Journal: Cancers

Article Title: Metabolic Crosstalk in Triple-Negative Breast Cancer Lung Metastasis: Differential Effects of Vitamin D and E in a Co-Culture System

doi: 10.3390/cancers18020294

Figure Lengend Snippet: ( A ) MDA-MB-231 and MRC-5 cells were co-cultured for 72 h and both cell lines were assessed via Western blot for CAF activation markers (α-SMA and FAP) and master metabolic regulators (HIF-1 α and c-Myc); ( B ) A schematic representation depicting HIF-1α as the master regulator of metabolism, regulating key metabolic pathways and showing its differential expression in cancer cells and fibroblasts within the co-culture system; ( C ) A general scheme of cellular energy metabolism is shown, highlighting key enzymes (indicated in boxes) whose expression levels were evaluated in the co-culture experiments. Co-cultures were assessed for ( D ) glutamine metabolic enzymes (GLUL, GDH and ASCT2); ( E ) TCA cycle enzymes (Aconitase, SDHA, CS, IDH2, fumarase and MPC2); ( F ) glycolysis and other metabolic enzymes (GLUT1, HKII, PKM2, LDHA, PDH, MCT1, and MCT4). ( A , D – F ) Results are represented as mean ± SD ( n = 3). Significance relative to control cells: * p < 0.05, ** p < 0.01, *** p < 0.001. The uncropped original Wester blotting images can be found in .

Article Snippet: The primary antibodies used in this study, including α-SMA (Cat.#:19245S), FAP (Cat.#:66562S), GLUL (Cat.#:80636S), GDH (Cat.#:12793S), ASCT2 (Cat.#:5345S), Aconitase (Cat.#:6571T), SDHA (Cat.#:11998T), CS (Cat.#:14309T), IDH2 (Cat.#:56539T), Fumarase (Cat.#:4567T), MPC2 (Cat.#:46141S), GLUT1 (Cat.#:73015S), HKII (Cat.#:2867S), PKM2 (Cat.#:4053T), PDH (Cat.#:3205S), LDHA (Cat.#:2012S), MCT1 (Cat.#:76508S), HIF-1α (Cat.#:36169S), and c-Myc (Cat.#:5605S) were from Cell Signalling Technologies (CST; Danvers, MA, USA).

Techniques: Cell Culture, Western Blot, Activation Assay, Quantitative Proteomics, Co-Culture Assay, Expressing, Control

Journal: Genes & Diseases

Article Title: Cancer-associated fibroblasts derived fibronectin extra domain A promotes sorafenib resistance in hepatocellular carcinoma cells by activating SHMT1

doi: 10.1016/j.gendis.2024.101330

Figure Lengend Snippet: CAFs promote sorafenib resistance by activating NF-κB in HCC cells. (A) Immunofluorescence analysis was performed to assess the expression of FAP and α-SMA on primary CAFs. Scale bars, 50 μm. (B) Co-culture with CAFs significantly reduced apoptosis of HepG2 and Huh7 upon sorafenib treatment. (blue bar: sorafenib-treated tumor cells cultured alone; red bar: sorafenib-treated tumor cells co-cultured with CAFs). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. (C) Enrichment score of the “I-κB kinase/NF-κB signaling” in sorafenib-resistant group versus sorafenib-sensitive group after sorafenib treatment, analyzed by Gene Set Enrichment Analysis based on the RNA-seq data (after performing log 2 transformation, normalization, and mean value calculation) obtained from the GEO database (GSE182593). (D) Western blot analyses of the protein level of P-p65 in the indicated HCC cells with different treatments. Results are representative of three experiments. (E) Inhibition of the NF-κB signaling pathway in HCC cells induced apoptosis significantly upon sorafenib treatment (blue bar: sorafenib-treated group; red bar: sorafenib and BAY11-7082-treated (100 μM) group). Student's t -test of variance, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. CAFs, cancer-associated fibroblasts; NF-κB, nuclear factor kappa B; HCC, hepatocellular carcinoma; FAP, fibroblast activation protein; α-SMA, alpha-smooth muscle actin.

Article Snippet: The cells on slides were fixed with 4% paraformaldehyde for 10 min and permeabilized in phosphate buffer saline for 20 min. Then, cells were blocked with goat serum at room temperature for 60 min and incubated with primary antibodies against FAP (fibroblast activation protein; R&D system, #FAB3715A, RRID: AB_2884010) (1:200) and α-SMA (alpha-smooth muscle actin; R&D system, #MAB1420, RRID: AB_262054) (1:200) for 2 h. Then, the cells on slides were reheated and incubated with the corresponding secondary antibody at 37 °C in the dark for 2 h. Nuclei were counter-stained with DAPI.

Techniques: Immunofluorescence, Expressing, Co-Culture Assay, Cell Culture, RNA Sequencing, Transformation Assay, Western Blot, Inhibition, Activation Assay