Cell culture and antibodies
The endometriotic epithelial cell line (11Z) was established by Professor Anna Strazinski-Powitz [18]. The human endometrial stromal cell line (ESC) was established by Dr. Krikun [19]. All cell lines were cultured in Dulbecco’s Modified Eagle Medium/Ham’s F-12 50/50 Mix (DMEM/F-12) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) with 100mg/mL penicilin and 100mg/mL streptomycin at 37℃ and 5% CO2.
Mouse anti-β-actin (A1978) was from Sigma-Aldrich, and dilution: 1:5000. Mouse anti-HSF1 (sc-17757) was from Santa Cruz, and dilution: 1:1000. Rabbit anti-PFKFB3 (ab181861) was purchase from ABCAM, and dilution: 1:2000. KRIBB11 were obtained from Med Chem Express (MCE), 50mg/kg.
SiRNA and transfection
The sequence of small interfering (si) RNAs against HSF1 was 5’- GCAGGUUGUUCAUAGUCAGAA-3’. The sequence of siRNA-NC (Negative Control) was 5’-UUCUCCGAACGGUCACGU-3’ [20]. The method was performed as described previously [21].
Western blot
The indicated cells were collected and lysed on ice using lysis buffer, and were centrifuged at 12000rpm at 4℃ for 15min. Then, 5´loading buffer was added to the sample, and boiled for 10min. The western blot method was performed as described previously [22].
Quantitative real-time PCR
The isolation of total RNA from cells and the synthesis of cDNA were described above [23]. Performing quantitative real-time PCR using SYBR Green PCR Master Mix (Takara) with CFX96 Real-Time PCR detection system (Bio-Rad, shanghai, China).
Cell proliferation assay
The indicated cells were transfected with the indicated plasmids, and reseeded in 24-well plates. The cell numbers were counted every 24hr for 4 days [22].
Colony-formation assay
The 500 indicated cells were seeded in six-well plates, and cultured at 37℃ in 5% CO2 for 10-14 days. After the clones were formed, the culture medium was removed and fixed at room temperature with 4% paraformaldehyde for 15min. After the fixation, the cells were stained with crystal violet, and then photographed [24].
Wound healing assay
The indicated cells were inoculated in 6-well plates and cultured in medium until overgrown. The pipette tip was used to draw a fine line and washed with PBS. After 24hr, cells were photographed again [25].
Glucose consumption and lactate production
The indicated cells were seeded in 6-well plates, and the culture mediums were collected after 24hr to determine the concentration of glucose and lactic acid. The methods were performed as described previously [22, 24].
Animal experiments
Animal experiments have been approved by ethics Committee of Weifang Medical University. We used 5-week-old BALB/c female mice, and the donor mice (n=5) were injected with estradiol benzoate to promote endometrial development. Estradiol benzoate was diluted with oil and injected intramuscularly into the thigh of donor mice, 3mg/mouse, 2 times for one week. After one week, the uterus of donor mice was cut into pieces and intraperitoneally injected into experimental (n=7) and control mice (n=7). After one week, the mice in the experimental group were intraperitoneally injected HSF1 inhibitor KRIBB11, and the mice in the control group were injected with normal saline in the same amount 2 times a week for one month. Then, the mice were sacrificed to observe the endometrial lesion.
Tissue Collection and Immunohistochemistry
All tissues were derived from mice model of endometriosis. The sections were embedded in paraffin, dried and dewaxed with xylene. The immunohistochemistry was performed as described previously [21]. The immunostaining intensity was quantified using the Image J [26].
Statistical analysis
All statistical analyses were used Graphad Prism 5.0 software. The statistical analyses were presented as mean ± SEM, and performed by two-tailed unpaired Student’s t-test. P<0.05 is significant (*p<0.05). n.s.= not significant.