Ly294002

MC3T3-E1 cells were
cultured in various media for 3 days to assess the effects of CA,
as follows: (1) NC group: cells were cultured in regular medium (α-MEM
containing 10% FBS); (2) HGHL group: cells were cultured in HGHL medium
whose main ingredients are regular medium containing 25 mM glucose
and 200 mM sodium PA (Alladin Scientific, China) to mimic diabetic
conditions; and (3) HGHL + CA group: cells were cultured in HGHL medium
containing CA (1, 10, or 100 μM of CA; Alladin Scientific, China).
For the experiments employing LY294002 (inhibitor of PI3K; Cell Signaling
Technology, USA), the cells were pretreated with LY294002 (50 μM)
for 1 h, followed by the designated treatments for an additional 3
days; the experiments detailed in the subsequent sections were then
carried out.

Immortalized HLEB-3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), placed in a humidified atmosphere containing 5% CO₂ at 37°C. The cells were washed with phosphate-buffered saline (PBS), and dissociated with 0.05% trypsin and 0.02% EDTA. As in our previous experiments (7 (link)), when the cell cultures reached a confluence of 80%, the cells were divided into three groups (1×10⁶ cells/ml each). In the first group, the cells were stimulated with 10 ng/ml recombinant human TGF-β₂ (Peprotech, Inc., Rocky Hill, NJ, USA) for 24 h in serum-free medium to induce EMT. This was termed the TGF-β₂ group. To determine the effect of PI3K inhibition on EMT, the cells were pretreated with LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA) at an appropriate concentration for 1 h prior to being co-treated with 10 ng/ml TGF-β₂ for 24 h. This was termed the LY294002+TGF-β₂ group. The control group consisted of cells, which were incubated under conventional conditions without the presence of either TGF-β₂ or LY294002 in the medium. Following treatment, the cells were collected for western blot analysis and confocal immunofluorescence assays.

The in vitro culture of ovaries was performed, as previously reported (14 (link)). Briefly, gonads were isolated from E12.5 embryos and cultured on Transwell-COL membranes (Coaster) supplied with αMEM [Minimum Essential Medium α (Invitrogen) containing 2% fetal calf serum (FCS), 55 μM 2ME (2-mercaptoethanol), 1× penicillin/streptomycin, 1× GlutaMAX, and 150 μM ascorbic acid]. The medium was changed every 2 days. At 4 days of culture, the medium was changed to S10 medium [StemPro (Invitrogen) containing 10% fetal bovine serum (FBS), 55 μM 2ME, 1× penicillin/streptomycin, 1× GlutaMAX, and 150 μM ascorbic acid]. From days 9 to 13, 500 nM ICI182780 (Tocris) was added to the culture. For LY294002 treatment, 25 μM LY294002 (Cell Signaling Technology) was added to the culture from days 6 to 16. In the case of CD and LMB treatments, 25 μM of CD (Merck) and 20 nM of LMB (Cell Signaling Technology) were added to the culture for 6 hours at the end of day 16. On day 16, the cultured ovaries were fixed and sectioned, and then immunohistochemical analysis was performed, as described above. Ovaries from P3 newborn pups were also cultured on Transwell-COL membranes (Coaster) with S10. To supply air pressure to the culture, culture dishes were placed in a gas-pressurized cell culture chamber (AGP-3001S, STREX), and the pressure was adjusted to 33.3 kPa (≒ 250 mmHg).

To identify upregulated genes at early time points, we first calculated the sum of the expression values at the seven measured time points (0 and every hour up to 6 h), for each condition as below. Following this, we obtained the average gene expression value by dividing the sum by 7, for each condition. Each condition is as follows: N, no treatment; D, treatment with 1 μg/ml DHMEQ (NF-κB inhibitor); LY, treatment with 1 μm LY294002 (PI3K inhibitor) (Cell Signaling Technology, Beverly, MA, USA); H, stimulation with 100 ng/ml HRG; HD, stimulation with HRG and treatment with DHMEQ; and HLY, stimulation with HRG and treatment with LY294002. The potentially upregulated genes downtream of HER2/HER3-PI3K-NF-κB pathway were selected that satisfied the following criteria: H ≧1.25 N, H ≧1.25 D, H ≧1.25 LY, H ≧1.11 HD and H ≧1.11 HLY. We obtained data at various time points; we considered the differences in expression levels to be significant, even when they were <1.5-fold. Similarly, to identify upregulated genes throughout the time course, we calculated the average gene expression values at the 17 measured time points (0, and every hour up to 16 h), for the aforementioned conditions (GEO accession number GSE64073).