SUPPLEMENTARY MATERIAL

Estradiol promotes pentose phosphate pathway addiction and cell survival via reactivation of Akt in mTORC1 hyperactive cells

Running Title: Estradiol enhances glucose utilization via Akt

Yang Sun1, Xiaoxiao Gu2, Erik Zhang1, Mi-Ae Park1, Ana M. Pereira1, Shuyan Wang1, Tasha Morrison1, Chenggang Li1, John Blenis2, Victor H. Gerbaudo1, Elizabeth Petri Henske1, and Jane J. Yu1

1Brigham and Women's Hospital and Harvard Medical School, One Blackfan Circle, 6th Floor, Boston, MA 02115

2Harvard Medical School, Boston, MA 02115

Corresponding author:

Jane J. Yu

Division of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Harvard Medical School, One Blackfan Circle, Boston MA 02115

Tel: 617 355 9018 Email:

Supplementary materials and methods:

Immunoblot analysis. Cells were lysed in M-PER buffer (Pierce) supplemented with protease inhibitors and phosphatase inhibitor cocktails. Cleared cell lysates were obtained by centrifugation at 14,000 x rpm for 10 min at 4℃ and then subjected to immunoblotting.

G6PD mature-mRNA/Pre-mRNA ratio. TSC2-deficient cells were treated with estradiol or vehicle control for 24 hr. Total RNA was extracted using the RNeasy mini kit (Qiagen). cDNA was synthesized from 500 ng of total RNA using a high-capacity cDNA reverse transcription kit (Life Technologies) with random primers, according to the manufacturer's protocol. SYBR green real-time PCR Master Mixes were purchased from Life Technologies. The primers for amplification of mature-mRNA and Pre-mRNA of G6PD1, 2 are as follows: human G6PD Intron 11-Exon12: Forward: 5’-CATACCTGTGGGCTATGGGGTG-3’; Reverse: 5’-AT AAATATAGGGGATGGGCTTGGGC-3’. G6PD Exon 12-Intron 12: Forward: 5’-CTGTTCCG TGAGGACCAGATCT-3’; Reverse: 5’-TGAAGGTGAGGATAACGCAGGC-3’. 1, 2 The 2−ΔCt method was used to calculate the relative abundance of G6PD gene expression compared with Tubulin expression.

Supplementary Figure Legends:

Figure S1. Estradiol does not modify G6PD mature-mRNA/pre-mRNA ratio in TSC2- deficient cells. Cells were treated with estradiol or vehicle control for 24 hr. G6PD mature-mRNA and Pre-mRNA were measured using real-time RT-PCR in patient-derived TSC2-deficient or re-expression cells treated with estradiol or vehicle control. The ratio of G6PD mature-mRNA/Pre-mRNA was calculated.

Figure S2. The effect of estradiol in G6PD expression in TSC2 re-expressing cells. Cells were treated with 10 nM estradiol or vehicle control. (A) Immunoblot analysis of TSC2, G6PD, phospho-ERK (Thr202/Tyr204) and β-actin in patient-derived cells re-expressing TSC2. (B) Immunoblot analysis of TSC2, G6PD, phospho-ERK (Thr202/Tyr204) and β-actin in rat-derived cells re-expressing TSC2.

Reference

1. Hong X, Song R, Song H, Zheng T, Wang J, Liang Y, et al. PTEN antagonises Tcl1/hnRNPK-mediated G6PD pre-mRNA splicing which contributes to hepatocarcinogenesis. Gut 2013.

2. Tao H, Szeszel-Fedorowicz W, Amir-Ahmady B, Gibson MA, Stabile LP, Salati LM. Inhibition of the splicing of glucose-6-phosphate dehydrogenase precursor mRNA by polyunsaturated fatty acids. The Journal of biological chemistry 2002, 277(34): 31270-31278.