Fig 2: Boosted Tandem-HTRF thanks to the use of several “acceptor” antibodies.(a) Tandem-HTRF applied to the detection of Ntag-CycD1 from RAS-G12V/DNP53 transformed MEFs, using several combinations of “acceptor” antibodies together with FLAG “donor” (top schematic) or HA “donor” and the resulting HTRF signal obtained (bottom graph). Error bars = SD, n = 3. Of note, the plateau of HTRF signal is reached at 30 000 a.u in this experimental setting. (b) Tandem-HTRF applied to the detection of Ctag-CycD1 from RAS-G12V/DNP53 transformed MEFs, using several combinations of “acceptor” antibodies together with FLAG “donor” (top schematic) or HA “donor” and the resulting HTRF signal obtained (bottom graph). Error bars = SD, n = 3. (c) Tandem-HTRF controls obtained with lysates from RAS-G12V/DNP53 transformed wildtype MEFs, using several combinations of “acceptor” antibodies together with FLAG “donor” (right schematic) or HA “donor” (left graph). Note that in this configuration no donor antibody can work in tandem with any acceptor for energy transfer. Error bars = SD, n = 3.

Fig 3: Semi-quantification of Tagged-CycD1 by Tandem-HTRF.(a) Schematic representing the technological setting where lysates from 3 Ccnd1Ntag/Ctag hybrids E.13.5 embryonic brains (Tagged-CycD1) have been mixed with lysates from 3 Ccnd1+/+ E.13.5 embryonic brains (wildtype CycD1). (b) Graph showing the HTRF signal obtained following decreased dilutions series of Tagged-CycD1 together with increased dilution series of wildtype CycD1 as described in (a) (top panel), compared with the corresponding immunoblot using the same Tagged-CycD1/wildtype CycD1 proportions (bottom panel). Note the better precision of Tandem-HTRF for Tagged-CycD1 semi-quantification. Error bars = SD, n = 3. (c) Graph showing the HTRF signal obtained following decreased dilutions series of Tagged-CycD1 together with increased dilution series of wildtype CycD1 as described in (a) (top panel), compared with the corresponding immunoblot using the same Tagged-CycD1/wildtype CycD1 proportions where the tagged-CycD1 signal becomes almost undetectable (bottom panel). Note the precision of Tagged-CycD1 semi-quantification by Tandem-HTRF at levels where the protein is not detectable by immunoblot. Error bars = SD, n = 3.

Fig 4: In diabetic hyperinsulinemic mice, cyclin D1-CDK4 is dysregulated and hyperactivation of cyclin D1-CDK4 attenuates the diabetic phenotype. a) qRT-PCR analysis of gluconeogenic and Ccnd1 gene expression changes upon fasting and refeeding in Leprdb/+ (db/+) and Leprdb/db (db/db) mice livers (two-tailed unpaired t-test, n=3). b) Cyclin D1 protein is chronically elevated upon fasting and refeeding in livers of high fat diet fed mice compared to control diet fed mice. Nuclear (N) and cytoplasmic (C) liver extracts were used. c-d) Phosphorylation of GCN5 is elevated upon fasting in db/db or high fat diet fed (HFD) mice liver compared its respective control mice and it remains insensitive to fast-refed transitions. Western-blot analysis of GCN5 immnunoprecipiated by anti-phosphoS*P (pS*P) antibody using liver extracts from mice that were tail-vein injected with adenoviruses expressing GFP or GCN5 (F=16hr fast, R=4hr refed). e) Cyclin D1 and Rb phosphorylation levels in livers from db/db mice tail-vein injected with GFP, cyclin D1 T286A, or cyclin D1 K112E adenoviruses, shown in Fig. 4b-c. Nuclear and cytoplasmic liver extracts were used. f-g) Cyclin D1 T286A overexpression reduces gluconeogenic genes and glycemia in high fat diet fed mice (two-tailed unpaired t-test, n=6/GFP, n=7/D1 T286A). h) Cyclin D1 and Rb phosphorylation levels in livers of high fat diet fed mice that were tail-vein injected with adenoviruses expressing GFP or cyclin D1 T286A, shown in Extended Data Fig. 4f-g. i) Overall Model. Statistical significance is represented by asterisk corresponding to *P<0.05, **P<0.01, ***P<0.001. Data are shown as mean±S.E.M.

Fig 5: Downregulation of miR-181a decreases the expression of CCND1. (A) Protein expression levels of cyclin D1 in RPMI8226 cells transfected with the miR-181a inhibitor measured by western blot analysis. (B) Quantified protein expression levels of cyclin D1 in cells transfected with the miR-181a inhibitor compared with cells transfected with miR-NC. *P<0.05, inhibitor vs. NC. miR-181a, microRNA; NC, negative control.