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 levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

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 levels of total glutathione (T‐GSH) and GSSG in A549 cells were measured using a GSH/GSSG ratio detection kit (E‐BC‐K097‐M, Elabscience, China) following the manufacturer’s instructions.

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