pp65 rela ser536  (Cell Signaling Technology Inc)

Journal: PLOS ONE

Article Title: Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer

doi: 10.1371/journal.pone.0304194

Figure Lengend Snippet: (A) Immunohistochemical staining images of tumors positive for: a) ING4 (IHC score +1), b) pp65/RelA(Ser276) (IHC score +2), c) CD68 (IHC score +2), d) CD4 (IHC score +3), e) CD8 (IHC score +2), f) PD-1 (IHC score +3); black boxes in the top panel are presented in higher magnification images in the bottom panel; black scale bars denote 100 mm in length. (B) Percentage of tumors positive for each marker (solid dark bar): ING4 (154/246, 63%), pp65 (84/245, 34%), CD68 (171/193, 89%), CD4 (50/193, 26%), CD8 (75/189, 40%), and PD-1 (24/132, 18%). (C) Venn diagram and table showing the number of tumors positive for CD68, CD8, CD4, and/or PD-1. (D) No association between immune markers with ING4-low vs ING4-high (left graph) or NF-kB-low vs NF-kB-high (right graph) tumors. (E) Increased CD8+ tumors in ING4-low/pp65-high tumors (solid dark bar, 59% vs 32–38%); p values were determined by Fisher’s Exact test; ns , not significant. (F) High lymph node positivity in ING4-low/pp65-high tumors (LN+, solid dark bar): All, all tumors; ER-, estrogen receptor-negative.

Article Snippet: Antibodies used in Western blots were against: ING4 (BTIM-4 hybridoma supernatant, 1:4 [ ]), pp65/RelA (Ser536) (93H1, 1:1,000; CST), histone H3 (1:5000, CST), and a-tubulin (DM1A, 1:5000, Sigma-Aldrich).

Techniques: Immunohistochemical staining, Staining, Marker

Journal: PLOS ONE

Article Title: Tumor immune microenvironment permissive to metastatic progression of ING4-deficient breast cancer

doi: 10.1371/journal.pone.0304194

Figure Lengend Snippet: (A) Schematic diagram of the Ing4 genome with exons (open boxes); CRISPR/CAS9 gRNAs R1 and M1 mapped to exon 1 and exon 3 (red line). (B) Nuclear accumulation of pp65/RelA in Ing4 -deleted p53MT cells treated with cytokines: v2, the vector control cells; v2R1, R1 gRNA construct cells; v2M1, M1 gRNA construct cells; cells were treated with PBS (-), 10ng/ml TNFa (T), or 10ng/ml IL-1b (I), for 1 hour in serum-free media prior to cell fractionation; nuc, nuclear fraction; cyto, cytoplasmic fraction; Western blot for ING4 and phospho-p65/RelA (ser536); Histone H3 and a-tubulin antibodies were used as the loading controls. (C) Relative fold expression of the mouse Il6 gene in p53MT cells treated with (+) or without (-) TNFa (left panel) or IL-1b (right panel) for 4 hours in serum-free media. Numbers denote the average fold change of a minimum three replicates: open bar, v2; closed bar, v2R1; serrated bar, v2M1; RT-qPCR to quantify Il6 expression using Gapdh as the reference; p values were determined by two-tailed student t-test; ns , not significant. (D) Cytokine-induced migration of Ing4 -deleted p53MT cells: PBS (-),TNFa (T), or IL-1b (I); v2, v2R1, or v2M1 cells migrated through 8mm pores in the transwell inserts were stained with DAPI and visualized under a fluorescent microscope. Cell numbers were determined by averaging a minimum of 4 images per experiment from at least 3 independent experiments; p values were determined by two-tailed student t-test; *, p < 0.001; **, p < 0.05.

Article Snippet: Antibodies used in Western blots were against: ING4 (BTIM-4 hybridoma supernatant, 1:4 [ ]), pp65/RelA (Ser536) (93H1, 1:1,000; CST), histone H3 (1:5000, CST), and a-tubulin (DM1A, 1:5000, Sigma-Aldrich).

Techniques: CRISPR, Plasmid Preparation, Control, Construct, Cell Fractionation, Western Blot, Expressing, Quantitative RT-PCR, Two Tailed Test, Migration, Staining, Microscopy