a549 cells  (CLS Cell Lines Service GmbH)

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

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). a Transwell assay schematic: A549 cells in the lower well; AlgTA + lactate oxidase (LOx) + horseradish peroxidase (HRP) in the insert. Cell-secreted lactate diffuses into the insert and is oxidised by LOx to generate H 2 O 2 , which HRP uses to oxidatively couple AlgTA phenols and form a gel (see also Fig. ). Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 cells at days 1–3. Stains: Calcein AM (live, green), NucBlue (nuclei, blue), propidium iodide (dead, red), n = 3 independent experiments. Scale bars, 100 µm. c Resazurin-based metabolic activity ( n = 9 wells) from 3 independent experiments. Shown P -values: day 1, P = 0.054, day 2, P < 0.001; day 3, P < 0.001. d Relative proteome profiling of selected tumour-associated proteins in AlgTA/LOx/HRP-treated cultures at day 2 versus matched controls ( n = 3 biological replicates, 10 wells each, 2 on-membrane replicates). Shades of red indicate decreased, and shades of green indicate increased relative protein abundance. e Evolution of mechanics and transport: Young’s modulus E measured by nanoindentation ( n = 9 indentations from 3 gels; see the “Methods” section) and fluorescence correlation spectroscopy (FCS) diffusion-time ratio ( τ gel / τ buffer ) for gels formed at days 1–3 after equilibration in tracer-containing PBS ( n = 4 positions per gel). f Lactate concentration under the indicated conditions at days 1–3. ( n = 6 samples from 2 independent experiments). Shown P -values: day 1, P = 0.005; day 2, P < 0.001; day 3, P < 0.001. g Extracellular H 2 O 2 quantified by a fluorimetric hydrogen peroxide assay at days 1–3 ( n = 4 samples).

Article Snippet: A549 human lung adenocarcinoma cells (DSMZ, ACC 107) and 4T1 murine mammary carcinoma cells (ATCC, CRL-2539) were cultured at 37 °C and 5% CO 2 in DMEM high glucose (Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% penicillin/streptomycin.

Techniques: Two Tailed Test, Transwell Assay, Fluorescence, Activity Assay, Profiling, Membrane, Quantitative Proteomics, Spectroscopy, Diffusion-based Assay, Concentration Assay, H2O2 Assay

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. (day 2). Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). Formulation: 3 wt% AlgTA, 10 U mL −1 HRP, 1 U mL −1 LOx; control: DMEM supplemented with FBS. a Communication cascade: spheroid-secreted lactate is converted by LOx/HRP to H 2 O 2 , driving phenol coupling and in situ AlgTA gelation around the spheroid (schematic) and photograph of encapsulated spheroids. Scale bar, 2 mm. Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . b Representative fluorescence micrographs of A549 spheroids: F-actin (phalloidin, green) and nuclei (NucBlue, blue), n = 3 independent experiments. Top: control (regular medium). Bottom: AlgTA/LOx/HRP, showing actin-rich protrusions. Scale bars, 100 µm. c Calibration of hydrogel crosslinking with defined lactate concentrations (green). Correlation between the diffusion-time ratio of Alexa488-BSA ( τ gel / τ buffer ) and known concentrations of lactate (0–10 mM sodium lactate). d Spatial transport mapping by FCS: diffusion-time ratio ( τ gel / τ buffer ) and apparent lactate-equivalent concentration ( c lac , eq ) versus distance from the spheroid surface. Individual values were taken at three parallel offset distances and are shown with a line representing the mean. e Proteomics (3D, day 2): relative abundance of selected tumour-associated proteins in AlgTA/LOx/HRP versus control ( n = 3 biological replicates, 12 spheroids each, 2 on-membrane replicates). Red indicates decreased, and green indicates increased relative protein abundance. f Proteomic profiling of treated AlgTA/LOx/HRP 2D Transwell and 3D spheroid cultures. Filled bars indicate the condition showing the stronger relative response. 3D: 3 biological replicates, 12 spheroids each, 2 on-membrane replicates; 2D: n = 3 biological replicates, 10 wells each, 2 on-membrane replicates. g Representative spheroid diameter with and without AlgTA/LOx/HRP; 16 spheroids per condition. Shown P -values: AlgTA/LOx/HRP day 0 vs day 2, P = 0.546; control day 0 vs day 2, P < 0.001; AlgTA/LOx/HRP day 2 vs control day 0, P < 0.001, n = 3 experiments. h Metabolism under encapsulation ( n = 3 independent experiments): CellTiter-Glo® 3D (ATP, n = 9 wells, P < 0.001) and resazurin (NADH, n = 9 wells, P < 0.001) assays.

Article Snippet: A549 human lung adenocarcinoma cells (DSMZ, ACC 107) and 4T1 murine mammary carcinoma cells (ATCC, CRL-2539) were cultured at 37 °C and 5% CO 2 in DMEM high glucose (Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% penicillin/streptomycin.

Techniques: Two Tailed Test, Formulation, Control, In Situ, Spheroids, Fluorescence, Diffusion-based Assay, Concentration Assay, Membrane, Quantitative Proteomics, Profiling, Encapsulation

Journal: Nature Communications

Article Title: Cell-derived lactate triggers local hydrogelation and phenotype remodelling

doi: 10.1038/s41467-026-77702-x

Figure Lengend Snippet: Unless stated otherwise, data are mean ± s.d. (day 2). Statistics: unpaired two-tailed Welch’s t -test; P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), P > 0.05 (ns, not significant). Formulation: 3 wt% AlgTA, 10 U mL −1 HRP, 1 U mL −1 LOx; control: medium without AlgTA/LOx/HRP. a Representative live/dead staining micrographs of lactate-gated AlgTA-encapsulated A549 spheroids after 2 days of incubation ( n = 3 independant experiments). Stains: Calcein AM (live, green), NucBlue (nuclei, blue), propidium iodide (dead, red). Scale bars, 200 µm. b Representative live/dead staining micrographs of lactate-gated AlgTA-encapsulated 4T1 spheroids after 2 days of incubation ( n = 3 independant experiments). Stains: Calcein AM (live, green), NucBlue (nuclei, blue), propidium iodide (dead, red). Scale bars, 200 µm. c Formation of patient-derived heterogeneous glioblastoma spheroids. Following surgical tumour resection, glioblastoma specimens are dissociated into single cells, which are subsequently used to generate stable spheroids. Patient-derived material was obtained under institutional ethical approval and informed consent; donor/sample information is provided in the “Methods” section. Created in BioRender. Braun, M. (2026) https://BioRender.com/569s2eu . d Invasive area of patient-derived glioblastoma spheroids with and without AlgTA/LOx/HRP after up to 7 days; 6 spheroids per condition. Representative of n = 3 independent experiments from different donors. Shown P -values: control vs. AlgTA/LOx/HRP; day 1, P = 0.042; day 3, P = 0.003; day 7, P < 0.001. e Representative brightfield micrographs of patient-derived glioblastoma spheroids after up to 7 days of incubation ( n = 3 independent experiments). Purple areas indicate the invasive area. Scale bar, 200 µm.

Article Snippet: A549 human lung adenocarcinoma cells (DSMZ, ACC 107) and 4T1 murine mammary carcinoma cells (ATCC, CRL-2539) were cultured at 37 °C and 5% CO 2 in DMEM high glucose (Gibco 31053-028) supplemented with 10% dialysed foetal bovine serum (FBS, Gibco A33820-01) and 1% penicillin/streptomycin.

Techniques: Two Tailed Test, Formulation, Control, Staining, Spheroids, Incubation, Derivative Assay, Brightfield