Fig 2: Geminin/c-Abl overexpression decreases the overall survival and increase hazard ratio in breast cancer patients.(A) Kaplan-Meier analysis for disease free survival (DFS) of patients expressing high, middle or low levels of geminin in their breast tumors. (B) Kaplan-Meier analysis for DFS of patients expressing high, middle or low levels of c-Abl in their breast tumors. (C) Kaplan-Meier analysis for DFS of patients expressing high vs. middle vs. low levels of geminin + c-Abl in their breast tumors. (D) Multi-variants analysis of the hazard ratio (estimated mean survival with 95% CI) plotted against tumor size, age, grade 3, lymph-node (LN)-negativity, ER-positivity. Kaplan-Meier analysis for overall survival (OS) of patients expressing high, middle or low levels of geminin + c-Abl in their grade 1 (E) or grade 3 (F) breast tumors.

Fig 3: Hypoxic/necrotic core in GemOE tumors enhances Ac-HMGB1 secretion(A–C) Adjacent sections from a GemOE tumor IHC stained for geminin (A), c-Abl (B) and HMGB1 (C). (D) The level of circulating HMGB1 measured using specific ELISA assay performed on serum isolated from samples collected 7 weeks after mice were injected in mammary fat pads with naïve HME cells (n = 10, no tumors developed) or GemOE cells (n = 30, tumor-bearing, p = 0.00042). Two different sets (E, G, I and K) and (F, H, J and L) of adjacent sections from GemOE orthotopic mammary tumors stained with H & E (E and F), or IHC stained for geminin (G and H), HMGB1 (I and J) as well as HIF-1α (K) or hypoxyprobe (L). N denotes necrosis within these tumors that are shown adjacent to the hypoxic cells as indicated by high HIF-1α or hypoxyprobe staining. These cells are also expressing cytoplasmic HMGB1. (M) The levels of HMGB1 detected using specific ELISA assay released from iGem9, iG197, iG240 or iG257 cells grown under normoxic (N) or hypoxic (H) conditions. Experiments were done in triplicates 3 different times, **represents p < 0.001. (N) The level of acetylated HMGB1 passively diffused from naïve HME, iG197, iG240 or iG257 cells after repeated freeze and thaw cycles.

Fig 4: c-Abl knockout inhibits actin filament polymerization in response to ACh stimulation. a Mouse tracheal rings from c-Abl-lox and c-Ablsmko mice were treated with acetylcholine (ACh) (100 μM, 5 min) or left untreated (UT). F/G-actin ratios were evaluated using the fractionation assay. Data are mean ± SE (n = 5). b Representative images illustrating the effects of c-Abl knockout on F/G-actin ratios. Sections of trachealis from c-Abl-lox and c-Ablsmko mice were stained with DNase I (for G-actin) or phalloidin (for F-actin). c ACh-induced increases in F/G-actin ratios evaluated by fluorescent microscopy are reduced in c-Ablsmko mice (n = 4). *P < 0.05, **P < 0.01

Fig 5: Increases in the association and translocation of CAS with Pfn-1 upon acetylcholine stimulation are regulated by c-Abl. a Mouse tracheal rings from c-Abl-lox and c-Ablsmko mice were treated with acetylcholine (ACh) (100 μM, 5 min) or left untreated (UT) followed by CAS immunoprecipitation. CAS immunoprecipitates were separated by SDS-PAGE and blotted with antibodies against CAS and Pfn-1. Data are mean ± SE (n = 4). b Human airway smooth muscle (HASM) cells expressing control shRNA or c-Abl shRNA were stimulated with ACh (100 μM, 5 min) or left untreated. The cellular localization of CAS and Pfn-1 was evaluated by immunofluorescent microscopy. Arrows indicate a single line scan to analyze the fluorescent intensity for each cell. The inset plots represent fluorescent intensity of the line scan indicated by the arrows. c Fluorescent ratios of cell periphery over interior for CAS or Pfn-1 in cells were calculated. Data are mean ± SE (n = 26–27 cells from 4 independent experiments). UT, untreated. ** P < 0.01