hy 141809 Search Results


rsl3  (MedChemExpress)
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MedChemExpress rsl3
<t>RSL3-induced</t> oxidative stress downregulates aldosterone synthase gene expression and aldosterone production in human adrenocortical cells. A. HAC15 cell viability after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 0–24 h (n = 3 independent experiments). B and C. Cell death analysis of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h by cytoimmunofluorescence and flow cytometry. Representative figures are shown, n = 3 independent experiments. Scale bars = 25 μm. D and E. MDA cytoimmunofluorescence (n = 3) and quantification and ROS assay (n = 4) of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars, 20 μm (upper), 5 μm (lower). F. Representative flow cytometry analysis images and quantification of BODIPY C11-positive cells (lipid peroxidation; n = 3 independent experiments) after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. G. ROS assay of HAC15 cells treated with 0–50 μM Erastin or 0–8 μM GPX4-IN-3 . H and I. TfR1 cytoimmunofluorescence and TfR1 Western blot analysis(n = 3) in HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars = 10 μm. J. CYP11B2 (aldosterone synthase) gene expression analysis of HAC15 cells treated with 2 μM RSL3 or 2 μM GPX4-IN-3 for 6 h, or 25 μM Erastin for 24 h (n = 3 independent experiments). K and L. CYP11B2 and CYP11B1 (11β-hydroxylase) gene expression analysis and quantification of secreted aldosterone from HAC15 cells treated with 10 nM AngII, 10 nM AngII+2 μM RLS3 or 10 nM AngII+2 μM RLS3+10 μM Lip-1 for 14 h (n = 3 independent experiments). Data were presented as the mean ± standard error of mean. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.
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RSL3-induced oxidative stress downregulates aldosterone synthase gene expression and aldosterone production in human adrenocortical cells. A. HAC15 cell viability after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 0–24 h (n = 3 independent experiments). B and C. Cell death analysis of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h by cytoimmunofluorescence and flow cytometry. Representative figures are shown, n = 3 independent experiments. Scale bars = 25 μm. D and E. MDA cytoimmunofluorescence (n = 3) and quantification and ROS assay (n = 4) of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars, 20 μm (upper), 5 μm (lower). F. Representative flow cytometry analysis images and quantification of BODIPY C11-positive cells (lipid peroxidation; n = 3 independent experiments) after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. G. ROS assay of HAC15 cells treated with 0–50 μM Erastin or 0–8 μM GPX4-IN-3 . H and I. TfR1 cytoimmunofluorescence and TfR1 Western blot analysis(n = 3) in HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars = 10 μm. J. CYP11B2 (aldosterone synthase) gene expression analysis of HAC15 cells treated with 2 μM RSL3 or 2 μM GPX4-IN-3 for 6 h, or 25 μM Erastin for 24 h (n = 3 independent experiments). K and L. CYP11B2 and CYP11B1 (11β-hydroxylase) gene expression analysis and quantification of secreted aldosterone from HAC15 cells treated with 10 nM AngII, 10 nM AngII+2 μM RLS3 or 10 nM AngII+2 μM RLS3+10 μM Lip-1 for 14 h (n = 3 independent experiments). Data were presented as the mean ± standard error of mean. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Journal: Redox Biology

Article Title: EGR1 regulates oxidative stress and aldosterone production in adrenal cells and aldosterone-producing adenomas

doi: 10.1016/j.redox.2025.103498

Figure Lengend Snippet: RSL3-induced oxidative stress downregulates aldosterone synthase gene expression and aldosterone production in human adrenocortical cells. A. HAC15 cell viability after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 0–24 h (n = 3 independent experiments). B and C. Cell death analysis of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h by cytoimmunofluorescence and flow cytometry. Representative figures are shown, n = 3 independent experiments. Scale bars = 25 μm. D and E. MDA cytoimmunofluorescence (n = 3) and quantification and ROS assay (n = 4) of HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars, 20 μm (upper), 5 μm (lower). F. Representative flow cytometry analysis images and quantification of BODIPY C11-positive cells (lipid peroxidation; n = 3 independent experiments) after treatment with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. G. ROS assay of HAC15 cells treated with 0–50 μM Erastin or 0–8 μM GPX4-IN-3 . H and I. TfR1 cytoimmunofluorescence and TfR1 Western blot analysis(n = 3) in HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Scale bars = 10 μm. J. CYP11B2 (aldosterone synthase) gene expression analysis of HAC15 cells treated with 2 μM RSL3 or 2 μM GPX4-IN-3 for 6 h, or 25 μM Erastin for 24 h (n = 3 independent experiments). K and L. CYP11B2 and CYP11B1 (11β-hydroxylase) gene expression analysis and quantification of secreted aldosterone from HAC15 cells treated with 10 nM AngII, 10 nM AngII+2 μM RLS3 or 10 nM AngII+2 μM RLS3+10 μM Lip-1 for 14 h (n = 3 independent experiments). Data were presented as the mean ± standard error of mean. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: ROS levels in HAC15 cells treated with RSL3, GPX4-IN-3 (MedChemExpress, HY-141809) or Erastin (TOCRIS, 5449) were assessed using the DCFDA/H2DCFDA kit (Abcam, ab113851) following the manufacturer's protocol.

Techniques: Expressing, Flow Cytometry, ROS Assay, Western Blot

Identification and validation of oxidative stress-related genes in oxidative stress-induced human adrenocortical cells. A. Principal component analysis (PCA) of RNA-sequencing data of HAC15 cells treated with RSL3 (0–4 μM) for 3 h (three biologically independent samples for each condition). B. Heatmap showing the differentially expressed genes (DEGs) of RNA-sequencing. C. Volcano plot highlighting genes of interest (GOIs) in HAC15 cells induced by 4 μM RLS3 for 3 h compared to DMSO. The color scale bar indicates log 10 (adjusted p value). D, Related pathways in GSEA enrichment analysis in HAC15 cells treated with 4 μM RSL3. E, Venn diagram highlighting overlapping DEGs of interest induced by 4 μM RLS3 versus DMSO vehicle-only control (RNA sequencing) and in APA versus paired adjacent cortex (spatial transcriptomics). F, Expression of genes of interest in APA and adjacent adrenal cortex from GEO dataset GSE60042 (7 patients). G, Venn diagram showing common genes between the GOIs shown in panel F and ferroptotic genes from the FerrDb database. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Journal: Redox Biology

Article Title: EGR1 regulates oxidative stress and aldosterone production in adrenal cells and aldosterone-producing adenomas

doi: 10.1016/j.redox.2025.103498

Figure Lengend Snippet: Identification and validation of oxidative stress-related genes in oxidative stress-induced human adrenocortical cells. A. Principal component analysis (PCA) of RNA-sequencing data of HAC15 cells treated with RSL3 (0–4 μM) for 3 h (three biologically independent samples for each condition). B. Heatmap showing the differentially expressed genes (DEGs) of RNA-sequencing. C. Volcano plot highlighting genes of interest (GOIs) in HAC15 cells induced by 4 μM RLS3 for 3 h compared to DMSO. The color scale bar indicates log 10 (adjusted p value). D, Related pathways in GSEA enrichment analysis in HAC15 cells treated with 4 μM RSL3. E, Venn diagram highlighting overlapping DEGs of interest induced by 4 μM RLS3 versus DMSO vehicle-only control (RNA sequencing) and in APA versus paired adjacent cortex (spatial transcriptomics). F, Expression of genes of interest in APA and adjacent adrenal cortex from GEO dataset GSE60042 (7 patients). G, Venn diagram showing common genes between the GOIs shown in panel F and ferroptotic genes from the FerrDb database. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: ROS levels in HAC15 cells treated with RSL3, GPX4-IN-3 (MedChemExpress, HY-141809) or Erastin (TOCRIS, 5449) were assessed using the DCFDA/H2DCFDA kit (Abcam, ab113851) following the manufacturer's protocol.

Techniques: RNA Sequencing Assay, Control, Expressing

EGR1 expression in oxidative stress-induced adrenocortical cells and aldosterone-producing adenomas versus adjacent adrenal cortex. A. Gene expression analysis of EGR1 in HAC15 cells treated with 4 μM RSL3 or 2 μM GPX4-IN-3 for 4 h or 25 μM Erastin for 24 h (n = 3 independent experiments). B and C. Cytoimmunofluorescence and Western blot and gene expression analysis of EGR1 expression in HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Representative figures are shown, n = 3 independent experiments. Scale bars = 20 μm. D. Gene expression analysis of EGR1 in APA and adjacent cortex of 10 patients with and 10 without KCNJ5 mutated APAs. E. CYP11B2 (aldosterone synthase) and EGR1 immunohistochemistry was performed on 18 adrenal glands, comprising nine with KCNJ5 -mutated APAs and nine with wild-type KCNJ5 APAs and EGR1-immunopositive cells were quantified. Representative immunostainings are shown (left) and the proportion of EGR1-positive cells is also shown (right). Scale bars, 200 μm (left), 50 μm (right). F. Representative CYP11B2-EGR1 double-immunofluorescence of an APA and an APM, with quantification in nine APAs and nine APMs. Scale bar = 10 μm. G. Spatial transcriptome data showing inverse CYP11B2 and EGR1 gene expression profiles in an adrenal sample with an APA. H. Integrated CYP11B2 and EGR1 gene expression profiles from spatial transcriptome data of six adrenal samples with an APA and attached adjacent adrenal cortex in the same captured area. ∗ P < 0.05, ∗∗ P < 0.01.

Journal: Redox Biology

Article Title: EGR1 regulates oxidative stress and aldosterone production in adrenal cells and aldosterone-producing adenomas

doi: 10.1016/j.redox.2025.103498

Figure Lengend Snippet: EGR1 expression in oxidative stress-induced adrenocortical cells and aldosterone-producing adenomas versus adjacent adrenal cortex. A. Gene expression analysis of EGR1 in HAC15 cells treated with 4 μM RSL3 or 2 μM GPX4-IN-3 for 4 h or 25 μM Erastin for 24 h (n = 3 independent experiments). B and C. Cytoimmunofluorescence and Western blot and gene expression analysis of EGR1 expression in HAC15 cells treated with RSL3 (0–4 μM) or 10 μM Lip-1+4 μM RLS3 for 4 h. Representative figures are shown, n = 3 independent experiments. Scale bars = 20 μm. D. Gene expression analysis of EGR1 in APA and adjacent cortex of 10 patients with and 10 without KCNJ5 mutated APAs. E. CYP11B2 (aldosterone synthase) and EGR1 immunohistochemistry was performed on 18 adrenal glands, comprising nine with KCNJ5 -mutated APAs and nine with wild-type KCNJ5 APAs and EGR1-immunopositive cells were quantified. Representative immunostainings are shown (left) and the proportion of EGR1-positive cells is also shown (right). Scale bars, 200 μm (left), 50 μm (right). F. Representative CYP11B2-EGR1 double-immunofluorescence of an APA and an APM, with quantification in nine APAs and nine APMs. Scale bar = 10 μm. G. Spatial transcriptome data showing inverse CYP11B2 and EGR1 gene expression profiles in an adrenal sample with an APA. H. Integrated CYP11B2 and EGR1 gene expression profiles from spatial transcriptome data of six adrenal samples with an APA and attached adjacent adrenal cortex in the same captured area. ∗ P < 0.05, ∗∗ P < 0.01.

Article Snippet: ROS levels in HAC15 cells treated with RSL3, GPX4-IN-3 (MedChemExpress, HY-141809) or Erastin (TOCRIS, 5449) were assessed using the DCFDA/H2DCFDA kit (Abcam, ab113851) following the manufacturer's protocol.

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence

EGR1 promotes oxidative stress and inhibits aldosterone production in human adrenocortical cells. A and B. Gene expression (n = 6 independent experiments) and Western blot analysis (n = 3) of EGR1 in HAC15 cells after EGR1 gene silencing or overexpression. NC, negative control. EV, empty vector. Cell viability in HAC15 cells after EGR1 silencing ( A , n = 4) and EGR1 -overexpression ( B , n = 3) treated with 2 μM RSL3 or 10 μM Lip-1+2 μM RLS3 for 4 h. ROS level in the EGR1 -silenced and EGR1 -overexpressing HAC15 cells treated with RSL3 or Lip-1+ RLS3 for 4 h ( A, B, n = 4 independent experiments). C, Representative MDA staining in the HAC15 EGR1 -silenced HAC15 cells treated with 4 μM RSL3 for three and 4 h, and EGR1 -overexpressing HAC15 cells treated with 4 μM RSL3 for three and 4 h. Scale bars = 25 μm. D and E. Gene expression analysis of CYP11B2 after EGR1 silencing or overexpression in HAC15 cells (n = 6 independent experiments). CYP11B2 gene expression and aldosterone production after EGR1 silencing of HAC15 cells treated with 10 nM AngII, 2 μM RSL3 or 10 nM AngII+2 μM RSL3 for 12 h ( D , n = 3 independent experiments). CYP11B2 gene expression and aldosterone production in EGR1 overexpressing HAC15 cells treated with and without 10 nM AngII for 12 h ( E , n = 3 independent experiments). Data were presented as the mean ± standard error of mean. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Journal: Redox Biology

Article Title: EGR1 regulates oxidative stress and aldosterone production in adrenal cells and aldosterone-producing adenomas

doi: 10.1016/j.redox.2025.103498

Figure Lengend Snippet: EGR1 promotes oxidative stress and inhibits aldosterone production in human adrenocortical cells. A and B. Gene expression (n = 6 independent experiments) and Western blot analysis (n = 3) of EGR1 in HAC15 cells after EGR1 gene silencing or overexpression. NC, negative control. EV, empty vector. Cell viability in HAC15 cells after EGR1 silencing ( A , n = 4) and EGR1 -overexpression ( B , n = 3) treated with 2 μM RSL3 or 10 μM Lip-1+2 μM RLS3 for 4 h. ROS level in the EGR1 -silenced and EGR1 -overexpressing HAC15 cells treated with RSL3 or Lip-1+ RLS3 for 4 h ( A, B, n = 4 independent experiments). C, Representative MDA staining in the HAC15 EGR1 -silenced HAC15 cells treated with 4 μM RSL3 for three and 4 h, and EGR1 -overexpressing HAC15 cells treated with 4 μM RSL3 for three and 4 h. Scale bars = 25 μm. D and E. Gene expression analysis of CYP11B2 after EGR1 silencing or overexpression in HAC15 cells (n = 6 independent experiments). CYP11B2 gene expression and aldosterone production after EGR1 silencing of HAC15 cells treated with 10 nM AngII, 2 μM RSL3 or 10 nM AngII+2 μM RSL3 for 12 h ( D , n = 3 independent experiments). CYP11B2 gene expression and aldosterone production in EGR1 overexpressing HAC15 cells treated with and without 10 nM AngII for 12 h ( E , n = 3 independent experiments). Data were presented as the mean ± standard error of mean. ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: ROS levels in HAC15 cells treated with RSL3, GPX4-IN-3 (MedChemExpress, HY-141809) or Erastin (TOCRIS, 5449) were assessed using the DCFDA/H2DCFDA kit (Abcam, ab113851) following the manufacturer's protocol.

Techniques: Expressing, Western Blot, Over Expression, Negative Control, Plasmid Preparation, Staining

Suppression of oxidative stress under conditions of hyperaldosteronism. A. Immunofluorescence and quantification showing increased expression of the oxidative stress marker COX-2 in HAC15 cells treated with the ferroptosis inducer RSL3 but not with an inducer of apoptosis staurosporine (STS) (n = 3 independent experiments). B. CYP11B2 and COX-2 immunohistochemistry showing reduced COX-2 immunostaining in the APA compared to the adjacent cortex. Scale bars, 2 mm (upper left), 50 μm (lower right). C. CYP11B2, COX-2, 4-HNE and MDA immunohistochemistry and EGR1 and MDA immunofluorescence of an APA and its adjacent cortex. The immunostainings are from the same adrenal sample. Scale bars, 50 μm. D. MDA immunostaining in patients with APA. Scale bars, 2 mm (upper left), 200 μm (upper right), 50 μm (lower right). E. KCNJ5, EGR1, and 4-HNE immunohistochemistry and KCNJ5-EGR1 double immunofluorescence of adrenal glands from pigs fed a low or high sodium diet. Quantification of EGR1 expression was performed in two regions from each pig adrenal gland. KCNJ5 is a marker of aldosterone-producing outer zona glomerulosa layer in pig adrenal glands. Sodium restriction activates the renin-angiotensin-aldosterone system and stimulates expansion of the adrenal zona glomerulosa layer and aldosterone production. Immunostaining of EGR1 and the oxidative stress marker 4-HNE are markedly reduced in adrenals of pigs with hyperaldosteronism caused by low sodium compared to a high sodium diet. Scale bars, 1 mm (upper), 50 μm (lower). Representative figures are shown.

Journal: Redox Biology

Article Title: EGR1 regulates oxidative stress and aldosterone production in adrenal cells and aldosterone-producing adenomas

doi: 10.1016/j.redox.2025.103498

Figure Lengend Snippet: Suppression of oxidative stress under conditions of hyperaldosteronism. A. Immunofluorescence and quantification showing increased expression of the oxidative stress marker COX-2 in HAC15 cells treated with the ferroptosis inducer RSL3 but not with an inducer of apoptosis staurosporine (STS) (n = 3 independent experiments). B. CYP11B2 and COX-2 immunohistochemistry showing reduced COX-2 immunostaining in the APA compared to the adjacent cortex. Scale bars, 2 mm (upper left), 50 μm (lower right). C. CYP11B2, COX-2, 4-HNE and MDA immunohistochemistry and EGR1 and MDA immunofluorescence of an APA and its adjacent cortex. The immunostainings are from the same adrenal sample. Scale bars, 50 μm. D. MDA immunostaining in patients with APA. Scale bars, 2 mm (upper left), 200 μm (upper right), 50 μm (lower right). E. KCNJ5, EGR1, and 4-HNE immunohistochemistry and KCNJ5-EGR1 double immunofluorescence of adrenal glands from pigs fed a low or high sodium diet. Quantification of EGR1 expression was performed in two regions from each pig adrenal gland. KCNJ5 is a marker of aldosterone-producing outer zona glomerulosa layer in pig adrenal glands. Sodium restriction activates the renin-angiotensin-aldosterone system and stimulates expansion of the adrenal zona glomerulosa layer and aldosterone production. Immunostaining of EGR1 and the oxidative stress marker 4-HNE are markedly reduced in adrenals of pigs with hyperaldosteronism caused by low sodium compared to a high sodium diet. Scale bars, 1 mm (upper), 50 μm (lower). Representative figures are shown.

Article Snippet: ROS levels in HAC15 cells treated with RSL3, GPX4-IN-3 (MedChemExpress, HY-141809) or Erastin (TOCRIS, 5449) were assessed using the DCFDA/H2DCFDA kit (Abcam, ab113851) following the manufacturer's protocol.

Techniques: Immunofluorescence, Expressing, Marker, Immunohistochemistry, Immunostaining