SUPPLEMENTAL DATA. Estrogen receptors interact with the alpha catalytic subunit of AMPactivated. Running title: Estrogen receptors interact with AMPK

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1 SUPPLEENTL T Estrogen receptors teract with the alpha catalytic subunit of Pactivated prote kase Runng title: Estrogen receptors teract with PK Yulia Lipovka,, Hao hen,, Josef Vagner, Theodore J. Price 5,7, Tsu-Shuen Tsao 6, John P. Konhilas, * epartent of olecular and ellular iology, epartent of Physiology, Sarver olecular ardiovascular Research Progra, The IO5 Research Institute, 5 epartent of Pharacology, 6 epartent of iocheistry and olecular iophysics, University of rizona, Tucson, Z urrent address: School of ehavioral and ra Sciences, University of Texas at allas, JO., 8 W apbell Rd, Richardson TX 758 *To who correspondence should be addressed: John P. Konhilas, Ph University of rizona epartent of Physiology, Sarver olecular ardiovascular Research Progra, edical Research uildg, roo Tucson, Z 857- Tel: (5) Fax: (5) E-ail: konhilas@arizona.edu

2 Figure S. OSU 5 E μ pt7 T7 yotubes E μ n µ E μ µ pt7 pt PN PPT n μ n E μ μ T7 / * * PN µ E µ T PN n PP µ PP n co nt ro l T E μ PPT μ pt7 T7 / 6 * E Figure S: Estradiol activates PK. () T7 cells were treated with E centrations (µ) for, 5, 5,, and 6 utes. n crease pt7 was observed after 5 utes of E exposure. OSU 5: PK activator. () Tie and dosedependent activation of PK yotubes as dicated by an crease pt7 by Western blot analysis was perfored on total cell lysate. () T7 cells were treated with oncentrations ( n or µ) of PPT or PN. Western blot (top panel) and bar graph representation (botto panel) of / ratio. (*P <.5 fro trol.) () T7 cells were treated with µ PPT for 5, 5, and 6 utes; a Western blot analysis was perfored on total cell lysate. Western blot (top panel) and bar graph representation (botto panel) of / ratio. (*P <.5 fro trol.)

3 Figure S. RN expression levels T7 -- 9T ERα ERβ trol OSU 5 p T7 p T7 / -- trol OSU 5 ERβ RN expression trol ERβ sirn O5 RN expression trol O5 sirn Figure S: Interrogation of ER specificity underlyg estradiol-dependent PK activation. () ERα and ERβ RN levels T7, -- and 9T cells detered by RT-PR. There was no detectable ERα expression -- and 9T cells. However, ERβ RN was detectable all cell les with 9T cells showg the highest experssion levels (-fold over T7) while -- cells showed the lowest with expression leves about 5% that of T7 cells. () PK signalg reas tact -- cells. -- cells were treated with µ OSU-5 for hour and then analyzed for PK activity. Western blot analysis of p T7 /PK showed a significant crease usg this treatent strategy dicatg, despite lackg ERα, PK signalg -- cells reas functional. () ERβ RN levels T7 cells transfected with ERβ sirn detered by RT-PR. () O5 RN levels T7 cells transfected with O5 sirn detered by RT-PR.

4 Figure S. W: flag yc-gfp - + yc- + - flag-erβ + + W: egfp yc-gfp - + yc- + - egfp-erα ka IP: flag 75 ka IP: egfp Figure S: Validation of co-iunoprecipitation. 9-T cells were co-transfected with structs expressg flag-erβ, egfp-erα, yc- and yc-gfp. ell lysates were iunoprecipitated with anti-flag (IP: flag) or anti-egfp (IP: egfp) antibody, followed by Western blot analysis usg anti-flag (, W: flag) or anti-egfp (, W: egfp) antibody.

5 5 Figure S. W: O5 E W: O5 E 75 k 75 k 5 k 5 k 7 k 7 k IP: ERβ Total lysate W: O5 E W: O5 E 75 k 75 k 5 k 5 k 7 k 7 k IP: ERβ Total lysate Figure S: ERβ teracts with O5 cells and neonatal rat cardioyocytes (NR). () cells were treated with µ E for hour. ell lysates were iunoprecipitated with anti-erβ (IP: ERβ) antibody, followed by Western blot analysis usg anti-o5 (W: O5) antibody. () The sae lysates were iunoblotted directly usg anti-o5 (W: O5) antibody. () NR were treated with µ E for hour. ell lysates were iunoprecipitated with anti-erβ (IP: ERβ) antibody, followed by Western blot analysis usg anti-o5 (W: O5) antibody. () The sae lysates were iunoblotted directly usg anti-o5 (W: O5) antibody.

6 6 Figure S5. trol E ct ct+e trol E ct ct+e p T7 p T7 / 8 6 *" trol E ct ct+e / #" #" ontrol E ct ct+e trol E G5 G5+E trol E G5 G5+E p T7 p T7 / 8 *" ontrol E G5 G5+E / #" #" ontrol E G5 G5+E Figure S5: Estradiol activates PK through non-genoic pathway and dependent of GPER. T7 cells were treated with µ E the presence or absence of µ ctoyc (ct); Western blot analysis was perfored on total cell lysate. () Western blot (top panel) and bar graph representation (botto panel) of p T7 / ratio and () / ratio. T7 cells were pre-treated with 5n G5, a GPER antagonist for 5 utes and then treated with µ E for hour; a Western blot analysis was perfored on total cell lysate. () Western blot (top panel) and bar graph representation (botto panel) of p T7 / ratio and () / ratio. (*P <.5 fro trol group; #P <.5 fro trol and G5- or ct-treated group.)

7 7 Figure S6. LK O5 Figure S6: PK pathway prote expression levels T7, -- and NR cells. Western blot analysis was perfored on total cell lysates usg anti-, LK and O5 antibodies.