rabbit anti chac1  (Proteintech)

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Cell viability of eight human cell lines, treated with DMSO or 1, 5, 10, 20, and 40 μM erastin, was measured by a CellTiter-Glo luminescent cell viability assay. b : Quantification of GSSG in eight human cell lines treated with DMSO, or 10 μM or 20 μM erastin. c: H1299 and SKOV3 cells transfected with CHAC1 siRNA and then treated with 10 μM erastin. The mRNA level of CHAC1 was analysed by RT-qPCR.

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Cell Viability Assay, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : S-glutathionylated proteins in eight human cell lines treated with DMSO, or 10 μM or 20 μM erastin were analysed using western blotting with anti-glutathione. Ponceau S staining were used as internal references. b : Quantification of GSH and GSSG in eight human cell lines treated with DMSO, or 10 μM or 20 μM erastin. c : CHAC1 mRNA and protein expression in eight human cell lines treated with DMSO or 10 μM, 20 μM erastin for 24 h were analysed using RT-qPCR and western blotting respectively. β-ACTIN were used as internal references for western blotting. d : GLRX mRNA expression in eight human cell lines treated with DMSO, or 10 μM or 20 μM erastin for 24 h. e : H1299 cells transfected with CHAC1 small interfering RNA (siRNA) and then treated with 10 μM erastin. The levels of GSH and GSSG were quantified by GSSG/GSH Quantification Kit. CHAC1 and S-glutathionylated proteins were analysed by Western blots and quantified. β-ACTIN and Ponceau S staining were used as internal references. f : SKOV3 cells transfected with CHAC1 siRNA and then treated with 10 μM erastin. The levels of GSH and GSSG were quantified by GSSG/GSH Quantification Kit. CHAC1 and S-glutathionylated proteins were analysed by Western blots and quantified. β-ACTIN and Ponceau S staining were used as internal references. g : Cell viability of H1299 cells transfected with CHAC1 siRNA and then treated with DMSO, 10 μM, or 20 μM erastin for 24 h was measured by CellTiter-Glo® luminescent cell viability assay. h : Cell viability of SKOV3 cells transfected with CHAC1 siRNA and then treated with DMSO, 10 μM, or 20 μM erastin for 24 h. i : Cell viability of PMHs and Hepa1-6 cells treated with DMSO, or 10 or 20 μM erastin for 24 h. j : S-glutathionylated proteins in PMHs and Hepa1-6 cells treated with DMSO or 10 μM, 20 μM erastin was analysed by Western blots. Ponceau S staining was used as an internal reference. k : Chac1 mRNA expression in PMHs and Hepa1-6 cells treated with DMSO or 10 μM erastin for 12 h was analysed by RT-qPCR. l : Cell viability of Ad-GFP or Ad-CHAC1 PMH and Hepa1-6 treated with DMSO, or 10 μM or 20 μM erastin for 24 h. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Western Blot, Staining, Expressing, Quantitative RT-PCR, Transfection, Small Interfering RNA, Cell Viability Assay

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Volcano plots shows upregulated and downregulated genes from RNA transcriptome data of group treated with 750 mg/kg APAP for 6 h compared to the saline group (Fold change ≥ 1.5, P < 0.05; n = 3 mice/group). b : Chac1 mRNA expression in mouse liver tissues treated with saline or 300 and 750 mg/kg APAP for 3, 6, or 12 h. c : Chac1 mRNA expression in PMHs with or without 20 mM APAP challenge for 1, 3, 6, and 12 h was analysed by RT-qPCR. d : Immunohistochemical staining of CHAC1 in liver sections from healthy controls ( n = 9) and patients with AILI ( n = 9), followed by immunohistochemical score of liver tissue. The black arrow indicates positive staining. Scale bar = 100 μm. e, f : Quantification of GSH and GSSG in the liver tissues of Chac1 + /+ Ad-GFP, Chac1 - /- Ad-GFP, and Chac1 - /- Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 and 6 h. g : S-glutathionylated proteins in the liver tissues of Chac1 + /+ Ad-GFP and Chac1 - /- Ad-GFP mice treated with 300 mg/kg APAP for 2 and 6 h were analysed by western blots with anti-glutathione. Ponceau S staining was used as an internal reference. The relative expression levels of S-glutathionylated proteins after APAP treatment for 6 h were quantified. h : S-glutathionylated proteins and CHAC1-FLAG protein in the liver tissues of Chac1 - /- Ad-GFP and Chac1 - /- Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h were analysed by western blotting. i : Serum levels of ALT and AST in Chac1 + /+ Ad-GFP, Chac1 - /- Ad-GFP, and Chac1 - /- Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 6 h. j : H&E staining, TUNEL staining and 4-HNE protein adducts staining in liver tissues of Chac1 + /+ Ad-GFP, Chac1 - /- Ad-GFP and Chac1 - /- Ad-CHAC1 mice treated with 300 mg/kg APAP for 6 h. Scale bars = 200 μm. The hepatocyte necrosis area, TUNEL-positive area, and immunohistochemical score for 4-HNE protein adduct staining were quantified. AILI, APAP-induced liver injury; ALT, alanine aminotransferase; APAP, acetaminophen; AST, aspartate aminotransferase; H&E, haematoxylin and eosin; PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Saline, Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Western Blot, TUNEL Assay

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : The heatmap shows the relative levels of upregulated and downregulated genes in the liver tissue of mice treated with 300 mg/kg APAP for 3 h, 750 mg/kg APAP for 3 h, 300 mg/kg APAP for 6 h, and 750 mg/kg APAP for 6 h compared to the saline group. The heatmap was ranked by fold change of 750 mg/kg APAP for 3 h group to saline for 3 h group. (Fold change ≥ 1.5, P < 0.05) ( n = 3 mice/group). b : Volcano plots shows upregulated and downregulated genes from RNA transcriptome data of groups treated with 300 mg/kg APAP for 3 h, 750 mg/kg APAP for 3 h, and 300 mg/kg APAP for 6 h compared to the saline group. (Fold change ≥ 1.5, P < 0.05) (n = 3 mice/group). c : Strategies for Chac1 knockout and gene identification. The Chac1 gene (NM_026929) has three exons, with the ATG start codon in exon 1 and the TGA stop codon in exon 3. Exons 1–3 were selected as the target sites. Cas9 and gRNA were co-injected into fertilized eggs to produce for Chac1 knockout (KO) mice. d : Genotyping of experimental mice. e : Chac1 mRNA expression in liver tissues from Chac1 + /+ or Chac1 - /- mice treated with saline or 300 mg/kg APAP for 6 h analysed by RT-qPCR ( n = 3 mice/group, t test). f . Chac1 mRNA expression in PMHs from Chac1 + /+ or Chac1 - /- mice treated with DMEM or 20 mM APAP for 2 h, 6 h, and 12 h analysed by RT-qPCR. g : The ratio of GSH/GSSG in liver tissues of Chac1 + /+ Ad-GFP, Chac1 - /- Ad-GFP and Chac1 - /- Ad-CHAC1 mice treated with saline or 300 mg/kg APAP for 2 h and 6 h. h : S-glutathionylated proteins in liver tissues of Chac1 + /+ Ad-GFP and Chac1 - /- Ad-GFP mice treated with saline or 300 mg/kg APAP for 2 h. Ponceau S staining was used as internal reference. APAP, acetaminophen; PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Saline, Knock-Out, Injection, Expressing, Quantitative RT-PCR, Staining

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : S-glutathionylated proteins in PMHs infected with Ad-GFP or Ad-CHAC1 adenovirus with MOI=0.1, 2.5, 5, and 10 for 36 h and then treated with 20 mM APAP for 6 h were analysed by western blotting with anti-glutathione. Ponceau S staining was used as internal reference. b : The ratio of GSH/GSSG in PMHs infected with Ad-GFP or Ad-CHAC1 adenovirus with MOI=0.1, 2.5, 5, 10 for 12 h and then treated with 20 mM APAP for 6 h. c : Quantification of GSH and GSSG and the ratio of GSH/GSSG in PMHs infected with Ad-GFP or Ad-CHAC1 with MOI=0.1, 2.5, 5, and 10 for 36 h and then treated with 20 mM APAP for 6 h. APAP, acetaminophen; GSH, glutathione; GSSG, oxidised glutathione

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Infection, Western Blot, Staining

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : S-glutathionylated proteins in PMHs from Chac1 + /+ and Chac1 - /- mice treated with 20 mM APAP for 3 h were analysed by western blotting with anti-glutathione. Ponceau S staining was used as an internal reference. b : S-glutathionylated proteins in PMHs infected with Ad-GFP or Ad-CHAC1 at a multiplicity of infection (MOI)=0.1, 2.5, 5, and 10 for 12 h and then treated with 20 mM APAP for 6 h were analysed by western blotting with anti-glutathione. Ponceau S staining was used as an internal reference. c : Quantification of GSH and GSSG in PMHs infected with Ad-GFP or Ad-CHAC1 adenovirus at MOI=0.1, 2.5, 5, and 10 for 12 h and then treated with 20 mM APAP for 6 h ( n = 2, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 via t test). d: PMHs were isolated from Chac1 + /+ and Chac1 - /- mice and treated with 20 mM APAP for 12 h. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay ( n = 5, t test). e : Isolated Chac1 + /+ and Chac1 - /- PMHs were infected at MOI of 10 for 36 h and then treated with 20 mM APAP. After 6 h, TUNEL staining was performed for nuclear DNA fragmentation (scale bars = 200 μm). f : Determination of malondialdehyde (MDA) levels in PMHs infected with Ad-GFP or Ad-CHAC1 adenoviruses at MOI=10 for 36 h and then treated with 20 mM APAP for 12 h. g : PMHs from Chac1 + /+ and Chac1 - /- mice were treated with 20 mM APAP for 12 h. Representative images of C11 Bodipy 581/591 fluorescence. Red fluorescence represents non-lipid oxidation, and green fluorescence represents lipid oxidation. The statistical chart shows the ratio of green to red fluorescence (scale bars = 200 μm). APAP, acetaminophen; GSH, glutathione; GSSG, oxidised glutathione; PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Western Blot, Staining, Infection, Isolation, Cell Viability Assay, TUNEL Assay, Fluorescence

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Flowchart outlining the key experimental procedures for proteomic analysis of S-glutathionylation. b : Overview of the identification of modification sites. c : The histogram shows the distribution of differential modification sites/proteins among different comparison groups (Coefficient of Variation (CV) < 0.1, fold change ≥ 1.2). d : Scatter plot showing the distribution of differential modification sites sorted by the ratio of Ad-GFP + APAP/Ad-GFP. Red dots indicating upregulation of significant differences, blue dots indicating significant differences down-regulation, and grey indicating no significant differences (CV < 0.1, fold change ≥ 1.2). e : Scatter plot showing the distribution of differential modification sites sorted by the ratio of Ad-CHAC1 + APAP/Ad-GFP + APAP. Red dots indicating up-regulation of significant differences, blue dots indicating significant differences down-regulation, and grey indicating no significant differences (CV < 0.1, fold change ≥ 1.2). f : Venn diagram shows differentially modified sites both under APAP stimulation and CHAC1 overexpression (Fold change ≥ 1.2). g : The heatmap shows the union of differential modification sites in Ad-GFP, Ad-GFP + APAP, Ad-CHAC1, and Ad-CHAC1 + APAP comparison groups (CV < 0.1, fold change ≥ 1.2). h : The scatter plot shows differentially modified sites both under APAP stimulation and CHAC1 overexpression; the order was sorted by the ratio of Ad-GFP + APAP / Ad-GFP (CV < 0.1, fold change ≥ 1.2). PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Modification, Comparison, Over Expression

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: GO enrichment of differentially modified glutathionylated proteins both under APAP stimulation and CHAC1 overexpression.

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Modification, Over Expression

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Two-stage mass spectrometry of the glutathionylated peptide from ARF6. The secondary mass spectrum shows fragment ion information of the ARF6 C90 peptide segment. b : Histogram showing the relative modification abundance of ARF6 C90 in different treatment groups, with glutathionylated peptides identified and quantified by LC-MS/MS (all values were standardised by the mean of the AdGFP-CON group). c : Illustrations of predicted 3D structures of ARF6 and Cys90 glutathionylated ARF6 from PyMOL. Structural alignment of modelled ARF6 and modelled Cys90 glutathionylated ARF6 (cyan: before modification; green: after modification). d : Regions with significant changes in the predicted 3D structures of ARF6 and Cys90 glutathionylated ARF6 from PyMOL. Structural alignment of the modelled ARF6 and Cys90 glutathionylated ARF6 (blue: before modification; red: after modification). e : Docking score between ARF6 and GTP and the docking score between Cys90 glutathionylated ARF6 and GTP. f : PMHs were isolated from C57BL/6 mice and infected with Ad-CHAC1 or Ad-GFP at an MOI of 10 for 36 h, followed by treatment with 20 mM APAP for 6 h. Proteins were extracted and fractionated into cytosolic and membrane fractions. The ARF6 protein was analysed using western blotting. β-ACTIN marks the cytosol, and Na + /K + -ATPase marks the membrane. Statistical chart shows the ratio of ARF6 in the plasma membrane to ARF6 in the cytosol. g : PMHs were isolated from Chac1 + /+ or Chac1 - /- mice and treated with 20 mM APAP for 6 h. Proteins were extracted and fractionated into cytosolic and membrane fractions. Protein ARF6 was analysed using western blotting. β-ACTIN marks the cytosol, and Na + /K + -ATPase marks the membrane. Statistical chart shows the ratio of ARF6 in the plasma membrane to ARF6 in the cytosol. h : Pull-down assay showing the expression of activated ARF6 in Ad-CHAC1 or Ad-GFP PMHs treated with 20 mM APAP for 6 h (pull-down, ARF6-GTP; IB, ARF6). Whole cell lysates to confirm the expression of ARF6. i : Expression of the ARF6-Myc-tag protein in AML12 cells transfected with CON, WT, C90A, or C90D plasmids and treated with APAP for 6 h was analysed using western blotting. GAPDH marks the cytosol and Na + /K + -ATPase marks the membrane. Statistical chart shows the ratio of ARF6 in the plasma membrane to ARF6 in the cytosol. j : The viability of AML12 cells transfected with CON, WT, C90A, or C90D plasmids and treated with APAP for 12 h was quantified using the CellTiter-Glo luminescent cell viability assay. PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Mass Spectrometry, Modification, Liquid Chromatography with Mass Spectroscopy, Isolation, Infection, Membrane, Western Blot, Clinical Proteomics, Pull Down Assay, Expressing, Transfection, Cell Viability Assay

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Arf6 mRNA expression in PMHs transfected with Arf6 siRNA for 48 h and then treated with DMEM or 20 mM APAP, as analysed by RT-qPCR. b : PMHs were transfected with Arf6 siRNA and treated with 20 mM APAP. Cell viability was measured using the CellTiter-Glo luminescent cell viability assay. The ratio of dead to live cells was measured using calcein-AM/PI live/dead cell staining. c : Representative images of C11 Bodipy 581/591 fluorescent probe, which was used for detecting the formation of lipid peroxides (Red: non-lipid oxidation; green: lipid oxidation, Scale bars = 200 μm). The statistical chart shows the ratio of green to red fluorescence. d : Representative images of the FerroOrange fluorescent probe used to detect labile ferrous ions (Red: FerroOrange; blue: DAPI, Scale bars = 200 μm). The statistical chart shows the relative Fe 2+ fluorescence intensity. e : ARF6 and TFRC protein expression in PMHs transfected with Arf6 siRNA and treated with 20 mM APAP was analysed using western blotting. β-ACTIN marks the cytosol, and Na + /K + -ATPase marks the membrane. The statistical chart shows the ratio of TFRC in the plasma membrane to TFRC in the cytosol. f : ARF6 and TFRC protein expression in PMHs infected with Ad-GFP or Ad-CHAC1 adenovirus and treated with 20 mM APAP was analysed by western blotting. β-ACTIN marks the cytosol, and Na + /K + -ATPase marks the membrane. The statistical chart shows the ratio of TFRC in the plasma membrane to TFRC in the cytosol. g : Immunofluorescence staining (with ARF6 and TFRC) in PMHs infected with Ad-GFP or Ad-CHAC1 adenovirus and then treated with 20 mM APAP (Red: ARF6; purple: TFRC; blue: DAPI, Scale bars = 200 μm). h : Representative images of the transferrin fluorescent probe (red: transferrin; blue: DAPI, Scale bars = 200 μm). The statistical chart shows the relative transferrin content. i : Representative images of the FerroOrange fluorescent probe (Red: FerroOrange; green: GFP, Scale bars = 200 μm). The statistical chart shows the relative Fe 2+ fluorescence intensity. PMH, primary mouse hepatocyte

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Cell Viability Assay, Staining, Fluorescence, Western Blot, Membrane, Clinical Proteomics, Infection, Immunofluorescence

Journal: bioRxiv

Article Title: Protein S-glutathionylation confers cell resistance to ferroptosis

doi: 10.1101/2024.05.03.592374

Figure Lengend Snippet: a : Expression of the ARF6-Myc-tag protein in 293T cells transfected with CON, WT, C90A, or C90D plasmids and treated with 10 μM erastin was analysed using western blotting. GAPDH marks the cytosol, and Na + /K + -ATPase marks the membrane. Statistical chart shows the ratio of ARF6-Myc in the plasma membrane to ARF6-Myc in the cytosol. b : Expression of the ARF6-Myc-tag protein in H1299 cells transfected with CON, WT, C90A, or C90D plasmids and treated with 10 μM erastin was analysed by western blotting. GAPDH marks the cytosol, and Na + /K + -ATPase marks the membrane. Statistical chart shows the ratio of ARF6-Myc in the plasma membrane to ARF6-Myc in the cytosol. c : The viability of H1299 cells transfected with CON, WT, C90A, or C90D plasmids and treated with 10 μM erastin was quantified using the CellTiter-Glo luminescent cell viability assay. d : RNA-seq analysis using ARF6-myc (WT) or ARF6-C90A-myc transfected H1299 cells, with or without erastin treatment. Venn plot, GO and KEGG enrichment for genes upregulated only in the ARF6-C90A group but not in the WT group, genes downregulated only in the ARF6-C90A group but not in the WT group, genes only upregulated in WT group but not in the ARF6-C90A group upon 10 μM erastin treatment. e : Schematic presentation of the effect of decreased protein S-glutathionylation caused by CHAC1-induced glutathione deprivation on cell susceptibility to ferroptosis.

Article Snippet: Sections were incubated with an anti-rabbit CHAC1 (Proteintech, Cat. # 15207-1-AP) and anti-rabbit 4HNE (Abcam, Cat. # ab46545) antibody at 4°C overnight.

Techniques: Expressing, Transfection, Western Blot, Membrane, Clinical Proteomics, Cell Viability Assay, RNA Sequencing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Differential expression of CHAC1 in KIRC. A, Pan‐cancer analysis of the expression of CHAC1. B, The expression of CHAC1 in KIRC samples and normal samples. C, The expression of CHAC1 in KIRC samples and their paired normal samples. D, The expression of CHAC1 in KIRC samples with different T stages. E, The expression of CHAC1 in KIRC samples with different grades. F, The expression of CHAC1 in KIRC samples with different total stages. G, The overall survival curve of KIRC. (* P < .05; ** P < .01; *** P < .001)

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques: Quantitative Proteomics, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Establish of predict model for the prognosis of KIRC. A, Univariate cox analysis of the factors associated with overall survival of KIRC. B, Multivariate cox regression analysis of the factors associated with overall survival of KIRC. C, Multi‐ROC analysis of CHAC1 and the conventional prognostic factors. D, The nomogram model for predicting the prognosis of KIRC. E‐G, The calibration curves of the nomogram model for predicting patients' 1‐y survival, 3‐y survival and 5‐y survival. H‐J, The ROC curves of nomogram‐based model for predicting patients' 1‐y survival, 3‐y survival and 5‐y survival of KIRC

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Immunological features related to CHAC1 in KIRC. A, The correlation analysis of CHAC1 expression and neoantigens. B, The correlation analysis of CHAC1 expression and MSI. C, The correlation analysis of CHAC1 expression and TMB. D, ESTIMATE: the correlation analysis of CHAC1 expression and tumour microenvironment. E, TIMER: the correlation analysis of CHAC1 expression and infiltration of immune cells. F, The correlation analysis of CHAC1 expression and acknowledged markers of immune pathway. G, The correlation analysis of CHAC1 expression and checkpoint genes. (* P < .05; ** P < .01; *** P < .001)

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques: Expressing, Immunopeptidomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Correlation analyses of CHAC1 and MMR genes as well as methylation transferases in KIRC. A, The correlation analysis of CHAC1 expression and MMR genes. B, The correlation analysis of CHAC1 expression and methylation transferases. (* P < .05; ** P < .01; *** P < .001)

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques: Methylation, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Gene Set Enrichment Analysis of CHAC1 in KIRC. A, 'cardiac muscle contraction'. B, 'proteasome'. C, 'glycosaminoglycan biosynthesis chondroitin sulfate'. D, Summarizing of three signalling pathways

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Ferroptosis‐related gene CHAC1 is a valid indicator for the poor prognosis of kidney renal clear cell carcinoma

doi: 10.1111/jcmm.16458

Figure Lengend Snippet: Evaluate the function of CHAC1 in vitro. A, The qRT‐PCR and western blot of CHAC1 mRNA and protein expression in KIRC cell lines 786‐0 and CAKI‐1 and renal tubular epithelial cell HK‐2. B, The qRT‐PCR and western blot of CHAC1 mRNA and protein expression in KIRC cell lines 786‐0 and CAKI‐1 transfected with CHAC1 overexpression vector or negative control vector. C, CCK‐8 experiments of 786‐0 and CAKI‐1 transfected with CHAC1 overexpression vector or negative control vector. D, Cell migration and transwell experiments of 786‐0 and CAKI‐1 transfected with CHAC1 overexpression vector and negative control vector. E, The qRT‐PCR and western blot of CHAC1 mRNA and protein expression in KIRC samples and pericarcinous tissues extracted from patients who underwent surgical treatment for KIRC. F, Immunohistochemistry of CHAC1 in KIRC samples and pericarcinous tissues extracted from patients who underwent surgical treatment for KIRC. ( # P > .05; * P < .05; ** P < .01; *** P < .001)

Article Snippet: After antigen retrieval, samples were blocked in 10% BSA and incubated with primary rabbit antibodies against CHAC1 (Proteintech) for 30 minutes followed with incubation of biotinylated secondary antibodies (CST) for 30 minutes.

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Negative Control, CCK-8 Assay, Migration, Immunohistochemistry