Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: RNLS adapts upward in the APA and promotes aldosterone production in vitro. (a) Distribution of RNLS immunoreactivity in different types of adrenocortical pathology sections. NFA: n = 7; APA: n = 14; AAG: n = 14; scale bar = 50µm. Localised magnification is shown in the box below. (b) Quantitative statistical analysis of RNLS shown in the positive area of a. (c) NCI-H295R cells were stimulated with RNLS (0.5, 1, 2, or 4μg/ml) for 12 h, and the levels of aldosterone production in the cell supernatant were measured. (d) Cells were treated with 4 μg/ml RNLS, and the level of aldosterone production in the supernatant was measured at different times. (e) EdU detection of cell proliferation after treatment with 4μg/ml RNLS. Nuclei are shown in blue, and red-stained EdU-positive cells with blue-stained nuclei in the merged layers are shown in purple; scale bar = 50 μm. (f) Statistical analysis of the percentages of EdU-positive cells in e. (g) CCK-8 assay for determining cell activity. (h) Photograph of plate colony formation after three weeks of 4 μg/ml RNLS treatment. (i) Statistical analysis of the plate colony formation rate in h. NFA: non-functioning adenoma; APA: aldosterone-producing adenoma; AAG: APA-adjacent adrenal gland.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: In Vitro, Staining, CCK-8 Assay, Activity Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Effect of RNLS on aldosterone synthase expression in human adrenocortical cells. (a) Schematic diagram illustrating the process of aldosterone synthesis. The orange ovals represent mitochondria, and the blue mesh represents the endoplasmic reticulum. (b) WB analysis of the protein levels of aldosterone synthesis-related enzymes after NCI-H295R cells were treated with RNLS for 16h. The protein blots in b were statistically analysed for (c) CYP11B2, (d) CYP21A2, (e) HSD3B2, (f) StAR, and (g) CYP11A1. The qRT-PCR was performed to measure the mRNA levels of aldosterone synthesis-related enzymes, including (h) CYP11B2, (i) CYP21A2, (j) HSD3B2, (k) StAR, and (l) CYP11A1.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Impact of RNLS on the level and activity of aldosterone synthase-related transcription factors. (a) Schematic representation of regulatory element sites within 1000 bp upstream of the HSD3B2, CYP21A2, and CYP11B2 promoters. Transcription factors known to bind to each regulatory element are shown in the table below. (b) The transcription factor NR4A2 was detected via qRT-PCR following 16 h of RNLS intervention. (c–e) NR4A2 was silenced for 48 h with small interfering RNA in NCI-H295R cells, followed by treatment with 4μg/ml RNLS for 16 h. The mRNA levels of related aldosterone synthases, including (c) CYP11B2, (d) CYP21A2, and (e) HSD3B2, were subsequently detected via qRT-PCR. (f) The protein expression of ATF/CREB family members and their phosphorylation levels were detected via WB. Quantitative statistical analysis of the phosphorylated protein to total protein ratios in f for (g) ATF1, (h) ATF2, (i) CREB, and (j) CREM. The members of the ATF/CREB family were silenced with the corresponding small interfering RNA, followed by the addition of RNLS, and the mRNA levels of the relevant aldosterone synthases, including (k) CYP11B2, (l) CYP21A2, and (m) HSD3B2, were detected via qRT-PCR.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Activity Assay, Quantitative RT-PCR, Small Interfering RNA, Expressing, Phospho-proteomics

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Activation of cAMP/PKA signalling induced by RNLS. (a) GO enrichment analysis and (b) KEGG enrichment analysis of DEGs between the human-derived APA and AAG transcriptomes. (c) PKACA and PKA phospho-substrate protein levels detected by WB. (d–e) Quantification of the PKACA and p-PKA substrates in c.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Activation Assay, Derivative Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Effect of cAMP signalling inhibition on the RNLS regulation of aldosterone synthase expression and aldosterone production. NCI-H295R cells were pre-treated with the PKA inhibitor H-89 and then incubated with 4 μg/ml RNLS for 16 h. (a) Cellular supernatant aldosterone levels were assayed by ELISA. The mRNA levels of related aldosterone synthases, including (b) CYP11B2, (c) CYP21A2, and (d) HSD3B2, were detected by qRT-PCR. (e) WB was used to examine the protein expression of the relevant aldosterone synthases. The relative expression of proteins in e was quantified, and the results are shown for (f) CYP11B2, (g) CYP21A2, and (h) HSD3B2. (i) WB assay for PKA phospho-substrate proteins. (j) Quantification of the p-PKA substrate in i. (k) qRT-PCR analysis of NR4A2. (l) WB detection of ATF/CREB family member proteins and their phosphorylated proteins. The phosphorylated protein to total protein ratio of ATF/CREB family members in l was quantified and statistically analysed, as shown for (m) ATF1, (n) ATF2, (o) CREB, and (p) CREM.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Inhibition, Expressing, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: RNLS colocalizes and interacts with PMCA4b. (a) Protein interaction networks associated with RNLS. The connecting lines indicate the presence of possible interactions, and the colour of the connecting line denotes the source of evidence: purple for experimentally determined, light blue for curated databases, dark blue for gene co-occurrence, light green for text mining, dark green for gene neighbourhood, and black for co-expression. (b) Immunofluorescence assay, with PMCA4b in red, RNLS in green, and nuclei in blue; scale bar = 50μm. (c) Co-IP was used to detect the interaction of RNLS with PMCA4b.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Expressing, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Renalase stimulates aldosterone production via PMCA4b/cAMP in NCI-H295R cells

doi: 10.1080/14756366.2025.2610034

Figure Lengend Snippet: Role of PMCA4b in aldosterone production stimulated by RNLS. NCI-H295R cells were pre-treated with siPMCA4b and then incubated with 4μg/ml RNLS for 16 hours. (a) Cellular supernatant aldosterone levels were assayed by ELISA. The mRNA levels of related aldosterone synthases, including (b) CYP11B2, (c) CYP21A2, and (d) HSD3B2, were detected via qRT-PCR. (e) WB was used to examine the protein expression of aldosterone synthase. The relative expression of proteins in e was quantified, and the results are shown for (f) CYP11B2, (g) CYP21A2, and (h) HSD3B2. (i) WB assay for PKA phospho-substrate proteins and PKACA. (j,k) Quantification of the p-PKA substrate and PKACA in i. (l) qRT-PCR analysis of NR4A2. (m) WB detection of ATF/CREB family member proteins and their phosphorylated proteins. The phosphorylated protein to total protein ratios of the ATF/CREB family members were quantified and statistically analysed as shown for (n) ATF1, (o) ATF2, (p) CREB, and (q) CREM.

Article Snippet: The cells were starved for 12 h and further treated with exogenous recombinant RNLS protein (Proteintech, Ag13061) or drugs such as H-89 (Beyotime, S1643).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: Wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice of both C57BL/6 and BalB/C backgrounds were subjected to 7-day 2.5% DSS administration in the drinking water. ( A ) Schematic of construction of DSS-induced colitis model. ( B ) Disease activity index (DAI), n = 25 per group. ( C ) Survival rate of WT ( n = 25) and Ccl5 -KO ( n = 25) mice was observed for 2 weeks, and the corresponding survival curves were plotted. ( D and E ) Gross anatomy of colons ( D ) and colon length ( E ) were monitored. Colon length was measured in both the C57BL/6 and BalB/C mouse strains after 7 days of 2.5% DSS treatment. The results are shown as the mean ± SEM, with statistical significance assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. ( F and G ) Representative H&E staining ( F ) of colon sections from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration (scale bars: 50 μm, 100 μm). Histological score ( G ) was quantified. Results shown are the mean ± SEM (ns, nonsignificant; ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Knock-Out, Activity Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A and B ) Flow cytometric analysis of number of CD4 + and CD8 + T cells in intestinal lymphoid tissues from mice with indicated genotypes 4 days after 7-day 2.5% dextran sulfate sodium salt (DSS) administration. Graphs ( B ) show the relative percentage of, respectively, CD4 + and CD8 + T cells ( n = 6 per group). ( C and D ) Representative CD4 staining of distal colon sections from wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice 4 days after 7-day 2.5% DSS administration ( C ; scale bars, 20 μm), with quantitative analysis ( D , n = 50); black arrows indicate CD4-positive cells. ( E and F ) Flow cytometric plots ( E ) of FOXP3 + CD4 + T cell population in intestines from control or DSS-treated mice with indicated genotypes ( n = 6 per group). Percentage of FOXP3 + population among CD4 + T cells is shown ( F , n = 6). ( G and H ) Representative FOXP3 staining of distal colon sections from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration ( G ; scale bars: 10 μm, 50 μm), with quantitative analysis ( H , n = 50); red arrows indicate FOXP3-positive cells. ( I ) Immunoblotting of FOXP3 expression in colonic tissues from WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. Results shown are the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Staining, Knock-Out, Control, Western Blot, Expressing

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Enzyme-linked immunosorbent assay (ELISA) analysis of the level of IL-33 in colon explant cultures at the indicated time points (days 0, 2, 4, 6, 8) during the 7-day dextran sulfate sodium salt (DSS) treatment in wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice ( n = 6 per group). ( B ) Immunoblotting of IL-33 and ST2 in intestinal epithelial cells of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( C ) Immunoblotting of NF-κB ( P65 ) related pathway in intestinal epithelium of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( D ) Immunofluorescence staining for P65 (red) in colonic sections of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration; DNA (DAPI, blue); scale bars, 50 μm. White arrows indicate P65-positive cells with nuclear translocation. ( E ) Immunoblotting of PI3K/Akt pathway in intestinal epithelium of WT and Ccl5 -KO mice 4 days after 7-day 2.5% DSS administration. ( F ) ELISA analysis of IL-33 levels in colon explant cultures of WT and Ccl5 -KO mice at the indicated time points (days 0, 2, 4, 6, 8) during 7-day DSS treatment with CCL5 small protein interventions ( n = 6 per group). ( G ) Colon length measurements on day 12 in Ccl5 -KO mice treated with different drug groups (control, CCL5 small protein, CCL5 small protein + BAY 11-7082, CCL5 small protein + Capivasertib); n = 6 per group. ( H ) Recording of DAI different time points ( D0, D3, D6, D9, D12 ) in different drug treatment groups during the treatment period. ( I ) Immunoblot analysis of corresponding protein levels in the intestinal epithelial tissues of Ccl5 -KO mice after treatment with different drug groups. ( J ) Immunofluorescence staining analysis of P65-positive (red) cells in the intestines of Ccl5 -KO mice treated with different drug groups (control, CCL5 small protein, CCL5 small protein + Capivasertib); DNA (DAPI, blue), scale bars, 50 μm. White arrows indicate P65-positive cells with nuclear translocation. Results shown are the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Enzyme-linked Immunosorbent Assay, Knock-Out, Western Blot, Immunofluorescence, Staining, Translocation Assay, Control

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Immunoblotting of IL-33, ST2, forkhead box protein 3 (FOXP3), and JAK1/STAT5 signaling in intestinal CD4 + T cells of wildtype (WT) and CCL5 knockout ( Ccl5 -KO) mice 4 days after 7-day 2.5% dextran sulfate sodium salt (DSS) administration. ( B and C ) Mice received daily intraperitoneal injections of rIL-33 protein (10 ng/µL) at the onset of the DSS regimen. The intraperitoneal administration of different therapeutic agents continued for an additional 4 days after 7-day DSS treatment. Subsequently, the mice were killed, and the affected intestinal tissues were analyzed. Disease activity index (DAI) ( B ) and colon length ( C ) were monitored, n = 6 per group. ( D and E ) Representative H&E staining ( D ) of colon sections in mice from different treated groups (scale bars, 50 μm). Histological score ( E ) was quantified. ( F and G ) Representative FOXP3 staining of distal colon sections from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment ( F ; scale bars, 20 μm) and quantitative analysis ( G , n = 50). ( H and I ) Flow cytometric plots ( H ) of FOXP3 + CD4 + T cell population in intestines from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment and quantitative analysis ( I , n = 6). ( J ) Immunoblotting of FOXP3 and JAK1/STAT5 signaling in intestinal CD4 + T cells, respectively, from rescued DSS-treated mice with indicated genotypes after 8-day rIL-33/PBS treatment. The results are shown as the mean ± SEM (ns, nonsignificant; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments, with statistical significance assessed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Western Blot, Knock-Out, Activity Assay, Staining

Journal: Clinical Science (London, England : 1979)

Article Title: CCL5 deficiency aggravates acute DSS-induced colitis by restricting IL-33-induced formation of Tregs in intestinal tract

doi: 10.1042/CS20256734

Figure Lengend Snippet: ( A ) Immunoblotting ( n = 12) of CCL5 and FOXP3 expression in inflamed tissue in ulcerative colitis (UC) patients. ( B ) Quantitative polymerase chain reaction (qPCR) analysis ( n = 32) of CCL5 expression, respectively, in intestinal epithelial (IECs) and stromal cells (SCs) from inflamed and adjacent normal tissue in UC patients. ( C and D ) Representative H&E staining (right), CCL5 (middle), and FOXP3 (left) staining of inflamed intestinal tissue from UC patients with different levels of CCL5 expression (CCL5 [high] vs. CCL5 [low]), ( C ; scale bars, 20 μm, 100 μm) and quantitative analysis of CCL5 and FOXP3 expression ( D , n = 32 per group) in inflamed (UC) and adjacent normal tissue (NC). ( E ) Correlation analysis between the proportion of CCL5-positive cells and FOXP3-positive cells in inflamed (UC) tissue from UC patients ( n = 32). ( F ) Quantitative real-time polymerase chain reaction (RT-qPCR) analysis of the correlation between CCL5 and FOXP3 expression levels in inflamed intestinal tissue ( n = 32) from UC patients with different levels of CCL5 expression. ( G ) Flow cytometric plots (right) of FOXP3 + CD4 + T cell population in intestines from CCL5-low and CCL5-high UC patient tissues with quantitative analysis (left, n = 6). Results shown are the mean ± SEM (ns, nonsignificant; *** P <0.001, **** P <0.0001) of triplicate determination from three independent experiments.

Article Snippet: To rescue the phenotypes of enteritis, exogenous recombinant murine CCL5 protein (HY- P71890 , MCE) [ ] was intraperitoneally injected at a concentration of 350 ng/kg from the onset of DSS treatment.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Staining, Quantitative RT-PCR