Ddit3

The ovarian cancer tissue samples were embedded in paraffin. After deparaffinization and rehydration with graded alcohols, slices were rinsed twice with PBS for 10 min. Next, slices were incubated overnight with rabbit polyclonal primary antibody of CD1a (Cat# ab108309, 1/1000, Abcam, Cambridge, MA, USA), CD4 (Cat# ab133616, 1/500, Abcam), CHMP5 (Cat# ab96273, 1/500, Abcam) and DDIT3 (Cat# ab11419, 1/100, Abcam), followed by incubation at 37℃ for 30 min with 45µl secondary antibody horseradish peroxidase-conjugated goat polyclonal anti-rabbit or mouse IgG H&L (HRP) (Cat# ab6721 and ab205719 1:1000, Abcam). After staining with 3, 3’-diaminobenzidine (DAB) for 3 min, slices were rinsed with water for 10 min. After counterstaining with hematoxylin, slices were rinsed with water for 10 min, and then dehydrated and cleared. Lastly, a light microscope was employed to observe and photograph slices. IHC score was calculated by photographing at least 5 random fields of each specimen. We employed a combined score system based on both intensity and extent for calculating the IHC score. 1) Staining intensity is graded as 0, 1, 2, or 3 (for negative, weak, moderate, and strong). 2) The percentage of positive cells is scored as follows: 0, < 5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, > 76%. The final IHC score is the product of two indicators.

Western blot and qRT-PCR were performed as described in our previous study [16 (link)]. Primary antibodies for Western blot were: PHF5A (1:500; Invitrogen); IGFBP3 (1:500; Abcam); PIK3CB (1:500; CST); AKT2 (1:500; Abcam); DDIT3 (1:200; Abcam); Skp2 (1:500; Abcam); P53 (1:1000; CST); GAPDH (1:2000; Santa Cruz Biotechnology). The primers used for qRT-PCR are listed in Additional file 1: Table S1. Gene and protein expression levels were normalized to those of the internal control GAPDH.

After tumor collection, tissue was processed for immunohistochemical analysis following our published procedures [49 (link),52 (link)]. Antigen detection was performed using the following antibodies: Ki67 (ab15580; Abcam, Cambridge, MA, USA), p21 (2947; Cell Signaling, Danvers, MA, USA), cleaved Casp-3 (9661, Cell Signaling), phospho-eIF2α (3398; Cell Signaling), DDIT3 (179823; Abcam), and HO-1 (ab13248; Abcam). Slides were scored manually using a 20 × objective. Average histologic scores (H-score) were calculated as previously reported [55 (link)]. Based on the percentage of cells staining with 3+ (strong), 2+ (moderate), 1+ (weak), or O (absent) intensity, an H-score (range 0–3) was calculated by summing the percentages of cells staining at each intensity multiplied by the weighted intensity of staining: H-score = (% weakly stained cells × 1) + (% moderately stained cells × 2) + (% strongly stained cells × 3).