NSDHL in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). D The typical staining and IHC score of SQLE in in normal tissues and HNSCC tissues from recurrent HNSCC patients (recurrence group) and first diagnosed HNSCC patients surviving at least 5 years (high survival group). Data were shown as mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001)

Article Snippet: Slides were incubated overnight at 4 °C with the following primary antibodies: anti-ENO1 rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-PFKFBS rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-SQLE rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China), anti-NSDHL rabbit polyclonal antibody (1:200; Proteintech, Wuhan, China).

Techniques: Expressing, Staining