antibodies against hdac4  (Proteintech)

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Primer sequences.

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies against HDAC4 (1:1000, 17449‐1‐AP, Proteintech, USA), SLC38A2 (1:2000, 25928‐1‐AP, Proteintech, USA), HIF‐1α (1:1000, A26889, ABclonal, China), Nrf2 (1:1000, ab62352, Abcam, UK), HO‐1 (1:1000, 66743‐1‐Ig, Proteintech, USA), NQO1 (1:5000, 67240‐1‐Ig, Proteintech, USA), and β‐actin (1:1000, ab8227, Abcam, UK) at 4°C overnight.

Techniques: Sequencing

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Correlations of HDAC4 expression with cancer stemness and metabolic markers in NSCLC. (A) Heatmap showing the correlation between HDAC4 and cancer stemness/metabolic markers in TCGA‐LUAD and TCGA‐LUSC cohorts. (B) External validation of the correlation between HDAC4 and key markers in multiple GEO NSCLC datasets ( GSE18842 , GSE19188 , and GSE19804 ). (C) Scatter plots with fitted trend lines showing the correlations of HDAC4 with SLC38A2, SOX2, and SLC7A5 in TCGA‐LUAD cohort. (D) Scatter plots with fitted trend lines showing the correlations of HDAC4 with SOX2, SLC7A5, and ALDH1A1 in TCGA‐LUSC cohort. HDAC4, histone deacetylase 4; NSCLC, nonsmall cell lung cancer; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; GEO, Gene Expression Omnibus; SLC38A2, solute carrier family 38 member 2; SOX2, SRY‐box transcription factor 2; SLC7A5, solute carrier family 7 member 5; ALDH1A1, aldehyde dehydrogenase 1 family member A1.

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies against HDAC4 (1:1000, 17449‐1‐AP, Proteintech, USA), SLC38A2 (1:2000, 25928‐1‐AP, Proteintech, USA), HIF‐1α (1:1000, A26889, ABclonal, China), Nrf2 (1:1000, ab62352, Abcam, UK), HO‐1 (1:1000, 66743‐1‐Ig, Proteintech, USA), NQO1 (1:5000, 67240‐1‐Ig, Proteintech, USA), and β‐actin (1:1000, ab8227, Abcam, UK) at 4°C overnight.

Techniques: Expressing, Biomarker Discovery, Histone Deacetylase Assay, Gene Expression

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Knocking down HDAC4 inhibits glutamine metabolism in A549 cells. (A) Glutamine uptake levels in A549 cells. (B) Glutamate production levels in A549 cells. (C) α‐Ketoglutarate production levels in A549 cells. (D) Measurement of GLS activity in A549 cells. (E) DCFH‐DA staining was used to measure the level of ROS in cells. (F) A kit was used to measure the GSH/GSSG ratio. (G) Western blotting was used to detect the expression of Nrf2, HO‐1, and NQO1. ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)‒(G). GLS, glutaminase; DCFH‐DA, 2 ′ ,7 ′ dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, oxidized glutathione; Nrf2, nuclear factor erythroid 2 related factor 2; HO‐1, heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1. The original, uncropped western blot images for Figure are provided in the Supporting Information.

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies against HDAC4 (1:1000, 17449‐1‐AP, Proteintech, USA), SLC38A2 (1:2000, 25928‐1‐AP, Proteintech, USA), HIF‐1α (1:1000, A26889, ABclonal, China), Nrf2 (1:1000, ab62352, Abcam, UK), HO‐1 (1:1000, 66743‐1‐Ig, Proteintech, USA), NQO1 (1:5000, 67240‐1‐Ig, Proteintech, USA), and β‐actin (1:1000, ab8227, Abcam, UK) at 4°C overnight.

Techniques: Activity Assay, Staining, Western Blot, Expressing, Analysis

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: HDAC4 promotes SLC38A2 expression through HIF‐1α. (A) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 knockdown. (B) Western blot detection of SLC38A2 expression in A549 cells with HDAC4 overexpression. (C) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 knockdown. (D) Western blot detection of HIF‐1α expression in A549 cells with HDAC4 overexpression. (E) JASPAR analysis of the binding sites of HIF‐1α and the promoter of SLC38A2. (F) ChIP‐qPCR detection of the enrichment level of HIF‐1α at the SLC38A2 promoter in A549 cells. (G) A promoter‐luciferase reporter assay was used to verify the binding of HIF‐1α to the SLC38A2 promoter. (H) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α overexpression. (I) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α overexpression. (J) Western blot detection of SLC38A2 expression in A549 cells. (K) RT‐qPCR detection of HIF‐1α mRNA expression in A549 cells with HIF‐1α knockdown. (L) Western blot detection of HIF‐1α expression in A549 cells with HIF‐1α knockdown. (M) Western blot detection of SLC38A2 expression in A549 cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with three independent biological replicates. Statistical analysis was conducted using Student’s t ‐test for panels (A)–(D), (F)–(I) and (K)–(L) and one‐way ANOVA for panels (J) and (M). HIF‐1α, hypoxia‐inducible factor‐1 subunit alpha; ChIP‐qPCR, chromatin immunoprecipitation quantitative polymerase chain reaction; IgG, immunoglobulin G; OE‐HIF‐1α, overexpression HIF‐1α; sh‐HIF‐1α, short hairpin RNA targeting HIF‐1α. The original, uncropped Western blot images for Figure –D, I,J, L,M are provided in the Supporting Information.

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies against HDAC4 (1:1000, 17449‐1‐AP, Proteintech, USA), SLC38A2 (1:2000, 25928‐1‐AP, Proteintech, USA), HIF‐1α (1:1000, A26889, ABclonal, China), Nrf2 (1:1000, ab62352, Abcam, UK), HO‐1 (1:1000, 66743‐1‐Ig, Proteintech, USA), NQO1 (1:5000, 67240‐1‐Ig, Proteintech, USA), and β‐actin (1:1000, ab8227, Abcam, UK) at 4°C overnight.

Techniques: Expressing, Western Blot, Knockdown, Over Expression, Analysis, Binding Assay, ChIP-qPCR, Luciferase, Reporter Assay, Quantitative RT-PCR, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, shRNA

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: HDAC4 Promotes Cancer Stemness in Nonsmall Cell Lung Cancer by Upregulating SLC38A2 Expression via HIF‐1α to Enhance Glutamine Metabolism

doi: 10.1155/ancp/2525721

Figure Lengend Snippet: Knockdown of HDAC4 inhibits tumor growth and stemness in vivo. (A) Tumor morphology in nude mice. (B) Tumor volume in nude mice. (C) Tumor weight in nude mice. (D) Immunohistochemical detection of HIF‐1α, SLC38A2, SOX2, and OCT4 expression in tumor tissues; scale bar: 25 μm. ∗∗∗ p < 0.001. Data are presented as mean ± SD. All experiments were performed with five independent biological replicates. Statistical analysis was conducted using two‐way ANOVA for panel (B) and Student’s t ‐test for panel (C).

Article Snippet: The membranes were blocked with 5% skim milk at room temperature for 1.5 h and incubated with primary antibodies against HDAC4 (1:1000, 17449‐1‐AP, Proteintech, USA), SLC38A2 (1:2000, 25928‐1‐AP, Proteintech, USA), HIF‐1α (1:1000, A26889, ABclonal, China), Nrf2 (1:1000, ab62352, Abcam, UK), HO‐1 (1:1000, 66743‐1‐Ig, Proteintech, USA), NQO1 (1:5000, 67240‐1‐Ig, Proteintech, USA), and β‐actin (1:1000, ab8227, Abcam, UK) at 4°C overnight.

Techniques: Knockdown, In Vivo, Morphology, Immunohistochemical staining, Expressing, Analysis