Supplementry Figure S Connexin4 TroponinI Merge Plsm memrne Met Intrcellulr Met Supplementry Figure S H9c rt crdiomyolsts cell line. () Immunofluorescence of crdic mrkers: Connexin4 (green) nd TroponinI (red). DAPIstined nuclei in lue. () Plsm memrne (green) nd intrcellulr (red) immunofluorescence of Met (see Mterils nd methods section). Nuclei in lue. Br: 5µm.
Supplementry Figure S. (AAy)/A.. VSVG. 4h 48h 7h c MTT % Reltive Asornce Vlue 8 6 4 CoCl VSVG nm VSVG nm % Reltive Numer Cells 8 6 4 CoCl VSVG nm VSVG nm Supplementry Figure S The crdioprotective ctivity medited y Met gonist DN nd DO4 is specific. () Wound heling ssy in cells not treted (, white) or treted with VSVG ma (nm, grey) for 4, 48 or 7 hours. Ttest ws clculted etween treted vs smples t ech time point nd ws never sttisticlly significnt. (c) Cells were untreted (, white), treted with µm CoCl (lck) or CoCl VSVG (nm: light grey, nm: drk grey). MA ws concomitntly dded to CoCl for 4h () nd 48h (c). Cell viility ws mesured y MTT ssy () nd cells were counted (c). Results re expressed s the percentge of MTT or cell count reduction reltive to. Vlues re the men ± SD clculted in three independent experiments. For Ttest ech group of smples ws compred to CoCl treted cells, except for (). p<.5 nd p<.5.
Supplementry Figure S h HIF 6h h 4h c Glut mrna Reltive Expression.5.5 HIF mrna Reltive Expression d CAXII mrna Reltive Expression.5.5 Tu 8 6 4 h h 4h 48h e Met mrna Reltive Expression.5 h h 4h 48h f GAPDH Reltive Densitometry.5 h h 4h 48h h h 4h 48h GAPDH Tu h 4h 48h CoCl Supplementry Figure S CoCl induces HIF protein stiliztion nd mrna upregultion of its trget genes in H9c crdiomyolsts. Cells were treted with µm of CoCl (lck) for different lengths of time. () WB of HIF protein. Tuulin is showed s loding control. HIF (), GLUT (c), CAXII (d) nd Met (e) mrna expression levels were nlysed nd normlized using the βctin. (f) WB of GAPDH protein, normlized using tuulin. The vlues re expressed s fold reltive to (white) nd re the men ± SD of three independent experiments. Ttest ws clculted etween treted smples vs. In (e) onetiled Ttest ws significnt. p<.5, p<. nd p<.5.
Supplementry Figure S4 PAkt Reltive Densitometry.5 PErk Reltive Densitometry 8 6 4 PAkt PErk Tu CoCl (48h) HGF (h) c Tu CoCl (48h) HGF (h) PProtein Reltive densitometry.5 Pp7S6K Tu CoCl (48h) HGF (h) P4EBP Tu Supplementry Figure S4 CoCl tretment decreses the prosurvivl pthwys Akt, Erk nd mtor nd impirs the response to HGF. H9c cells were untreted (white) or treted with µm CoCl (lck), HGF (.5nM, light grey) or HGFCoCl (drk grey). Cells were treted for 48h nd HGF ws dded in the lst h. PAkt (), P Erk (), Pp7S6K nd P4EBP (c) protein levels were nlysed y WB densitometry. Tuulin ws used s loding control. Representtive imges re shown elow ech grph. The vlues re expressed s fold reltive to nd re the men ± SD of three independent experiments. Ttest ws clculted etween vs CoCl nd HGF vs HGFCoCl. p<.5, p<. nd p<.5.
Supplementry Figure S5 A B CoCl CoCl MA CoCl Bf C D Supplementry Figure S5 CoCl induces utophgosome formtion nd mturtion. Immunofluorescence imges of LC utophgic mrker (green) in H9c cells untreted (A) or treted with µm CoCl lone (B), CoCl MA (C) or CoCl Bf (D) for 48h. MA nd Bf were dded in the lst h nd 6h, respectively. DAPIstined nuclei in lue. Br: 7µm.
Supplementry Tle S. Antiodies used throughout the study. Primry Antiody Compny Dilution Product n. Exp Met Snt Cruz : SP6 IP G Millipore :/ 5ug 6579 IP PMet (TyR 4/5) Upstte : IP PTyrosines Snt Cruz : PY99 IP Bx Snt Cruz : sc748 WB Bcl BD Biosciences : ps7 WB Totl/Cleved Cspse Cell Signling : 966 WB Prp/ Snt Cruz : sc75 WB Redd (RTP8) Sigm : PRS459 WB Bnip cm : AN4 WB PAMPK thr7 Sigm : SAB45754 WB AMPK Sigm : SAB459 WB PP7s6K thr89 Cell Signling : 95 WB p7s6k New Englnd BioLs : WB P4ep ser65 Cell Signling : 945 WB 4ep Snt Cruz : sc9977 WB Beclin Sigm : B686 WB LCB Sigm :/: L754 WB/IF p6 Sigm : P67 WB HIFα Novus Biologicls : NB479 WB GAPDH Open iosystems : TAB WB αtuulin Sigm : B5 WB Met Zymed : D4 IF Connexin 4 Sigm : C69 IF TroponinI Millipore : 69 IF Secondry Antiody Got ntimouse IG Amershm : 4 WB Got ntirit IG Amershm : 46 WB Alex Fluor 488 ntimouse Moleculr Proes/Invitrogen :5 IF Alex Fluor 488 ntirit Moleculr Proes/Invitrogen :5 IF Alex Fluor 546 ntimouse Moleculr Proes/Invitrogen :5 IF
Supplementry Tle S. Primers used throughout the study. Gene Forwrd Reverse Tm ( C) Size (p) ReddRTPCR 5'GCCGGAGGAAGACTCCTCATA 5'CATCAGGTTGGCACACAGGT 6 97 Bnip RTPCR 5'GCCATTGGATTGGGGATCTAC 5'ACTGTGTGAGCAGAAGGCAG 6 9 Redd SQPCR 5'CTTGTCCGCAATCTTCGCTG 5'TATGAGGAGTCTTCCTCCGGC 6 98 Bnip SQPCR 5'TACCTCTCAGTGGTCACTTC 5'GTGGGTGTCAATTTCAGCTC 6 97 Beclin 5 CGCCTCCTATTCCATCAAAA 5 AACTGTGAGGACACCCAAGC 6 LC 5'CACTGCTCTGTCTTGTGTAGGTTG 5'TCGTTGTGCCTTTATTAGTGCATC 65 7 p6 5'CCCAGTGTCTTGGCATTCTT 5'AGGGAAAGCAGAGGAAGCTC 6 54 HIFα 5'GCTTGGTGCTGTTTGTGAACC 5'GCATCCTGTACTGTCCTGTGGTG 64 67 GLUT 5'CAGAAGGTAATTGAGGAGTTCTACA 5'ACAAAGGCCAACAGGTTCATCATC 6 6 CAXII 5'CAGCCATCAAGAAGGAGG 5'ACTCCTGCCCCCTAGTTC 58 56 Met 5'ACACCGTGGCGTGCCAACAT 5'TCGCCGCCGTTCAGCTTCAG 55 64 βctin SQPCR 5'CACGATGGAGGGGCCGGACTCAT 5'TAAAGACCTCTATGCCAACACAG 6 4 βctin RTPCR 5'CGCGAGTACAACCTTCTTGC 5'CGTCATCCATGGCGAACTGG 6 7