Figure 7 ( B ) . USP36 is required for rRNA processing . HeLa cells transfected with scr or USP36 siRNA ( left panels ) or infected with scr or USP36 shRNA lentiviruses ( right panels ) were assayed for rRNA processing by Northern blot using 5 ' - ETS , ITS1 and ITS2 probes as indicated . Knockdown of USP36 inhibits translation . HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O - propargyl - puromycin ( OPP ) followed by Click - iT OPP protein synthesis assays . Representative images ( C ) and quantification from three independent experiments ( D ) . Data were presented as mean ± SD , n = 3 biological replicates . * * * P < 0 . 001 , compared to scr control ( Student ' s t - test ) . Scale bar = 50 μM .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B). USP36 is required for rRNA processing. HeLa cells transfected with scr or USP36 siRNA (left panels) or infected with scr or USP36 shRNA lentiviruses (right panels) were assayed for rRNA processing by Northern blot using 5'- ETS, ITS1 and ITS2 probes as indicated.Knockdown of USP36 inhibits translation. HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O-propargyl-puromycin (OPP) followed by Click-iT OPP protein synthesis assays. Representative images (C) and quantification from three independent experiments (D). Data were presented as mean ± SD, n=3 biological replicates. ***P<0.001, compared to scr control (Student's t-test). Scale bar = 50 μM.


Figure 1 . Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( A ) Age - matched control Atg7 littermates ( Atg7flox / + ; nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) or Atg7 KO ( Atg7flox / flox ; nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) are shown . Accumulation of Ub in autophagy - deficient brain . Ub profile from the 1 % Triton X - 100 - soluble and - insoluble fraction of autophagy - deficient brain homogenates captured with P2UBA resin . ( B ) Control Atg5 littermates ( Atg5flox / + ; nestin - Cre ; denoted + ) at 16 ( open circles ; n = 4 ) and 26 wk ( open diamonds ; n = 3 ) or Atg5 KO ( Atg5flox / flox ; nestin - Cre ; denoted − ) at 16 ( closed circles ; n = 4 ) and 26 wk ( closed diamonds ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (A) Age-matched control Atg7 littermates (Atg7flox/+;nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4) or Atg7 KO (Atg7flox/flox;nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3) are shown.Accumulation of Ub in autophagy-deficient brain. Ub profile from the 1% Triton X-100-soluble and -insoluble fraction of autophagy-deficient brain homogenates captured with P2UBA resin. (B) Control Atg5 littermates (Atg5flox/+;nestin-Cre; denoted +) at 16 (open circles; n = 4) and 26 wk (open diamonds; n = 3) or Atg5 KO (Atg5flox/flox;nestin-Cre; denoted −) at 16 (closed circles; n = 4) and 26 wk (closed diamonds; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 2 . Suppressed accumulation of Ub in Atg7 - / p62 - deficient brains . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured total ( A ) and isopeptide - linked ( B ) Ub from the soluble and insoluble fractions of autophagy - deficient brains . Age - matched control Atg7 littermates ( Atg7flox / + : nestin - Cre : p62 + / − ; denoted + ) at 6 wk ( open circles ; n = 4 ) , p62 KO ( Atg7flox / flox : p62 − / − ; denoted − ) at 6 wk ( closed circles ; n = 3 ) , Atg7 KO ( Atg7flox / flox : nestin - Cre : p62 + / − ; denoted − ) at 6 wk ( closed diamonds ; n = 3 ) , or Atg7 - / p62 - DKO ( Atg7flox / flox : nestin - Cre : p62 − / − ) at 6 wk ( closed triangles ; n = 3 ) are shown . Each symbol represents one animal . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2.Suppressed accumulation of Ub in Atg7-/p62-deficient brains. (A and B) Quantitative mass spectrometry analysis of P2UBA captured total (A) and isopeptide-linked (B) Ub from the soluble and insoluble fractions of autophagy-deficient brains. Age-matched control Atg7 littermates (Atg7flox/+:nestin-Cre:p62+/−; denoted +) at 6 wk (open circles; n = 4), p62 KO (Atg7flox/flox:p62−/−; denoted −) at 6 wk (closed circles; n = 3), Atg7 KO (Atg7flox/flox:nestin-Cre:p62+/−; denoted −) at 6 wk (closed diamonds; n = 3), or Atg7-/p62-DKO (Atg7flox/flox:nestin-Cre:p62−/−) at 6 wk (closed triangles; n = 3) are shown. Each symbol represents one animal. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 3 . Suppressed accumulation of Ub in Atg7 - / Nrf2 - deficient livers . ( A and B ) Quantitative mass spectrometry analysis of P2UBA captured p62 ( A ) , total ( A ) , and isopeptide - linked ( B ) Ub from the soluble and insoluble fraction of autophagy - deficient livers ( 28 d after pIpC injection ) . Control littermates ( F / F or F / F : Nrf2 + / − ; denoted + ; open circles ; n = 4 ) , Nrf2 KO ( F / F : Nrf2 − / − ; denoted − ; closed circles ; n = 4 ) , Atg7 KO ( F / F : Mx1 or F / F : Mx1 : Nrf2 + / − ; denoted − ; closed diamonds ; n = 6 ) , or Atg7 - / Nrf2 - DKO ( F / F : Mx1 : Nrf2 − / − ; closed triangles ; n = 6 ) are shown . Each symbol represents one animal . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.Suppressed accumulation of Ub in Atg7-/Nrf2-deficient livers. (A and B) Quantitative mass spectrometry analysis of P2UBA captured p62 (A), total (A), and isopeptide-linked (B) Ub from the soluble and insoluble fraction of autophagy-deficient livers (28 d after pIpC injection). Control littermates (F/F or F/F:Nrf2+/−; denoted +; open circles; n = 4), Nrf2 KO (F/F:Nrf2−/−; denoted −; closed circles; n = 4), Atg7 KO (F/F:Mx1 or F/F:Mx1:Nrf2+/−; denoted −; closed diamonds; n = 6), or Atg7-/Nrf2-DKO (F/F:Mx1:Nrf2−/−; closed triangles; n = 6) are shown. Each symbol represents one animal. **, P ≤ 0.01; ***, P ≤ 0.005.


Figure 4 . ( B ) Elevated Ub mRNA levels from Atg5 - deficient brains . Total RNA was extracted from control Atg5 littermates ( Atg5flox / + ; nestin - Cre ) at 16 wk or Atg5 KO ( Atg5flox / flox ; nestin - Cre ) at 16 wk ( n = 4 for each genotype ; * * , P ≤ 0 . 01 ) and the amount of transcript from Ubc , Ubb , Uba52 , and Uba80 genes was quantified using real - time RT - PCR and normalized to actin . Error bars indicate SEM . au , arbitrary unit .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(B) Elevated Ub mRNA levels from Atg5-deficient brains. Total RNA was extracted from control Atg5 littermates (Atg5flox/+;nestin-Cre) at 16 wk or Atg5 KO (Atg5flox/flox;nestin-Cre) at 16 wk (n = 4 for each genotype; **, P ≤ 0.01) and the amount of transcript from Ubc, Ubb, Uba52, and Uba80 genes was quantified using real-time RT-PCR and normalized to actin. Error bars indicate SEM. au, arbitrary unit.


Figure 6 . ( B ) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line , m5 - 7 , as measured by the decreased levels of phosphatidylethanolamine - modified LC3 ( LC3 - II ) . ( C ) Western blot ( WB ) analysis of the accumulation of htt ( Q ) - GFP after autophagy shutoff . At steady - state ( t = 0 h ) , the levels of both htt ( Q25 ) - GFP and htt ( Q47 ) - GFP were virtually undetectable . After autophagy shutoff , there was a robust increase in the level of htt ( Q47 ) - GFP , as detected by GFP Western blot , whereas there was very little or no detectable increase in htt ( Q25 ) - GFP , GAPDH , LDH , or chFP . ( D ) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells . The ASI is the ratio of the relative increase in green and red fluorescence , as assessed by two - color flow cytometry , in response to dox . In control experiments , the ASI of two soluble , nonaggregating proteins , htt ( Q25 ) - GFP and chFP , was 1 . In contrast , the ASI of the aggregation - prone protein , htt ( Q47 ) - GFP , compared with chFP after autophagy shutoff ( + dox ; 100 h ) was 2 . Data represent three independent experiments performed on different days . * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 005 . ( E ) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff ( + dox ) . The degree of cell death correlated directly with increased htt ( Q47 ) - GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt ( Q47 ) - GFP compared with htt ( Q25 ) - GFP ( * * * , P ≤ 0 . 005 ) . Data represent three independent experiments performed on different days . ( F ) AQUA using heavy isotope - labeled GFP standard in addition to the routine Ub standards after P2UBA pull - down of soluble and insoluble lysates from htt ( Q ) - GFP - IRES - chFP bicistronic expressing cells before ( − dox ) and after autophagy shutoff ( + dox ) . The experimental GFP peptide was only detected in two of the four experiments , and the data shown are representative of these . In the other two experiments , the experimental GFP peptide was below the threshold of detection .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(B) The addition of dox for 96 h decreased autophagy in the bicistronic stably expressing cell lines similar to the parental line, m5-7, as measured by the decreased levels of phosphatidylethanolamine-modified LC3 (LC3-II).(C) Western blot (WB) analysis of the accumulation of htt(Q)-GFP after autophagy shutoff. At steady-state (t = 0 h), the levels of both htt(Q25)-GFP and htt(Q47)-GFP were virtually undetectable. After autophagy shutoff, there was a robust increase in the level of htt(Q47)-GFP, as detected by GFP Western blot, whereas there was very little or no detectable increase in htt(Q25)-GFP, GAPDH, LDH, or chFP.(D) Quantitative flow cytometry time course analysis measuring selective autophagy in live cells. The ASI is the ratio of the relative increase in green and red fluorescence, as assessed by two-color flow cytometry, in response to dox. In control experiments, the ASI of two soluble, nonaggregating proteins, htt(Q25)-GFP and chFP, was 1. In contrast, the ASI of the aggregation-prone protein, htt(Q47)-GFP, compared with chFP after autophagy shutoff (+dox; 100 h) was 2. Data represent three independent experiments performed on different days. **, P ≤ 0.01; ***, P ≤ 0.005.(E) Flow cytometry time course analysis of cell death at different time points after autophagy shutoff (+dox). The degree of cell death correlated directly with increased htt(Q47)-GFP levels after autophagy shutoff and was significantly enhanced in cells expressing htt(Q47)-GFP compared with htt(Q25)-GFP (***, P ≤ 0.005). Data represent three independent experiments performed on different days.(F) AQUA using heavy isotope-labeled GFP standard in addition to the routine Ub standards after P2UBA pull-down of soluble and insoluble lysates from htt(Q)-GFP-IRES-chFP bicistronic expressing cells before (−dox) and after autophagy shutoff (+dox). The experimental GFP peptide was only detected in two of the four experiments, and the data shown are representative of these. In the other two experiments, the experimental GFP peptide was below the threshold of detection.


Figure 3 ( E ) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals : MN2a axons projecting to the MHE muscle , representing the wild - type ( wt ) innervation pattern ( magenta ) ; MN2a axons projecting to muscles other than the MHE termed abnormal innervation ( light grey ) ; abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects ( dark grey ) . Note , each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS - RFP or UAS - GFPRNAi . Each experiment was performed in triplicates , innervation rates were calculated from n = 56 for elav > RFP , n = 21 for elav > DfdRNAi , n = 10 for elav > hthRNAi and n = 7 for elav > mirrRNAi . In the case of Dfd knock - down , two different driver lines were used , the pan - neural elav - GAL4 and the motoneuron - specific OK6 - GAL4 drivers , respectively . Both result in similar phenotypes , highlighting that the elav - GAL4 driven effects are specific to MNs . p - values between two genetic conditions were calculated by a two - sided Fisher test . * * * : p < 0 . 005 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(E) Quantification of different phenotypes manifesting after RNA interference in comparison to control animals: MN2a axons projecting to the MHE muscle, representing the wild-type (wt) innervation pattern (magenta); MN2a axons projecting to muscles other than the MHE termed abnormal innervation (light grey); abnormal synaptic morphologies at MN2a derived axon terminals termed terminal defects (dark grey). Note, each genetic experiment was performed in parallel to an adequate control experiment using the same driver line crossed to a line that controls expression of either UAS-RFP or UAS-GFPRNAi. Each experiment was performed in triplicates, innervation rates were calculated from n=56 for elav>RFP, n=21 for elav>DfdRNAi, n=10 for elav>hthRNAi and n=7 for elav>mirrRNAi. In the case of Dfd knock-down, two different driver lines were used, the pan-neural elav-GAL4 and the motoneuron-specific OK6-GAL4 drivers, respectively. Both result in similar phenotypes, highlighting that the elav-GAL4 driven effects are specific to MNs. p-values between two genetic conditions were calculated by a two-sided Fisher test. ***: p < 0.005.


Figure 4 ( B ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd ( purple ) , Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and animals mis - expressing the Hox TFs Lab , Dfd and Scr by means of the elav - GAL4 driver . A zoom on the projections ( FasII staining ) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right side represents a schematic drawing of the confocal image shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and mis - expression conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and animals mis-expressing the Hox TFs Lab, Dfd and Scr by means of the elav-GAL4 driver. A zoom on the projections (FasII staining) of MN2a and MN3 to the MHE and MHD muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


Figure 6 ( A ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and in animals with reduced neuronal DIPkappa ( elav > DIPkappaRNAi ) as well as in animals , which mis - express DIPgamma or dpr1 in neuronal cells ( elav > DIPgamma , elav > dpr1 ) . Single channels focusing on the projections ( FasII staining ) of MN2a and / or MN3 to the MHE , MHD or LR muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right represents a schematic drawing of the confocal images shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and mis - expression conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced neuronal DIPkappa (elav>DIPkappaRNAi) as well as in animals, which mis-express DIPgamma or dpr1 in neuronal cells (elav>DIPgamma, elav>dpr1). Single channels focusing on the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right represents a schematic drawing of the confocal images shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and mis-expression conditions.


Figure 8 ( A ) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd ( purple ) , Myosin in muscles ( blue ) and FasII in axonal projections ( green ) in control ( elav > RFP ) animals and in animals with reduced Dfd expression in neurons ( elav > DfdRNAi ) or in muscles ( Mef2 > DfdRNAi ) or both in both tissues ( elav ; Mef2 > DfdRNAi ) . The single channels for the projections ( FasII staining ) of MN2a and / or MN3 to the MHE , MHD or LR muscles of an early stage 17 Drosophila embryo are shown . Asterisks highlight the location of MN2a and MN3 , respectively . MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN , which normally innervates the Dfd expressing MHE muscle ( as shown in the elav > RFP control ) , while MN3 is identified as the MN underneath MN2a , which normally innervates the MHD muscle ( as shown in the elav > RFP control ) . The panel on the right side represents a schematic drawing of the confocal image shown on the left side , summarizing the innervation of the anterior muscles ( LR , MHE , MHD ) by projections emerging from MN2a ( magenta ) or MN3 ( cyan ) in control and perturbation conditions .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(A) Representative confocal images of stage 17 embryonic heads highlighting the expression of Dfd (purple), Myosin in muscles (blue) and FasII in axonal projections (green) in control (elav>RFP) animals and in animals with reduced Dfd expression in neurons (elav>DfdRNAi) or in muscles (Mef2>DfdRNAi) or both in both tissues (elav;Mef2>DfdRNAi). The single channels for the projections (FasII staining) of MN2a and/or MN3 to the MHE, MHD or LR muscles of an early stage 17 Drosophila embryo are shown. Asterisks highlight the location of MN2a and MN3, respectively. MN2a is identified by the FasII expressing axonal projection emerging from a Dfd expressing MN, which normally innervates the Dfd expressing MHE muscle (as shown in the elav>RFP control), while MN3 is identified as the MN underneath MN2a, which normally innervates the MHD muscle (as shown in the elav>RFP control). The panel on the right side represents a schematic drawing of the confocal image shown on the left side, summarizing the innervation of the anterior muscles (LR, MHE, MHD) by projections emerging from MN2a (magenta) or MN3 (cyan) in control and perturbation conditions.


Figure 1 ( A ) Adult muscles were transfected with YFP ‐ LC3 and with pDsRed2 ‐ Mito . Animals were starved for 24 h or denervated for 7 days . Muscles were exposed and observed in situ using confocal in vivo microscopy . At least five animals per condition have been studied . Scale bar , 20 μm . ( B ) Adult muscles were transfected with YFP ‐ LC3 and with pDsRed2 ‐ Mito . Lysosomes were inhibited by treating mice with 50 mg / kg of chloroquine for 7 days . Chloroquine treatment started at the day of denervation and lasted for 7 days , whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h . Muscles were exposed and observed in situ using confocal in vivo microscopy . At least five animals per condition have been studied . Scale bar , 20 μm .

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Figure 1(A) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Animals were starved for 24 h or denervated for 7 days. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.(B) Adult muscles were transfected with YFP‐LC3 and with pDsRed2‐Mito. Lysosomes were inhibited by treating mice with 50 mg/kg of chloroquine for 7 days. Chloroquine treatment started at the day of denervation and lasted for 7 days, whereas for fasting experiments at the 6th day of chloroquine injection food was removed for 24 h. Muscles were exposed and observed in situ using confocal in vivo microscopy. At least five animals per condition have been studied. Scale bar, 20 μm.


Figure 2 ( A ) Imaging of FoxO3 ‐ mediated modification of the mitochondrial network in soleus muscle of living mice . Adult soleus muscles were transfected with pDsRed2 ‐ Mito and either c . a . FoxO3 or mock vector ( control ) . Two weeks later , muscles were exposed and observed in situ using two ‐ photon microscopy . At least five animals per condition were studied . ( B ) Overexpression of c . a . FoxO3 induces mitochondrial changes and removal through autophagy . Muscle fibres were transfected by electroporation with YFP ‐ LC3 , pDsRed2 ‐ Mito and either c . a . FoxO3 or mock vector . Two weeks later , fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy . A higher magnification of the square is depicted on the right panels . White arrows point autophagosomes engulfing the mitochondria . ( C ) Overexpression of c . a . FoxO3 induces reduction in myofibre size and decrease in SDH staining . Left image : immunostaining for FoxO3 shows atrophic fibres , which over ‐ expressed FoxO3 ( red ) and are labelled by asterisks . Nuclei are stained by Hoechst ( blue ) . Right image : histochemistry for succinate dehydrogenase ( SDH ) on serial section . Note that fibres positive for FoxO3 are negative for SDH staining ( asterisks ) . ( D ) Electron micrograph of a control and c . a . FoxO3 ‐ expressing fibres . Muscles were processed with standard fixation ‐ embedding procedure . Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria , respectively . Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres , whereas intermyofibrillar mitochondria are smaller and paler than control ones . Red arrowheads show a vesicle containing mitochondria . The image is representative of the FoxO3 ‐ transfected fibres .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) Imaging of FoxO3‐mediated modification of the mitochondrial network in soleus muscle of living mice. Adult soleus muscles were transfected with pDsRed2‐Mito and either c.a.FoxO3 or mock vector (control). Two weeks later, muscles were exposed and observed in situ using two‐photon microscopy. At least five animals per condition were studied.(B) Overexpression of c.a.FoxO3 induces mitochondrial changes and removal through autophagy. Muscle fibres were transfected by electroporation with YFP‐LC3, pDsRed2‐Mito and either c.a.FoxO3 or mock vector. Two weeks later, fluorescent fibres were analysed for mitochondrial distribution by confocal microscopy. A higher magnification of the square is depicted on the right panels. White arrows point autophagosomes engulfing the mitochondria.(C) Overexpression of c.a.FoxO3 induces reduction in myofibre size and decrease in SDH staining. Left image: immunostaining for FoxO3 shows atrophic fibres, which over‐expressed FoxO3 (red) and are labelled by asterisks. Nuclei are stained by Hoechst (blue). Right image: histochemistry for succinate dehydrogenase (SDH) on serial section. Note that fibres positive for FoxO3 are negative for SDH staining (asterisks).(D) Electron micrograph of a control and c.a.FoxO3‐expressing fibres. Muscles were processed with standard fixation‐embedding procedure. Black and white arrows indicate subsarcolemmal and intermyofibrillar mitochondria, respectively. Note that subsarcolemmal mitochondria are reduced in FoxO3 overexpressing fibres, whereas intermyofibrillar mitochondria are smaller and paler than control ones. Red arrowheads show a vesicle containing mitochondria. The image is representative of the FoxO3‐transfected fibres.


Figure 3 ( A ) RNAi ‐ mediated knockdown of Fis1 showed by western blot assay . MEF cells were transfected with vectors expressing four different Fis shRNAs . For in vivo experiments Fis ( 4 ) was used . Efficiency of protein knockdown is indicated in Supplementary Table S2 . ( B ) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting . Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 ( oligo 4 ) and mouse Bnip3 or co ‐ transfected and 8 days later mice were starved for 48 h . Cross ‐ sectional area ( CSA ) of transfected fibres , identified by GFP , was measured as described earlier ( * P0 . 01 ) n = 450 . ( C ) RNAi of Fis1 and Bnip3 significantly prevents MuRF ‐ 1 and atrogin ‐ 1 promoters activation during starvation . TA muscles were transfected with either 5 kb of the MuRF ‐ 1 promoter ( left panel ) or 3 . 5 kb of atrogin ‐ 1 promoter ( right panel ) with or without shRNAs vectors against Fis and Bnip3 . In fasting experiments , after 7 days from transfection mice were starved for 24 h . A renilla ‐ luciferase construct ( pRL ‐ TK ) was co ‐ transfected to normalized for transfection efficiency . The activation of MuRF ‐ 1 and atrogin ‐ 1 promoters was determined by firefly activity and normalize for the renilla . Each condition represents the average of at least three independent experiments ± s . e . m . ( * P0 . 05 ) . ( D ) Muscle atrophy induced by FoxO3 is reduced by d . n . DRP1 . Adult skeletal muscles were transfected with HA ‐ tagged c . a . FoxO3 with or without d . n . DRP1 and examined after 2 weeks . CSA of transfected fibres , identified by anti ‐ HA immunofluorescence , was measured as described earlier ( * P0 . 001 ) n = 400 . ( E ) Blockade of the fission machinery by d . n . DRP1 or Bnip3 inhibition by RNAi reduces FoxO3 ‐ mediated autophagosome formation . Animals were transfected with YFP ‐ LC3 , c . a . FoxO3 in presence or absence of d . n . DRP1 or shRNAs against Bnip3 . Twelve days later , muscles were collected and analysed for fluorescent vesicles formation ( * P0 . 001 ) n = 450 . ( F ) FoxO3 ‐ dependent activation of MuRF ‐ 1 and atrogin ‐ 1 is significantly reduced by inhibition of the fission machinery . MuRF ‐ 1 promoter ( left panel ) or atrogin ‐ 1 promoter ( right panel ) was co ‐ transfected into adult TA muscle with or without c . a . FoxO3 and in the presence or absence of d . n . DRP1 . A renilla ‐ luciferase vector was co ‐ transfected to normalize for transfection efficiency . Eight days later , firefly and renilla ‐ luciferase activity was determined . Each condition represents the average of at least three independent experiments ± s . e . m . ( * P0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) RNAi‐mediated knockdown of Fis1 showed by western blot assay. MEF cells were transfected with vectors expressing four different Fis shRNAs. For in vivo experiments Fis (4) was used. Efficiency of protein knockdown is indicated in Supplementary Table S2.(B) Blockade of Fis1 and of Bnip3 by RNAi reduces muscle loss during fasting. Adult skeletal muscles were transfected with specific shRNAs for mouse Fis1 (oligo 4) and mouse Bnip3 or co‐transfected and 8 days later mice were starved for 48 h. Cross‐sectional area (CSA) of transfected fibres, identified by GFP, was measured as described earlier (*P0.01) n=450.(C) RNAi of Fis1 and Bnip3 significantly prevents MuRF‐1 and atrogin‐1 promoters activation during starvation. TA muscles were transfected with either 5 kb of the MuRF‐1 promoter (left panel) or 3.5 kb of atrogin‐1 promoter (right panel) with or without shRNAs vectors against Fis and Bnip3. In fasting experiments, after 7 days from transfection mice were starved for 24 h. A renilla‐luciferase construct (pRL‐TK) was co‐transfected to normalized for transfection efficiency. The activation of MuRF‐1 and atrogin‐1 promoters was determined by firefly activity and normalize for the renilla. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).(D) Muscle atrophy induced by FoxO3 is reduced by d.n.DRP1. Adult skeletal muscles were transfected with HA‐tagged c.a.FoxO3 with or without d.n.DRP1 and examined after 2 weeks. CSA of transfected fibres, identified by anti‐HA immunofluorescence, was measured as described earlier (*P0.001) n=400.(E) Blockade of the fission machinery by d.n.DRP1 or Bnip3 inhibition by RNAi reduces FoxO3‐mediated autophagosome formation. Animals were transfected with YFP‐LC3, c.a.FoxO3 in presence or absence of d.n.DRP1 or shRNAs against Bnip3. Twelve days later, muscles were collected and analysed for fluorescent vesicles formation (*P0.001) n=450.(F) FoxO3‐dependent activation of MuRF‐1 and atrogin‐1 is significantly reduced by inhibition of the fission machinery. MuRF‐1 promoter (left panel) or atrogin‐1 promoter (right panel) was co‐transfected into adult TA muscle with or without c.a.FoxO3 and in the presence or absence of d.n.DRP1. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least three independent experiments±s.e.m. (*P0.05).


Figure 4Overexpression of DRP1 / Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo . In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3 , an atrophy ‐ related gene under FoxO3 control , and DRP1 / Fis1 proteins . Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein ( mtM13 ‐ YFP ) and either mock vector , Bnip3 , HA ‐ DRP1 , Fis1 or DRP1 / Fis1 . Seven days later ( for Bnip3 ) or 12 days later ( for DRP1 and Fis1 ) , muscles were observed in vivo with confocal microscopy . TMRM was injected into the muscles to monitor mitochondria , which retain membrane potential . Muscles transfected with mtM13 ‐ YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria ( upper panel ‐ IMF ) . Scale bar : 20 μm .

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Figure 4Overexpression of DRP1/Fis1 and Bnip3 proteins induces changes in the mitochondrial network in vivo. In vivo imaging of the mitochondrial network was performed in muscles expressing Bnip3, an atrophy‐related gene under FoxO3 control, and DRP1/Fis1 proteins. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and either mock vector, Bnip3, HA‐DRP1, Fis1 or DRP1/Fis1. Seven days later (for Bnip3) or 12 days later (for DRP1 and Fis1), muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Muscles transfected with mtM13‐YFP and mock vector showed the orderly pattern of intermyofibrillar and subsarcolemmal mitochondria (upper panel‐IMF). Scale bar: 20 μm.


Figure 5 ( A ) Adult TA muscles were transfected with either DRP1 , Fis1 , DRP1 and Fis1 or mock vector . Muscles were collected 12 or 21 days after transfection and cross ‐ sectional area of transfected fibres , identified by immunofluorescence , was measured as described earlier ( * P0 . 001 ) n = 400 . ( B ) Bnip3 or Bnip3 l expression for 7 days causes a 20 % reduction of the cross ‐ sectional area . TA muscles were transfected with Bnip3 , Bnip3l or mock vector . Cross ‐ sectional area of transfected fibres , identified by immunofluorescence , was analysed ( * P0 . 001 ) n = 500 . ( C ) Quantification of LC3 ‐ positive autophagic vesicles in adult muscles co ‐ tranfected with YFP ‐ LC3 and DRP1 , Fis1 or DRP1 / Fis1 expression plasmids or mock vector . ( D ) Mutations in BH3 domain of Bnip3 ( Bnip3BH3mut ) do not affect BNIP3 ‐ mediated autophagy . Adult muscles were co ‐ transfected with YFP ‐ LC3 and Bnip3 or Bnip3BH3mut expression plasmids and LC3 ‐ positive vesicles were quantified . ( E ) Bnip3 ‐ mediated atrophy is not modified by mutating the BH3 domain ( Bnip3BH3mut ) . Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross ‐ sectional area was quantified 7 days later and plotted as % of controls ( * P0 . 001 ) n = 440 .

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Figure 5(A) Adult TA muscles were transfected with either DRP1, Fis1, DRP1 and Fis1 or mock vector. Muscles were collected 12 or 21 days after transfection and cross‐sectional area of transfected fibres, identified by immunofluorescence, was measured as described earlier (*P0.001) n=400.(B) Bnip3 or Bnip3 l expression for 7 days causes a 20% reduction of the cross‐sectional area. TA muscles were transfected with Bnip3, Bnip3l or mock vector. Cross‐sectional area of transfected fibres, identified by immunofluorescence, was analysed (*P0.001) n=500.(C) Quantification of LC3‐positive autophagic vesicles in adult muscles co‐tranfected with YFP‐LC3 and DRP1, Fis1 or DRP1/Fis1 expression plasmids or mock vector.(D) Mutations in BH3 domain of Bnip3 (Bnip3BH3mut) do not affect BNIP3‐mediated autophagy. Adult muscles were co‐transfected with YFP‐LC3 and Bnip3 or Bnip3BH3mut expression plasmids and LC3‐positive vesicles were quantified.(E) Bnip3‐mediated atrophy is not modified by mutating the BH3 domain (Bnip3BH3mut). Adult TA muscles were transfected with Bnip3 or Bnip3BH3mut expression plasmids and cross‐sectional area was quantified 7 days later and plotted as % of controls (*P0.001) n=440.


Figure 6 ( A ) In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R . Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein ( mtM13 ‐ YFP ) and Fis1K148R . Twelve days later muscles were observed in vivo with confocal microscopy . TMRM was injected into the muscles to monitor mitochondria , which retain membrane potential . Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved . ( B ) FDB muscle fibres were transfected by electroporation in vivo . Eight days later adult fibres were isolated and placed in cell culture . Adult fibres were loaded with TMRM ( 5 nM ) for 30 min at 37 ° C . TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential . Oligomycin ( Olm , 5 μM ) and the protonophore FCCP ( 4 μM ) were added at the indicated time points . TMRM staining were monitored in at least 10 fibres per construct ( * P0 . 001 ) . ( C ) hFisK148R ‐ transfected muscles were collected 12 and 21 days after transfection . Cross ‐ sectional area of transfected fibres , identified by anti ‐ myc immunofluorescence , was quantified ( n = 550 ) . ( D ) Fis1 ‐ dependent activation of MuRF ‐ 1 . MuRF ‐ 1 promoter was co ‐ transfected into adult TA muscle with or without Fis1 or Fis1K148R . A renilla ‐ luciferase vector was co ‐ transfected to normalize for transfection efficiency . Eight days later , firefly and renilla ‐ luciferase activity was determined . Each condition represents the average of at least five independent experiments ± s . e . m . ( * P0 . 05 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A)In vivo imaging of the mitochondrial network was performed in muscles expressing Fis1K148R. Adult muscles were transfected with plasmids encoding mitochondrially targeted yellow fluorescent protein (mtM13‐YFP) and Fis1K148R. Twelve days later muscles were observed in vivo with confocal microscopy. TMRM was injected into the muscles to monitor mitochondria, which retain membrane potential. Mitochondrial network was greatly altered but mitochondrial membrane potential was conserved.(B) FDB muscle fibres were transfected by electroporation in vivo. Eight days later adult fibres were isolated and placed in cell culture. Adult fibres were loaded with TMRM (5 nM) for 30 min at 37°C. TMRM accumulates in the mitochondria that maintain mitochondrial membrane potential. Oligomycin (Olm, 5 μM) and the protonophore FCCP (4 μM) were added at the indicated time points. TMRM staining were monitored in at least 10 fibres per construct (*P0.001).(C) hFisK148R‐transfected muscles were collected 12 and 21 days after transfection. Cross‐sectional area of transfected fibres, identified by anti‐myc immunofluorescence, was quantified (n=550).(D) Fis1‐dependent activation of MuRF‐1. MuRF‐1 promoter was co‐transfected into adult TA muscle with or without Fis1 or Fis1K148R. A renilla‐luciferase vector was co‐transfected to normalize for transfection efficiency. Eight days later, firefly and renilla‐luciferase activity was determined. Each condition represents the average of at least five independent experiments±s.e.m. (*P0.05).


Figure 7 ( A ) Left panel : mitochondrial fission activates AMPK . HEK293 cells were transfected with a mock vector ( control ) , Bnip3 , DRP1 , Fis1 or co ‐ transfected with DRP1 and Fis1 . Cells were collected 24 h later and lysates were analysed by immunoblotting . Right panel : immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy . P ‐ AMPK / AMPK ratio analysed by densitometric analysis is indicated below the panels . ( B - E ) Inhibition of AMPK blocks Bnip3 ‐ and DRP1 / Fis1 ‐ mediated atrophy . ( B ) Adult TA muscles were transfected with Bnip3 together with d . n . AMPK or mock vector and cross ‐ sectional area was measured ( * P0 . 001 ) n = 480 . ( C ) Adult TA muscles were transfected with DRP1 and Fis1 together with d . n . AMPK or mock vector . Twelve days later muscles were collected and cross ‐ sectional area was quantified ( * P0 . 001 ) n = 400 . ( D , E ) RNAi ‐ mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3 ‐ and DRP1 / Fis1 ‐ dependent atrophy . Mouse TA muscles were co ‐ transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ ( control ) or AMPK . Twelve days later , TA muscles were collected and the cross ‐ sectional area was quantified ( * P0 . 001 ) n = 450 .

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Figure 7(A) Left panel: mitochondrial fission activates AMPK. HEK293 cells were transfected with a mock vector (control), Bnip3, DRP1, Fis1 or co‐transfected with DRP1 and Fis1. Cells were collected 24 h later and lysates were analysed by immunoblotting. Right panel: immunoblotting analysis shows AMPK phosphorylation in EDL and soleus muscles during muscle atrophy. P‐AMPK/AMPK ratio analysed by densitometric analysis is indicated below the panels. (B-E) Inhibition of AMPK blocks Bnip3‐ and DRP1/Fis1‐mediated atrophy.(B) Adult TA muscles were transfected with Bnip3 together with d.n.AMPK or mock vector and cross‐sectional area was measured (*P0.001) n=480.(C) Adult TA muscles were transfected with DRP1 and Fis1 together with d.n.AMPK or mock vector. Twelve days later muscles were collected and cross‐sectional area was quantified (*P0.001) n=400.(D, E) RNAi‐mediated knockdown of AMPK α 1 and α 2 inhibits Bnip3‐ and DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with Bnip3 or DRP1 and Fis1 and pSuper vectors expressing shRNAs against lacZ (control) or AMPK. Twelve days later, TA muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.


Figure 8 ( A ) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides ( see Supplementary Table 1 ) ( * P0 . 01 ) . ( B ) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3 . ( C ) FoxO3 binding to atrogin ‐ 1 and MuRF ‐ 1 promoters , as determined by ChIP , is induced by AICAR treatment . ( D ) RNAi ‐ mediated knockdown of FoxO3 inhibits DRP1 / Fis1 ‐ dependent atrophy . Mouse TA muscles were co ‐ transfected with DRP1 and Fis1 and pSuper vectors expressing shRNAs against either lacZ ( control ) or FoxO3 . Twelve days later , muscles were collected and the cross ‐ sectional area was quantified ( * P0 . 001 ) n = 450 .

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Figure 8(A) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides (see Supplementary Table 1) (*P0.01).(B) Immunoblotting analysis shows that AICAR treatment causes phosphorylation of AMPK and the downstream target ACC but does not affect phosphorylation of AKT and FoxO3.(C) FoxO3 binding to atrogin‐1 and MuRF‐1 promoters, as determined by ChIP, is induced by AICAR treatment.(D) RNAi‐mediated knockdown of FoxO3 inhibits DRP1/Fis1‐dependent atrophy. Mouse TA muscles were co‐transfected with DRP1 and Fis1 and pSuper vectors expressing shRNAs against either lacZ (control) or FoxO3. Twelve days later, muscles were collected and the cross‐sectional area was quantified (*P0.001) n=450.


Figure 1Representative PET , MRI and fused PET / MRI images of the thoracic region of a mouse that received the PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal ( n = 5 ) . * P = 0 . 0488 ( 20 mins ) , * P = 0 . 0236 ( 25 mins ) , * P = 0 . 0167 ( 30 mins ) , * * P = 0 . 00301 ( 35 mins ) , * * P = 0 . 00269 ( 40 mins ) , * * P = 0 . 00215 ( 45 mins ) , * * P = 0 . 00129 ( 50mins ) and * * P = 0 . 00141 ( 45mins ) using unpaired 2 - tailed Student ' s t tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative PET, MRI and fused PET/MRI images of the thoracic region of a mouse that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT and skeletal muscle throughout the dynamic scan was calculated and compared for each animal (n = 5). * P = 0.0488 (20 mins), * P = 0.0236 (25 mins), * P = 0.0167 (30 mins), ** P = 0.00301 (35 mins), ** P = 0.00269 (40 mins), ** P = 0.00215 (45 mins), ** P = 0.00129 (50mins) and ** P = 0.00141 (45mins) using unpaired 2-tailed Student's t tests.


Figure 2A . Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP - 10 ( 30mg / kg ) ( n = 3 ) or vehicle ( n = 4 ) prior to receiving [ 18F ] - FDG . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake value ( SUV ) of [ 18F ] - FDG in BAT after both treatments was calculated for each group . * * P = 0 . 0013 using unpaired 2 - tailed Student ' s t test . B . The percentage ( % ) change in core body temperature and associated area under the curve ( AUC ) of mice treated with MP - 10 ( 30mg / kg ) ( n = 4 ) or vehicle ( n = 4 ) prior to a cold challenge study for 4 hours at 8oC . * P = 0 . 0108 ( 1st hour ) , * P = 0 . 0218 ( 2nd hour ) , * P = 0 . 0121 ( 3rd hour ) , * P = 0 . 0168 ( 4th hour ) and * P = 0 . 0264 ( AUC ) using unpaired 2 - tailed Student ' s t tests . C . RT - qPCR analysis of relative mRNA expression of Pde10a in BAT , peri - ovarian visceral white adipose tissue ( VAT ) and inguinal subcutaneous white adipose tissue ( SAT ) of lean mice ( n = 5 ) . * * P = 0 . 0063 ( SAT vs VAT ) , * * P = 0 . 0011 ( SAT vs BAT ) and * * P = 0 . 0037 ( VAT vs BAT ) using unpaired 2 - tailed Student ' s t test . D Cyclic GMP ( cGMP ) and cyclic AMP ( cGMP ) concentrations normalized to protein concentration in SAT , VAT and BAT 30 minutes after treatment of lean mice with MP - 10 ( 30 mg / kg ) ( n = 6 ) or vehicle control ( n = 6 ) . * * P = 0 . 0085 ( cGMP in VAT ) , * * P = 0 . 0011 ( cGMP in BAT ) and * P = 0 . 04 ( cAMP in VAT ) using unpaired 2 - tailed Student ' s t tests . E RT - qPCR analysis of relative mRNA expression of thermogenic genes in SAT , VAT and BAT after treatment of lean mice treated with MP - 10 ( 30 mg / kg ) ( n = 7 ) or vehicle control ( n = 6 ) . * * * * P < 0 . 0001 ( Pgc1alpha in VAT ) , * * P = 0 . 0012 ( Ucp1 in VAT ) , * * P = 0 . 0071 ( Pgc1alpha in BAT ) and * P = 0 . 0486 ( Ucp1 in BAT ) using unpaired 2 - tailed Student ' s t tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Representative PET images of a sagittal view of the thoracic region of fasted lean mice treated with either MP-10 (30mg/kg) (n = 3) or vehicle (n = 4) prior to receiving [18F]-FDG. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-FDG in BAT after both treatments was calculated for each group. ** P = 0.0013 using unpaired 2-tailed Student's t test.B. The percentage (%) change in core body temperature and associated area under the curve (AUC) of mice treated with MP-10 (30mg/kg) (n = 4) or vehicle (n=4) prior to a cold challenge study for 4 hours at 8oC. * P = 0.0108 (1st hour), * P = 0.0218 (2nd hour), * P = 0.0121 (3rd hour), * P = 0.0168 (4th hour) and * P = 0.0264 (AUC) using unpaired 2-tailed Student's t tests.C. RT-qPCR analysis of relative mRNA expression of Pde10a in BAT, peri-ovarian visceral white adipose tissue (VAT) and inguinal subcutaneous white adipose tissue (SAT) of lean mice (n = 5). ** P = 0.0063 (SAT vs VAT), ** P = 0.0011 (SAT vs BAT) and ** P = 0.0037 (VAT vs BAT) using unpaired 2-tailed Student's t test.D Cyclic GMP (cGMP) and cyclic AMP (cGMP) concentrations normalized to protein concentration in SAT, VAT and BAT 30 minutes after treatment of lean mice with MP-10 (30 mg/kg) (n=6) or vehicle control (n = 6). ** P = 0.0085 (cGMP in VAT), ** P = 0.0011 (cGMP in BAT) and * P = 0.04 (cAMP in VAT) using unpaired 2-tailed Student's t tests.E RT-qPCR analysis of relative mRNA expression of thermogenic genes in SAT, VAT and BAT after treatment of lean mice treated with MP-10 (30 mg/kg) (n=7) or vehicle control (n = 6). **** P < 0.0001 (Pgc1alpha in VAT), ** P = 0.0012 (Ucp1 in VAT), ** P = 0.0071 (Pgc1alpha in BAT) and * P = 0.0486 (Ucp1 in BAT) using unpaired 2-tailed Student's t tests.


Figure 3Representative fused PET / MRI images of the transverse view of the thoracic region of lean ( n = 5 ) , diet induced obese ( DIO ) ( n = 4 ) and leptin deficient ( ob / ob ) ( n = 5 ) mice that received the PDE10A radioligand [ 18F ] - AQ28A . Interscapular brown adipose tissue ( BAT ) in each image is highlighted by the crosshairs . The mean standardized uptake ( SUV ) value of [ 18F ] - AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve ( AUC ) . * P = 0 . 0128 ( lean vs DIO ) and * * P = 0 . 0031 ( lean vs ob / ob ) using unpaired 2 - tailed Student ' s t test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Representative fused PET/MRI images of the transverse view of the thoracic region of lean (n = 5), diet induced obese (DIO) (n = 4) and leptin deficient (ob/ob) (n = 5) mice that received the PDE10A radioligand [18F]-AQ28A. Interscapular brown adipose tissue (BAT) in each image is highlighted by the crosshairs. The mean standardized uptake (SUV) value of [18F]-AQ28A in BAT throughout the dynamic scan was calculated with associated area under the curve (AUC). * P = 0.0128 (lean vs DIO) and ** P = 0.0031 (lean vs ob/ob) using unpaired 2-tailed Student's t test.


Figure 4A , B The percentage ( % ) change in body weight and cumulative food intake of diet induced obese ( DIO ) mice in response to daily treatment with MP - 10 ( 10mg / kg ) or vehicle for a week ( n = 4 per group ) . * P = 0 . 0264 using ( day 4 ) , * * * P = 0 . 0002 ( day 5 ) , * * * P < 0 . 0001 ( day 6 ) and * * * P < 0 . 0001 ( day 7 ) using Two - Way analysis of variance ( ANOVA ) with Sidak ' s post - hoc test . C . Upon completion of the chronic feeding study , an insulin tolerance test was performed on mice with single treatment of 0 . 75 U / kg insulin ( n = 4 per group ) . * P = 0 . 0246 and * P = 0 . 0305 using unpaired 2 - tailed Student ' s t tests . D , E , F Upon completion of the chronic feeding study , resting energy expenditure ( REE ) , respiratory quotient ( RQ ) and locomotor activity of DIO mice treated with MP - 10 ( 30mg / kg ) compared to vehicle control were measured in metabolic cage experiments ( n = 4 per group ) . * P = 0 . 0239 ( 02 : 00 , RQ ) , * P = 0 . 0424 ( 02 : 30 , RQ ) , * P = 0 . 0420 ( 03 : 00 , RQ ) , * P = 0 . 0230 ( 03 : 30 , RQ ) , * P = 0 . 0213 ( 04 : 00 , RQ ) , * * P = 0 . 0116 ( 00 : 00 , REE ) , * * P = 0 . 00823 ( 00 : 30 , REE ) , * * P = 0 . 00969 ( 01 : 00 , REE ) , * * P = 0 . 00274 ( 01 : 30 , REE ) , * * P = 0 . 00323 ( 02 : 00 , REE ) , * * P = 0 . 000441 ( 02 : 30 , RE ) , * * P = 0 . 00390 ( 03 : 00 , REE ) , * * P = 0 . 000211 ( 03 : 30 , REE ) , * * P = 0 . 00216 ( 04 : 00 , REE ) and * * P = 0 . 0185 ( 04 : 30 , REE ) using unpaired 2 - tailed Student ' s t tests . G . RT - qPCR analysis of relative mRNA expression of candidate genes in inguinal subcutaneous white adipose tissue ( SAT ) , periovarian visceral white adipose tissue ( VAT ) and interscapularbrown adipose tissue ( BAT ) ( n = 8 per group ) * * * P = 0 . 0004 ( Pgc1alpha VAT ) , * * P = 0 . 0036 ( Ucp1 VAT ) , * * P = 0 . 0068 ( Cidea VAT ) , * P = 0 . 0411 ( Tmem26 VAT ) and * * P = 0 . 0070 ( Pgc1alpha BAT ) using unpaired 2 - tailed Student ' s t tests . H . Representative sections of inguinal SAT , peri - ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP - 10 ( 10 mg / kg ) or vehicle control . Scale bars , 100µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A, B The percentage (%) change in body weight and cumulative food intake of diet induced obese (DIO) mice in response to daily treatment with MP-10 (10mg/kg) or vehicle for a week (n= 4 per group). * P = 0.0264 using (day 4), *** P = 0.0002 (day 5), *** P < 0.0001 (day 6) and *** P < 0.0001 (day 7) using Two-Way analysis of variance (ANOVA) with Sidak's post-hoc test.C. Upon completion of the chronic feeding study, an insulin tolerance test was performed on mice with single treatment of 0.75 U/kg insulin (n = 4 per group). * P = 0.0246 and * P = 0.0305 using unpaired 2-tailed Student's t tests.D, E, F Upon completion of the chronic feeding study, resting energy expenditure (REE), respiratory quotient (RQ) and locomotor activity of DIO mice treated with MP-10 (30mg/kg) compared to vehicle control were measured in metabolic cage experiments (n = 4 per group). * P = 0.0239 (02:00, RQ), * P = 0.0424 (02:30, RQ), * P = 0.0420 (03:00, RQ), * P = 0.0230 (03:30, RQ), * P = 0.0213 (04:00, RQ), ** P = 0.0116 (00:00, REE), ** P = 0.00823 (00:30, REE), ** P = 0.00969 (01:00, REE), ** P = 0.00274 (01:30, REE), ** P = 0.00323 (02:00, REE), ** P = 0.000441 (02:30, RE), ** P = 0.00390 (03:00, REE), ** P = 0.000211 (03:30, REE), ** P = 0.00216 (04:00, REE) and ** P = 0.0185 (04:30, REE) using unpaired 2-tailed Student's t tests.G. RT-qPCR analysis of relative mRNA expression of candidate genes in inguinal subcutaneous white adipose tissue (SAT), periovarian visceral white adipose tissue (VAT) and interscapularbrown adipose tissue (BAT) (n = 8 per group) *** P = 0.0004 (Pgc1alpha VAT), ** P = 0.0036 (Ucp1 VAT), ** P = 0.0068 (Cidea VAT), * P = 0.0411 (Tmem26 VAT) and ** P = 0.0070 (Pgc1alpha BAT) using unpaired 2-tailed Student's t tests.H. Representative sections of inguinal SAT, peri-ovarian VAT and interscapular BAT immunostained for UCP1 of mice chronically treated with MP-10 (10 mg/kg) or vehicle control. Scale bars, 100µm.


Figure 5Representative PET image of the upper body of a human subject who received the PDE10A radioligand [ 18F ] - MNI - 1869 . Supraclavicular brown adipose tissue ( BAT ) is highlighted by the crosshairs . The mean standardized uptake value ( SUV ) of [ 18F ] - MNI - 1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects ( n = 5 ) . * * P = 0 . 00210 ( 5 mins ) , * * * * P < 0 . 0001 ( 15 mins ) , * * * * P < 0 . 0001 ( 30 mins ) , * * * * P < 0 . 0001 ( 50 mins ) and * * P = 0 . 00462 ( 75 mins ) using unpaired 2 - tailed Student ' s t tests .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Representative PET image of the upper body of a human subject who received the PDE10A radioligand [18F]-MNI-1869. Supraclavicular brown adipose tissue (BAT) is highlighted by the crosshairs. The mean standardized uptake value (SUV) of [18F]-MNI-1869 in supraclavicular BAT and skeletal muscle was calculated in human subjects (n = 5). ** P = 0.00210 (5 mins), **** P < 0.0001 (15 mins), **** P < 0.0001 (30 mins), **** P < 0.0001 (50 mins) and ** P = 0.00462 (75 mins) using unpaired 2-tailed Student's t tests.


Figure 6A . RT - qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM and 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) and cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0278 vs ( 100nM MP - 10 vs control ; Pgc1alpha ) , * * P = 0 . 0097 ( 10µM cA + cG vs control ; Pgc1alpha ) , * * P = 0 . 0063 ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0017 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) , § § § § P < 0 . 0001 ( 10µM cA + cG vs 100nM MP - 10 ; Ucp1 ) , * P = 0 . 0112 vs ( 100nM MP - 10 vs control ; Cidea ) , * * * P = 0 . 0007 ( 1nM MP - 10 vs control ; Dio2 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Dio2 ) , # P = 0 . 0267 ( 1nM MP - 10 vs 10µM cAMP ; Dio2 ) , § § § P = 0 . 0001 ( 10µM cAMP vs 100nM MP - 10 ; Dio2 ) , § § § P = 0 . 0007 ( 10µM cGMP vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0232 ( 10µM cA + cG vs control ; Dio2 ) § § P = 0 . 0058 ( 10µM cA + cG vs 100nM MP - 10 ; Dio2 ) , * P = 0 . 0416 ( 10µM cGMP vs control ; Prdm16 ) and * P = 0 . 0105 ( 10µM cA + cG vs control ; Prdm16 ) using one - way analysis of variance ( ANOVA ) with Tukey ' s post - hoc test . B . RT - qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments ( 8 hours ) with DMSO ( control ) , MP - 10 ( 1nM and 100nM ) , cyclic AMP ( cAMP - 10µM ) , cyclic GMP ( cGMP - 10µM ) and cAMP + cGMP ( cA + cG - 10µM ( n = 4 separate cultures ) . * P = 0 . 0186 vs ( 1nM MP - 10 vs control ; Ucp1 ) , * * * * P < 0 . 0001 ( 100nM MP - 10 vs control ; Ucp1 ) , # # P = 0 . 0016 ( 1nM MP - 10 vs 100nM MP - 10 ; Ucp1 ) , * * * P = 0 . 0003 ( 10µM cAMP vs control ; Ucp1 ) , § § § P = 0 . 0002 ( 10µM cGMP vs 100nM MP - 10 ; Ucp1 ) and * * * P = 0 . 0003 ( cA + cG vs control ; Ucp1 ) using one - way analysis of variance ( ANOVA ) with Tukey ' s post - hoc test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human brown adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0278 vs (100nM MP-10 vs control; Pgc1alpha), **P = 0.0097 (10µM cA + cG vs control; Pgc1alpha), ** P = 0.0063 (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0017 (1nM MP-10 vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cAMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cGMP vs 100nM MP-10; Ucp1), §§§§ P < 0.0001 (10µM cA + cG vs 100nM MP-10; Ucp1), * P = 0.0112 vs (100nM MP-10 vs control; Cidea), *** P = 0.0007 (1nM MP-10 vs control; Dio2), **** P < 0.0001 (100nM MP-10 vs control; Dio2), # P = 0.0267 (1nM MP-10 vs 10µM cAMP; Dio2), §§§ P = 0.0001 (10µM cAMP vs 100nM MP-10; Dio2), §§§ P = 0.0007 (10µM cGMP vs 100nM MP-10; Dio2), * P = 0.0232 (10µM cA + cG vs control; Dio2) §§ P = 0.0058 (10µM cA + cG vs 100nM MP-10; Dio2), * P = 0.0416 (10µM cGMP vs control; Prdm16) and * P = 0.0105 (10µM cA + cG vs control; Prdm16) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.B. RT-qPCR analysis of relative mRNA expression of thermogenic genes in cultured primary human white adipocytes in response to chronic treatments (8 hours) with DMSO (control), MP-10 (1nM and 100nM), cyclic AMP (cAMP - 10µM), cyclic GMP (cGMP - 10µM) and cAMP + cGMP (cA + cG - 10µM (n = 4 separate cultures). * P = 0.0186 vs (1nM MP-10 vs control; Ucp1), **** P < 0.0001 (100nM MP-10 vs control; Ucp1), ## P = 0.0016 (1nM MP-10 vs 100nM MP-10; Ucp1), *** P = 0.0003 (10µM cAMP vs control; Ucp1), §§§ P = 0.0002 (10µM cGMP vs 100nM MP-10; Ucp1) and *** P = 0.0003 (cA + cG vs control; Ucp1) using one-way analysis of variance (ANOVA) with Tukey's post-hoc test.


Figure 1 Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( A ) Percentage body weight loss Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( B ) Relative amount of PR8 viral NP mRNA , by qPCR Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( C ) Histology of lungs by H & E staining with pathology scores . Scale bars = 200 μm Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( D ) Absolute number of live CD45 + cells in whole lungs and BAL , by flow cytometry Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Statistical significanc was determined with unpaired student ' s T - test Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( E ) Level of LDH in BAL , by LDH cytotoxicity assay Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( F ) Expression of PDPN in lungs , by western blot Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M Mice were infected with sub - lethal dose ( 25 pfu ) of influenza virus and assessed as follows ( G ) Total protein in BAL , by Bradford assay . ( H ) Concentration of albumin in BAL , by ELISA Data information : Data are representative of three independent experiments with 3 - 5 mice per group and represented as mean ± S . E . M . Statistical significanc was determined with unpaired student ' s T -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (A) Percentage body weight loss Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (B) Relative amount of PR8 viral NP mRNA, by qPCR Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (C) Histology of lungs by H&E staining with pathology scores. Scale bars = 200 μm Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (D) Absolute number of live CD45+ cells in whole lungs and BAL, by flow cytometry Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M Statistical significanc was determined with unpaired student's T-test Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (E) Level of LDH in BAL, by LDH cytotoxicity assay Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (F) Expression of PDPN in lungs, by western blot Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M  Mice were infected with sub-lethal dose (25 pfu) of influenza virus and assessed as follows (G) Total protein in BAL, by Bradford assay. (H) Concentration of albumin in BAL, by ELISA Data information: Data are representative of three independent experiments with 3-5 mice per group and represented as mean ± S.E.M. Statistical significanc was determined with unpaired student's T-test


Figure 2 ( A ) Absolute cell counts from lungs of sub - lethally influenza virus infected mice following analysis by flow cytometry . After gating out alveolar macrophages by forward and side scatter , immune cells are defined as follows : Neutrophil ( Ly6G + CD11b + SiglecF - Ly6Cint ) , Eosinophil ( SiglecF + CD11c - / + F4 / 80 - CD64 - ) , Monocyte ( Ly6G - CD11b + MHCII - SiglecF - ) , Natural killer cell ( NK , B220 - / intNK1 . 1 + ) , B cell ( B220hiNK1 . 1 - ) , CD8 + T cell ( TCRβ + CD8 + ) , CD4 + T cell ( TCRβ + CD4 + ) Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M . Statistical significance in ( A ) was determined with unpaired student ' s T - test . n . s . = not significant ( B ) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub - lethal influenza virus infection . Numbers in plots indicate frequency of gated immune cells in each throughput Data information : Data are representative of three independent experiments with 3 - 4 mice per group and represented as mean ± S . E . M

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 (A) Absolute cell counts from lungs of sub-lethally influenza virus infected mice following analysis by flow cytometry. After gating out alveolar macrophages by forward and side scatter, immune cells are defined as follows: Neutrophil (Ly6G+CD11b+SiglecF-Ly6Cint), Eosinophil (SiglecF+CD11c-/+F4/80-CD64-), Monocyte (Ly6G-CD11b+MHCII-SiglecF-), Natural killer cell (NK, B220-/intNK1.1+), B cell (B220hiNK1.1-), CD8+ T cell (TCRβ+CD8+), CD4+ T cell (TCRβ+CD4+) Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M. Statistical significance in (A) was determined with unpaired student's T-test. n.s.= not significant  (B) Flow cytometry plots of neutrophils and eosinophils in lungs at days 9 and 15 post sub-lethal influenza virus infection. Numbers in plots indicate frequency of gated immune cells in each throughput Data information: Data are representative of three independent experiments with 3-4 mice per group and represented as mean ± S.E.M


Figure 3Mice were superinfected as described in ( A ) ( B ) Survival of mice following bacterial superinfection Data information : Data from ( B ) are combined from two independent experiments , n = 13 mice per experimental group All data are represented as mean ± S . E . M . Statistical significance was determined by logrank test in ( BMice were superinfected as described in ( A ) ( C ) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M Statistical significance wa determined b Mann - Whitney U test in ( CMice were superinfected as described in ( A ) ( D ) Absolute immune cell counts in lungs , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significanc determined b unpaired student ' s T - test in ( DMice were superinfected as described in ( A ) ( E ) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) or following influenza virus infection ( + Flu ) Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E . M . Statistical significance was determined b Mann - Whitney U tes n . s . = not significantMice were superinfected as described in ( A ) ( F ) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S . pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5 - mice per experimental group . All data are represented as mean ± S . E . MMice were superinfected as described in ( A ) ( G ) Absolute neutrophil and eosinophil cell count in lungs infected as described in ( E ) , assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3 - mice per experimental group All data are represented as mean ± S . E . M . Statistical significance wa determined b ) , unpaired student ' s T - test i ( G ( E ) . n . s . = not

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Mice were superinfected as described in (A) (B) Survival of mice following bacterial superinfection Data information: Data from (B) are combined from two independent experiments, n=13 mice per experimental group All data are represented as mean ± S.E.M. Statistical significance was determined by logrank test in (BMice were superinfected as described in (A) (C) Pneumococcal cfu in BAL of mice Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M Statistical significance wa determined b Mann-Whitney U test in (CMice were superinfected as described in (A) (D) Absolute immune cell counts in lungs, assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significanc determined b unpaired student's T-test in (DMice were superinfected as described in (A) (E) Pneumococcal cfu in BAL of mice 12 h after infection with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu) or following influenza virus infection (+Flu) Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.M. Statistical significance was determined b Mann-Whitney U tes n.s. = not significantMice were superinfected as described in (A) (F) Pneumococcal cfu in BAL 36 h after infection of naïve mice with 2x105 cfu of S. pneumoniae serotype 19F alone Data information Dat are representative of at least two independent experiments wit 5- mice per experimental group. All data are represented as mean ± S.E.MMice were superinfected as described in (A) (G) Absolute neutrophil and eosinophil cell count in lungs infected as described in (E), assessed by flow cytometry Data information Dat are representative of at least two independent experiments with 3- mice per experimental group All data are represented as mean ± S.E.M. Statistical significance wa determined b ), unpaired student's T-test i (G (E). n.s. = not significant


Figure 4 ( A ) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub - lethal influenza virus infection as determined by protein bead array Data information : Data in ( A ) are obtained from one independent experiment with 4 mice per group , and results of selected analytes IFNγ , CXCL10 , IL - 6 , IL - 13 and IL - 17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student ' s T - test . n . s . = not significantMice were superinfected as described in Fig 3A . ( B ) Concentrations of IL - 17 and IL - 22 in BAL of mice , as determined by ELISA Data informatio Data in ( B are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significance i was determined by unpaired student ' s T - test . n . s . = not significantMice were superinfected as described in Fig 3A ( C ) Concentrations of IL - 17 and IL - 22 in BAL of mice 12 h after with 2x105 cfu of S . pneumoniae serotype 19F alone ( - Flu ) , or following superinfection ( + Flu ) , assessed by ELISA Data information Dat i ( C ) are representative of two independent experiments with 3 - 4 mice per group . All data are represented as mean ± S . E . M . Significanc was determined by unpaired student ' s T - test . n . s . = not

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 (A) Concentrations of cytokines or chemokines in the BAL of days 5 and 9 post sub-lethal influenza virus infection as determined by protein bead array Data information: Data in (A) are obtained from one independent experiment with 4 mice per group, and results of selected analytes IFNγ, CXCL10, IL-6, IL-13 and IL-17 are reconfirmed in a separate independent experiment by a customized protein bead array Significance i was determined by unpaired student's T-test. n.s. = not significantMice were superinfected as described in Fig 3A. (B) Concentrations of IL-17 and IL-22 in BAL of mice, as determined by ELISA Data informatio Data in (B are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significance i was determined by unpaired student's T-test. n.s. = not significantMice were superinfected as described in Fig 3A (C) Concentrations of IL-17 and IL-22 in BAL of mice 12 h after with 2x105 cfu of S. pneumoniae serotype 19F alone (-Flu), or following superinfection (+Flu), assessed by ELISA Data information Dat i (C) are representative of two independent experiments with 3-4 mice per group. All data are represented as mean ± S.E.M. Significanc was determined by unpaired student's T-test. n.s. = not significant


Figure 5Mice were superinfected as described in Fig 3A . ( A ) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL - 17 or αIL - 22 blocking antibody . Control wild - type and T - bet - / - groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined with Kruskal - Wallis test followed by Dunn " s multiple comparisons test n . s . = not significantMice were superinfected as described in Fig 3A ( B ) T - bet - / - mice were administered αIL - 17 or IgG isotype control antibody every other day from day - 2 post - secondary bacterial infection and monitored for survival Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit logrank test in ( BMice were superinfected as described in Fig 3A ( C ) Absolute neutrophil counts in the lungs following administration of αIL - 17 or IgG isotype control antibody , as determined by flow cytometry Data information Dat are representative of two independent experiments with 3 - 4 in ( C mice per group All data are represented as mean ± S . E . M . Statistical significance was determined wit unpaired - students t - test in ( CMice were superinfected as described in Fig 3A ( D ) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion . Control T - bet - / - mice were administered with IgG isotype control antibody Data information : Dat are combined from two independent experiments ( n = 8 - 12 mice per group ) All data are represented as mean ± S . E . M . Statistical significance was determined wit Mann - Whitney U test in ( DMice were superinfected as described in Fig 3A ( E ) mRNA expression of antimicrobial peptides in lungs following administration of αIL - 17 , IL - 22 or IgG control antibody , as determined by qPCR Data information Dat are representative of two independent experiments wit 4 - 6 in ( E ) mice per group . All data are represented as mean ± S . E . M . Statistical significance was determined wit one - way ANOVA followed by Tukey " s multiple comparisons test in ( E ) . n . s . = not

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Mice were superinfected as described in Fig 3A. (A) Bacterial load as determined by enumeration of cfu in BAL following administration of αIL-17 or αIL-22 blocking antibody. Control wild-type and T-bet-/- groups were administered with IgG isotype control antibody Data information Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined with Kruskal-Wallis test followed by Dunn"s multiple comparisons test n.s. = not significantMice were superinfected as described in Fig 3A (B) T-bet-/- mice were administered αIL-17 or IgG isotype control antibody every other day from day -2 post-secondary bacterial infection and monitored for survival Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit logrank test in (BMice were superinfected as described in Fig 3A (C) Absolute neutrophil counts in the lungs following administration of αIL-17 or IgG isotype control antibody, as determined by flow cytometry Data information Dat are representative of two independent experiments with 3-4 in (C mice per group All data are represented as mean ± S.E.M. Statistical significance was determined wit unpaired-students t-test in (CMice were superinfected as described in Fig 3A (D) Pneumococcal cfu in BAL following αLy6G antibody mediated neutrophil depletion. Control T-bet-/- mice were administered with IgG isotype control antibody Data information: Dat are combined from two independent experiments (n = 8-12 mice per group) All data are represented as mean ± S.E.M. Statistical significance was determined wit Mann-Whitney U test in (DMice were superinfected as described in Fig 3A (E) mRNA expression of antimicrobial peptides in lungs following administration of αIL-17, IL-22 or IgG control antibody, as determined by qPCR Data information Dat are representative of two independent experiments wit 4-6 in (E) mice per group. All data are represented as mean ± S.E.M. Statistical significance was determined wit one-way ANOVA followed by Tukey"s multiple comparisons test in (E). n.s. = not signif


Figure 6 Mice were superinfected as described in Fig 3A ( A ) Frequency of IFNγ + and IL - 17 + immune cells following intracellular cytokine staining and flow cytometry Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc was determined by unpaired student ' s T - test Mice were superinfected as described in Fig 3A ( B ) Representative flow cytometry plots of total and IL - 17 + lymphocyte subsets on day 3 post - secondary bacterial infected lungs following intracellular cytokine staining . Numbers in plots indicate the frequency of each IL - 17 + cell subset over total numbers of respective cell - types Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M Mice were superinfected as described in Fig 3A ( C ) Frequency over total IL - 17 + cells and absolute numbers of each IL - 17 producing lymphocyte subset on days 0 , 1 and 3 post - secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information : Data are representative of at least two independent experiments with 3 mice per group . All data are represented as mean ± S . E . M . Statistical significanc i ( C ) was determined by unpaired student ' s T -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 Mice were superinfected as described in Fig 3A (A) Frequency of IFNγ+ and IL-17+ immune cells following intracellular cytokine staining and flow cytometry Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc was determined by unpaired student's T-test Mice were superinfected as described in Fig 3A (B) Representative flow cytometry plots of total and IL-17+ lymphocyte subsets on day 3 post-secondary bacterial infected lungs following intracellular cytokine staining. Numbers in plots indicate the frequency of each IL-17+ cell subset over total numbers of respective cell-types Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M  Mice were superinfected as described in Fig 3A (C) Frequency over total IL-17+ cells and absolute numbers of each IL-17 producing lymphocyte subset on days 0, 1 and 3 post-secondary bacterial superinfected lungs as assessed by flow cytometry of intracellular cytokine stained cells Data information: Data are representative of at least two independent experiments with 3 mice per group. All data are represented as mean ± S.E.M. Statistical significanc i (C) was determined by unpaired student's T-test


Figure 1B . Semi - quantitative PCR of miR - 132 and negative control miRNAs in the hippocampus of antagomiR - 132 - injected mice ( ant - 132 ) in comparison to control - injected animals ( aCSF and scramble ) at six months of age . Sample size , n = 9 per group . C . FISH of miR - 132 and negative control miR - 124 in the hippocampus of ant - 132 - and scramble - injected mice . Scramble probes were used as FISH negative controls . Scale bar , 500μm . D . Semi - quantitative PCR of miR - 132 and miR - 212 in the hippocampus of 3 - month old miR - 132 mimic - injected mice ( miR - 132 ) compared to animals injected with a negative control oligonucleotide ( Ctr ) . Sample size , n = 6 per group .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Semi-quantitative PCR of miR-132 and negative control miRNAs in the hippocampus of antagomiR-132-injected mice (ant-132) in comparison to control-injected animals (aCSF and scramble) at six months of age. Sample size, n=9 per group.C. FISH of miR-132 and negative control miR-124 in the hippocampus of ant-132- and scramble-injected mice. Scramble probes were used as FISH negative controls. Scale bar, 500μm.D. Semi-quantitative PCR of miR-132 and miR-212 in the hippocampus of 3-month old miR-132 mimic-injected mice (miR-132) compared to animals injected with a negative control oligonucleotide (Ctr). Sample size, n=6 per group.