Fig 2: Immunohistochemistry analysis of the tumor tissues. After 19 days of heat-killed single or mixed strain treatment, tumor tissues were harvested and stained by (a) p-Akt, (b) p53, (c) Bax, (d) cleaved caspase-9, (e) cleaved caspase-3, and (f) cleaved PARP. The scale bar represents 100 px. Brown dots are expression of p-Akt, p53, Bax, cleaved caspase-9 and -3, PARP. Black arrows indicate the brown dots of IHC (magnification 200× or 400× as indicated).

Fig 3: The combination of lycorine hydrochloride and HA14–1 enhances the therapeutic effect on gastric cancer. a Apoptosis of MKN-45 and SGC-7901 cells treated with LH (20 μM), HA14–1(9 μM) or LH (20 μM) + HA14–1(9 μM) for 48 h were examined by trypan blue staining. DMSO was used as control. Apoptotic rate of MKN-45 and SGC-7901 cells was quantified. b Western blotting was used to detect the expression of apoptotic protein, including BCL2, MCL1, C-Caspase 9, C-Caspase 3, PARP and C-PARP in MKN-45 and SGC-7901 cells after 48 h of treatment with LH (20 μM), HA14–1 (9 μM) or LH (20 μM) + HA14–1 (9 μM). DMSO was used as control. c Tumor volume and weight of indicated mice. DMSO was used as control. The efficiency index (q) analysis of LH (30 mg/kg) combined with HA14–1 (2.5 mg/kg) treatment in the weight of PDX tumors through Jin’s formula. d The weight of the mice treated with DMSO, HA14–1, LH or LH + HA14–1 was measured. e IHC of MCL1 and Ki67 in indicated tumors. Scale bar =20 μm. Gray value of IHC positive signal in panel was quantified. All data were analyzed by unpaired Student’s t-tests and are shown as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Knockdown of PRDX2 sensitized GC cells to Cisplatin treatments.(A) and (B) ATP-Glo cell viability assay in AGS or SNU-1 cells with control or PRDX2 siRNA knockdown with or without CDDP treatment. (C) and (D) Western blot analysis of PARP, cleaved-PARP, PRDX2, P–H2A.X, H2A.X and β-actin in AGS or SNU-1 cells as in A and B. (E) and (F) CDDP IC50 was calculated in AGS and SNU-1 cells. (G) and (H) The clonogenic formation was performed in AGS and SNU-1 control or PRDX2 siRNA knockdown cells. A total of 500 cells were seeded in each well in a 6-well plate. *, P < 0.05, **, P < 0.01, ***, P < 0.001.

Fig 5: Forced overexpression of miR-422a induces neuronal cell death and apoptosis.SH-SY5Y cells were transduced with pre-miR-422a lentivirus (“lv-miR-422a”) or the miR control lentivirus (“lv-miR-C”), followed by puromycin selection, and the stable cells (“sL1” and “sL2”) were achieved. Expression of miR-422a (a), MAPKK6 and MEF2D mRNAs (b) was tested by qPCR; MAPKK6, MEF2D and Bcl-w proteins were shown as well (c). Cells were further cultured for applied time, and then cell viability (d), cell death (e), expression of cleaved-caspase-3/-PARP (e) and cell apoptosis (g, h) were tested by the assays mentioned in the text. The primary murine neurons were transfected with miR-422a mimic (100 pmol) or the miR nonsense control mimic (“C-mimic”, 100 pmol) for 48 h, and then miR-422a expression (i), listed proteins expression (j), medium LDH contents (k), cleaved-caspase-3/-PARP levels (l) and cell apoptosis (m) were tested, with results quantified. Expression of listed proteins was quantified and normalized to GAPDH (c, f, j and l). “Ctrl” stands for the parental control cells. *p < 0.05 vs. “lv-miR-C”/“C-mimic” cells. Data indicate standard deviation (SD, n = 5). Each experiment was repeated three times and similar results were obtained. Bar = 100 μm (g).