Life imaging of wild - type ( WT ) and α1 - tubulin ( - ) oocysts expressing ef1α : GFP ( early and late oocysts ) and csp : mCherry ( late oocysts ) to locate the oocyst cytoplasm . Microtubules were labeled with SiR - tubulin ( green ) and DNA with Hoechst ( blue ) . Oocysts are shown in a chronological order from early ( day 4 ) to late ( day 12 ) development . ( A , B ) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage ( A ) and subsequent DNA replication ( B ) , where some but not all DNA is co - stained by microtubules in the mutant . ( C ) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites . Note the strong SiR - tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant . Sb : sporoblast . ( D ) Oocyst ready to burst with fully formed sporozoites in WT oocysts . Note that α1 - tubulin ( - ) oocysts retained sporozoite nuclei predominantly within the sporoblast ( Sb ) during budding . Scale bars : 5 μm , magnification box in B : 1 µm , .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Life imaging of wild-type (WT) and α1-tubulin(-) oocysts expressing ef1α:GFP (early and late oocysts) and csp:mCherry (late oocysts) to locate the oocyst cytoplasm. Microtubules were labeled with SiR-tubulin (green) and DNA with Hoechst (blue). Oocysts are shown in a chronological order from early (day 4) to late (day 12) development. (A, B) Early oocyst development with remaining subpellicular microtubules of the preceding ookinete stage (A) and subsequent DNA replication (B), where some but not all DNA is co-stained by microtubules in the mutant. (C) Nuclear alignments to the invaginated plasma membrane and budding of sporozoites. Note the strong SiR-tubulin signal only seen in sporozoites of WT oocysts and complete absence from the mutant. Sb: sporoblast. (D) Oocyst ready to burst with fully formed sporozoites in WT oocysts. Note that α1-tubulin(-) oocysts retained sporozoite nuclei predominantly within the sporoblast (Sb) during budding. Scale bars: 5 μm, magnification box in B: 1 µm,.


3D reconstructions from spinning disc confocal microscopy z - stacks across an early oocyst labelled with the DNA dye Hoechst ( left ) . 50 and 19 images with a z - distance of 0 . 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Right : 3D reconstructions from late WT and α1 - tubulin ( - ) oocysts labelled with Hoechst ( blue ) and SiR - tubulin ( green ) . Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites . 10 and 28 images with a z - distance of 1 , 5 µm and 1 µm were collated for WT and α1 - tubulin ( - ) oocysts , respectively . Sb : sporoblast ; scale bars : 5 µm .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

3D reconstructions from spinning disc confocal microscopy z-stacks across an early oocyst labelled with the DNA dye Hoechst (left). 50 and 19 images with a z-distance of 0.5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Right: 3D reconstructions from late WT and α1-tubulin(-) oocysts labelled with Hoechst (blue) and SiR-tubulin (green). Note that the mutant does not form proper sporozoite shapes during budding and the few peripheral nuclei that were uptaken into budding sporozoites. 10 and 28 images with a z-distance of 1,5 µm and 1 µm were collated for WT and α1-tubulin(-) oocysts, respectively. Sb: sporoblast; scale bars: 5 µm.


TEM cross sections of WT and α1 - tubulin ( - ) oocysts during nuclear division . Spindle microtubules ( arrowheads ) emerging from centriolar plaques ( M ) were seen in WT oocysts . Although individual nuclei ( N ) were visible in α1 ( - ) oocysts , microtubules were absent even in dividing centriolar plaques ( M ) . Scale bars : 0 . 2 μm .

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

TEM cross sections of WT and α1-tubulin(-) oocysts during nuclear division. Spindle microtubules (arrowheads) emerging from centriolar plaques (M) were seen in WT oocysts. Although individual nuclei (N) were visible in α1(-) oocysts, microtubules were absent even in dividing centriolar plaques (M). Scale bars: 0.2 μm.


Fluorescence images showing the SiR - tubulin stained microtubules , nuclei ( Hoechst ) and cytoplasmic shape ( mCherry ) of the parasite lines . Note the small amount of DNA ( black arrowhead ) and the reduced level of SiR - tubulin fluorescence in the α1cm & ∆ introns - expressing sporozoites ( lower graph ) . See also Fig S6 . Quantitative data was derived from 96 - 120 sporozoites per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . Scale bar : 5 μm . * * * * indicates p < 0 . 0001 , Kruskal - Wallis - test . Note that α1cm & ∆ introns - expressing sporozoites do not express mCherry ; their autofluorescence ( auto - fluo ) indicates an aberrant shape .

O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Fluorescence images showing the SiR-tubulin stained microtubules, nuclei (Hoechst) and cytoplasmic shape (mCherry) of the parasite lines. Note the small amount of DNA (black arrowhead) and the reduced level of SiR-tubulin fluorescence in the α1cm&∆introns-expressing sporozoites (lower graph). See also Fig S6. Quantitative data was derived from 96-120 sporozoites per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. Scale bar: 5 μm. **** indicates p<0.0001, Kruskal-Wallis-test. Note that α1cm&∆introns-expressing sporozoites do not express mCherry; their autofluorescence (auto-fluo) indicates an aberrant shape.


qRT - PCR analysis of the three parasite lines at day 7 and 12 after infection . Error bars correspond to the standard deviation from two biological replicates .

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qRT-PCR analysis of the three parasite lines at day 7 and 12 after infection. Error bars correspond to the standard deviation from two biological replicates.


TEM reveals α1cm & ∆ introns - expressing sporozoites with smaller ( A and B ) and larger ( C ) diameters than wild type sporozoites . See also Fig S7 . Scale bar : 0 . 2 µm .

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TEM reveals α1cm&∆introns-expressing sporozoites with smaller (A and B) and larger (C) diameters than wild type sporozoites. See also Fig S7. Scale bar: 0.2 µm.


α1cm & ∆ introns - expressing sporozoites show a reduced number of subpellicular microtubules ( green arrowheads ) by TEM .

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α1cm&∆introns-expressing sporozoites show a reduced number of subpellicular microtubules (green arrowheads) by TEM.


Expression of different α1 - α2 chimeric α - tubulins instead of α1 - tubulin leads to sporozoite shape changes as revealed by SEM . See also Fig EV5 . Scale bar : 1 µm . α1 ∆ c - term lacks the last 3 amino acids of α1 - tubulin . α2 + parasites contain an α1 - tubulin with all amino acids that are different between the two α - tubulins changed into those of α2 - tubulin ; see also Fig S4 . α2 + + parasites contain α2 - tubulin ( exons , introns , 3 ' UTR ) and α2 + + + parasites express the exons of α2 - tubulin and introns of α1 - tubulin from the α1 - tubulin promoter .

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

Expression of different α1-α2 chimeric α-tubulins instead of α1-tubulin leads to sporozoite shape changes as revealed by SEM. See also Fig EV5. Scale bar: 1 µm. α1∆c-term lacks the last 3 amino acids of α1-tubulin. α2+ parasites contain an α1-tubulin with all amino acids that are different between the two α-tubulins changed into those of α2-tubulin; see also Fig S4. α2++ parasites contain α2-tubulin (exons, introns, 3'UTR) and α2+++ parasites express the exons of α2-tubulin and introns of α1-tubulin from the α1-tubulin promoter.


SiR - tubulin staining reveals differences in microtubule lengths and intensity . Between 69 and 156 sporozoites were analysed per line . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * , and * * * * indicate p < 0 . 001 and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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

SiR-tubulin staining reveals differences in microtubule lengths and intensity. Between 69 and 156 sporozoites were analysed per line. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. ***, and **** indicate p<0.001 and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


qRT - PCR analysis of the parasite lines at day 12 after infection shows different expression level . Note the subtly higher expression of α2 + + compared to α2 + + + . Error bars show standard deviation from the mean of two biological replicates .

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

qRT-PCR analysis of the parasite lines at day 12 after infection shows different expression level. Note the subtly higher expression of α2++ compared to α2+++. Error bars show standard deviation from the mean of two biological replicates.


TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α - tubulin chimeras . Green arrowheads point to microtubules . See also Fig S8 and S9 . Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier . Scale bars : 0 . 2 µm .

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TEM along sporozoites shows a decrease of microtubule numbers towards the nucleus in parasites expressing α-tubulin chimeras. Green arrowheads point to microtubules. See also Fig S8 and S9. Note that the WT control panel is shown already in Figure 4E to indicate that here we perform an extended analysis to the dataset generated earlier. Scale bars: 0.2 µm.


TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines . Each dot represents the numbers of microtubules counted per section . Red lines indicate median from 23 to 40 analysed sections per line .

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TEM reveals different numbers of microtubules at the apical end of sporozoites for the different lines. Each dot represents the numbers of microtubules counted per section. Red lines indicate median from 23 to 40 analysed sections per line.


Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2 + and α1 ∆ c - term parasites compared to wild type sporozoites .

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

Microtubule quantification along the sporozoite reveals the presence of fewer microtubules in central and nuclear sections of α2+ and α1∆c-term parasites compared to wild type sporozoites.


Semilog correlation of gene expression as determined by qRT - PCR with microtubule numbers as determine by TEM . Bars show range of values in x and standard deviation in y around the mean ( dots ) of two biological replicates ( x ) and all determined values ( y ) .

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

Semilog correlation of gene expression as determined by qRT-PCR with microtubule numbers as determine by TEM. Bars show range of values in x and standard deviation in y around the mean (dots) of two biological replicates (x) and all determined values (y).


The microtubule number increases with the increase in the concentration of tubulin dimers . Microtubule number scales with tubulin concentration at low expression . At high expression , microtubule number is limited by the number of nucleation sites . Shown are mean + / - standard deviation from all determined values .

The microtubule number increases with the increase in the concentration of tubulin dimers. Microtubule number scales with tubulin concentration at low expression. At high expression, microtubule number is limited by the number of nucleation sites. Shown are mean +/- standard deviation from all determined values.


The average microtubule length remains close to the maximum length 6 μm . The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data ( blue stars are for ( α2 + , α2 + + , α2 + + + ) strains ) . The error bar shows the standard deviation in the data and simulation results .

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

The average microtubule length remains close to the maximum length 6 μm. The circles are obtained by averaging results from 200 stochastic simulations of the model equations and stars are the experimental data (blue stars are for (α2+, α2++, α2+++) strains). The error bar shows the standard deviation in the data and simulation results.


For the WT expression level ( c0 = 36 . 6 ( m0 + p + n ) ) , the rate of nucleation is faster than the microtubule growth . Then the microtubule number is determined by the number of nucleation sites . The average microtubule length is around 1 μm ( shown by dashed arrow ) when the last nucleus forms . At intermediate tubulin numbers ( c0 = 16 ( m0 + p + n ) ) , corresponding to the α1c - term mutant , the microtubule formation rate is comparable to nucleation rate . The average microtubule length is around 4 μm ( shown by dashed arrow ) when the last nucleus forms .

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

For the WT expression level (c0 = 36.6 (m0+p+n)), the rate of nucleation is faster than the microtubule growth. Then the microtubule number is determined by the number of nucleation sites. The average microtubule length is around 1 μm (shown by dashed arrow) when the last nucleus forms. At intermediate tubulin numbers (c0 = 16 (m0+p+n)), corresponding to the α1c-term mutant, the microtubule formation rate is comparable to nucleation rate. The average microtubule length is around 4 μm (shown by dashed arrow) when the last nucleus forms.


Quantitative analysis of salivary gland sporozoite lengths from the different lines . n indicates numbers of investigated sporozoites . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * * * and * * * * indicate p < 0 . 001 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test ; scale bar : 1 µm .

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

Quantitative analysis of salivary gland sporozoite lengths from the different lines. n indicates numbers of investigated sporozoites. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. *** and **** indicate p<0.001, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test; scale bar: 1 µm.


Quantification of SiR - tubulin fluorescence from 69 - 127 salivary gland derived sporozoites . Note the decrease in the fluorescence intensity of sporozoites expressing α1 - tubulin without introns as well as the decreased intensity and length of the chimeras . Linear correlation of sporozoite length and microtubule length reveals a R2 = 0 . 94 . * , * * , and * * * * indicate p < 0 . 05 , p < 0 . 01 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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

Quantification of SiR-tubulin fluorescence from 69-127 salivary gland derived sporozoites. Note the decrease in the fluorescence intensity of sporozoites expressing α1-tubulin without introns as well as the decreased intensity and length of the chimeras. Linear correlation of sporozoite length and microtubule length reveals a R2=0.94. *, **, and **** indicate p<0.05, p<0.01, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass . Scale bar : 20 µm . Box plots represent 50 % ( boxes ) and 95 % ( bars ) of data with horizontal bar showing the median . * and * * * * indicate p < 0 . 05 , and p < 0 . 0001 , respectively ; ns : not significant ; Kruskal - Wallis - test .

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

Shorter sporozoites exhibit stronger curvatures and hence tighter circular motility on glass. Scale bar: 20 µm. Box plots represent 50% (boxes) and 95% (bars) of data with horizontal bar showing the median. * and **** indicate p<0.05, and p<0.0001, respectively; ns: not significant; Kruskal-Wallis-test.


Sporozoites with fewest microtubules ( α2 + + + ) show a motility defect . n indicates numbers of investigated sporozoites ; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites .

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Sporozoites with fewest microtubules (α2+++) show a motility defect. n indicates numbers of investigated sporozoites; Numbers on grey bars indicate percentage of partially and persistently moving sporozoites.


Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise ( CW ) direction . Numbers on black bars indicate percentage of CW moving sporozoites ; numbers in movie stills indicate time in seconds . CW moving sporozoites move at lower speed than CCW moving ones .

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

Sporozoites from lines with fewer microtubules move more frequently in apparent clockwise (CW) direction. Numbers on black bars indicate percentage of CW moving sporozoites; numbers in movie stills indicate time in seconds. CW moving sporozoites move at lower speed than CCW moving ones.


Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2 + + infected salivary glands . Green arrowheads point to microtubules . Scale bar : 0 . 2 µm . Error bars show the full range and boxes 50 % of values . Red crosses indicate the mean , red lines the median . Fisher ' s exact test ( two - tailed ) .

Salivary gland sporozoites contain at least 10 microtubules as revealed by TEM of α2++ infected salivary glands. Green arrowheads point to microtubules. Scale bar: 0.2 µm. Error bars show the full range and boxes 50% of values. Red crosses indicate the mean, red lines the median. Fisher's exact test (two-tailed).


( A - C ) Bone marrow derived mouse macrophages ( BMDMs ) from wild type C57BL / 6 mice were treated with 4µ8c ( 100 µM ) or vehicle ( dimethyl sulfoxide ; DMSO ) for 24 hours prior to sample processing for lipidomics analysis : ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP ratio are shown . Data represent mean values ± standard error of mean ( SEM ) of peak areas normalized to internal standards ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Bone marrow derived mouse macrophages (BMDMs) from wild type C57BL/6 mice were treated with 4µ8c (100 µM) or vehicle (dimethyl sulfoxide; DMSO) for 24 hours prior to sample processing for lipidomics analysis: (A) PI(3,4,5)P3 to PIP2; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP ratio are shown. Data represent mean values ± standard error of mean (SEM) of peak areas normalized to internal standards (n=3 biological replicates).


( D ) BMDMs were treated with 4µ8c ( 100 µM ) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates ; a representative blot is shown ) .

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(D) BMDMs were treated with 4µ8c (100 µM) or DMSO for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates; a representative blot is shown).


( E ) Wild type C57BL / 6 mice were injected with tunicamycin ( 1 mg / kg ) and 4µ8c ( 10 µg / kg ) for 8 hours prior to sacrifice and collection of thiogylcolate - elicited peritoneal macrophages . Protein lysates were analyzed by western blotting using specific antibodies against , pIRE1S742 , IRE1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin ( n = 3 biological replicates ; a representative blot is shown ) . Western blot quantifications are shown next to the figure .

O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Wild type C57BL/6 mice were injected with tunicamycin (1 mg/kg) and 4µ8c (10 µg/kg) for 8 hours prior to sacrifice and collection of thiogylcolate-elicited peritoneal macrophages. Protein lysates were analyzed by western blotting using specific antibodies against, pIRE1S742, IRE1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin (n=3 biological replicates; a representative blot is shown). Western blot quantifications are shown next to the figure.


( F ) IRE1 + / + and IRE1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742 , IRE1 , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 and β - actin . Western blot quantifications are shown next to the figure ( n = 3 biological replicates , a representative blot is shown ) .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) IRE1+/+ and IRE1-/- BMDMs were treated with PA (500 µM) for 6 hours and protein lysates were analyzed by western blotting using specific antibodies against pIRE1S742, IRE1, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1 and β-actin. Western blot quantifications are shown next to the figure (n=3 biological replicates, a representative blot is shown).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( B ) RNA lysates from the same experiment were analyzed by qRT - PCR for miR - 2137 expression .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (B) RNA lysates from the same experiment were analyzed by qRT-PCR for miR-2137 expression.


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( C ) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742 , IRE1 and β - actin ( n = 3 biological replicates ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (C) Protein lysates were analyzed by western blotting using specific antibodies for pIRE1S742, IRE1 and β-actin (n=3 biological replicates).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( D ) RNA lysates were analyzed by qRT - PCR for sXBP1 expression ( n = 3 biological replicates ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (D) RNA lysates were analyzed by qRT-PCR for sXBP1 expression (n=3 biological replicates).


IRE1 - / - and IRE1 + / + BMDMs were treated with PA ( 500 µM ) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro - array platform . ( E - F ) RNA lysates from the same experiment were analyzed by qRT - PCR for ( E ) BLOC1S1 and ( F ) SCARA3 expression ( n = 3 biological replicates ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IRE1-/- and IRE1+/+ BMDMs were treated with PA (500 µM) or vehicle for 6 hours prior to RNA isolation for miRNA analysis using a micro-array platform. (E-F) RNA lysates from the same experiment were analyzed by qRT-PCR for (E) BLOC1S1 and (F) SCARA3 expression (n=3 biological replicates).


( A - B ) BMDMs were treated with PA , steric acid ( SA ) , palmitoleic acid ( PAO ) or oleic acid ( OA ) ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for the expression of ( A ) miR - 2137 and ( B ) sXBP1 ( n = 3 biological replicates ) .

O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) BMDMs were treated with PA, steric acid (SA), palmitoleic acid (PAO) or oleic acid (OA) (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for the expression of (A) miR-2137 and (B) sXBP1 (n=3 biological replicates).


( C , D ) ( C ) C57BL / 6 ( WT ) and ( D ) Apoe - / - mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( WT ; n = 4 - 4 mice per group , Apoe - / - ; n = 5 - 5 mice per group ) .

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

(C, D) (C) C57BL/6 (WT) and (D) Apoe-/- mice were fed with chow or WD for 16 weeks followed by peritoneal macrophage isolation. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (WT; n=4-4 mice per group, Apoe-/-; n=5-5 mice per group).


( E - F ) Apoe - / - mice were fed with chow or WD for 12 weeks and injected with 4µ8c ( 10 mg / kg / day ) or vehicle ( DMSO ) in the final 6 weeks of the diet ( n = 5 - 7 mice per group ) : ( E ) RNA lysates from bone marrow were analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) . ( F ) Total RNA isolated from the aortic root plaques was analyzed by qRT - PCR for miR - 2137 expression ( n = 5 - 7 mice per group ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) Apoe-/- mice were fed with chow or WD for 12 weeks and injected with 4µ8c (10 mg/kg/day) or vehicle (DMSO) in the final 6 weeks of the diet (n=5-7 mice per group): (E) RNA lysates from bone marrow were analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group). (F) Total RNA isolated from the aortic root plaques was analyzed by qRT-PCR for miR-2137 expression (n=5-7 mice per group).


( A ) BMDMs were transfected with scrambled ( Scr ) or IRE1 - specific siRNA ( 40 nM ) and treated with palmitate ( PA , 500 µM ) or vehicle for 6 and 9 hours ( hr ) . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) BMDMs were transfected with scrambled (Scr) or IRE1-specific siRNA (40 nM) and treated with palmitate (PA, 500 µM) or vehicle for 6 and 9 hours (hr). RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( B ) BMDMs were treated with PA ( 500 µM ) and 4µ8c ( 100 µM ) or vehicle for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) . ( C ) BMDM cells were treated with tunicamycin ( Tun ; 5 µg / ml ) and 4µ8c ( 100 µM ) or vehicle ( DMSO ) for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O 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) BMDMs were treated with PA (500 µM) and 4µ8c (100 µM) or vehicle for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates). (C) BMDM cells were treated with tunicamycin (Tun; 5 µg/ml) and 4µ8c (100 µM) or vehicle (DMSO) for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( D ) IRE1 - / - MEFs were transfected with vector ( control ) , WT - IRE1 or K907A - IRE1 ( RNase dead mutant ) plasmids for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) IRE1-/- MEFs were transfected with vector (control), WT-IRE1 or K907A-IRE1 (RNase dead mutant) plasmids for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( E ) BMDMs were transfected with scrambled or XBP - 1 specific siRNA ( 70 nM ) for 24 hours , followed by treatment with PA ( 500 µM ) for 16 hours . RNA lysates were analyzed by qRT - PCR for miR - 2137 expression ( n = 3 biological replicates ) .

O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) BMDMs were transfected with scrambled or XBP-1 specific siRNA (70 nM) for 24 hours, followed by treatment with PA (500 µM) for 16 hours. RNA lysates were analyzed by qRT-PCR for miR-2137 expression (n=3 biological replicates).


( F ) XBP1 - / - MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . RNA lysates were analyzed for miR - 2137 expression by qRT - PCR ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) XBP1-/- MEFs were transfected with empty vector or sXBP1 plasmid for 24 hours, followed by PA (500 µM) treatment for 9 hours. RNA lysates were analyzed for miR-2137 expression by qRT-PCR (n=3 biological replicates).


( G ) IRE1 cleavage assay performed using synthetic pre - miR - 2137 ( 50 nM ) as substrate with active , recombinant IRE1 ( 100 ng ) at 37 ℃ for 2 hours , followed by sample separation in Urea - PAGE and detection with SYBR gold staining . M indicates micro RNA marker .

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

(G) IRE1 cleavage assay performed using synthetic pre-miR-2137 (50 nM) as substrate with active, recombinant IRE1 (100 ng) at 37℃ for 2 hours, followed by sample separation in Urea-PAGE and detection with SYBR gold staining. M indicates micro RNA marker.


( H - I ) BMDMs isolated from WT ( C57BL6 ) and DICER - / - mice were treated with PA ( 500 µM ) for 12 hours . RNA lysates were analyzed by qRT - PCR for ( H ) miR - 2137 and ( I ) sXBP1 expression ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H-I) BMDMs isolated from WT (C57BL6) and DICER-/- mice were treated with PA (500 µM) for 12 hours. RNA lysates were analyzed by qRT-PCR for (H) miR-2137 and (I) sXBP1 expression (n=3 biological replicates).


( B ) HEK293 cells were co - transfected with an INPPL1 3 ` UTR - luciferase plasmid and scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours . Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase ( SEAP ) reporter activity ( n = 3 biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were co-transfected with an INPPL1 3` UTR-luciferase plasmid and scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours. Protein lysates were analyzed for luciferase activity and normalized to secreted embryonic alkaline phosphatase (SEAP) reporter activity (n=3 biological replicates).


( C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) and miR - 205 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications are displayed in graph next to the blots ( n = 3 biological replicates ) .

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

(C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) and miR-205 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications are displayed in graph next to the blots (n=3 biological replicates).


( D ) BMDMs were transfected with negative control antagomir ( 200 nM ) or miR - 2137 antagomir ( 200 nM ) for 24 hours , followed by PA ( 500 µM ) treatment for 9 hours . Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β - actin . Protein quantifications shown in the graph next to blots ( n = 3 biological replicates ) .

O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) BMDMs were transfected with negative control antagomir (200 nM) or miR-2137 antagomir (200 nM) for 24 hours, followed by PA (500 µM) treatment for 9 hours. Protein lysates were analyzed by western blotting using specific antibodies for INPPL1 and β-actin. Protein quantifications shown in the graph next to blots (n=3 biological replicates).


( A - C ) BMDMs were transfected with scrambled miRNA mimic ( 40 nM ) , miR - 2137 mimic ( 40 nM ) or miR - 205 mimic ( 40 nM ) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section . The ratio of lipids from this analysis is shown for ( A ) PI ( 3 , 4 , 5 ) P3 to PIP2 . ; ( B ) PI ( 3 , 4 , 5 ) P3 to PI ; ( C ) PI ( 3 , 4 , 5 ) P3 to PIP . Data from LC / MS / MS using a Waters Xevo TQ - S MS / MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods . ( n = 3 biological replicates )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) BMDMs were transfected with scrambled miRNA mimic (40 nM), miR-2137 mimic (40 nM) or miR-205 mimic (40 nM) for 24 hours and cells were processed for lipidomics analysis as described in materials and methods section. The ratio of lipids from this analysis is shown for (A) PI(3,4,5)P3 to PIP2.; (B) PI(3,4,5)P3 to PI; (C) PI(3,4,5)P3 to PIP. Data from LC/MS/MS using a Waters Xevo TQ-S MS/MS in MRM mode represent mean values ± SEM of peak areas normalized to internal standards as described in Methods. (n=3 biological replicates)


BMDMs were transfected with ( D ) scrambled miRNA mimic ( 40 nM ) or miR - 2137 mimic ( 40 nM ) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected with (D) scrambled miRNA mimic (40 nM) or miR-2137 mimic (40 nM) for 48 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


BMDMs were transfected ( E ) with control antagomir ( 200 nM ) or miR - 2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1 , pAKTS473 , AKT , p70S6KT389 , p70S6K , pS6S235 / 236 , S6 , p4E - BP1S65 , 4E - BP1 , pmTORS2448 , mTOR and β - actin . Protein quantifications are shown next to the western blots ( n = 3 biological replicates ) .

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

BMDMs were transfected (E) with control antagomir (200 nM) or miR-2137 antagomir for 24 hours and protein lysates were analyzed by western blotting using specific antibodies against INPPL1, pAKTS473, AKT, p70S6KT389, p70S6K, pS6S235/236, S6, p4E-BP1S65, 4E-BP1, pmTORS2448, mTOR and β-actin. Protein quantifications are shown next to the western blots (n=3 biological replicates).


Overexpression of USP36 promotes SUMOylation in cells . Whole cell lysates ( WCL ) from H1299 ( A ) , U2OS and HeLa ( B ) cells transfected with control empty vector or Flag - USP36 were assayed by IB .

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

Overexpression of USP36 promotes SUMOylation in cells. Whole cell lysates (WCL) from H1299 (A), U2OS and HeLa (B) cells transfected with control empty vector or Flag-USP36 were assayed by IB.


H1299 cells transfected with WT USP36 or the indicated mutants and His - SUMO2 were subjected to Ni2 + - NTA agarose beads pull down ( PD ) followed by IB using anti - SUMO2 / 3 antibody .

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

H1299 cells transfected with WT USP36 or the indicated mutants and His-SUMO2 were subjected to Ni2+-NTA agarose beads pull down (PD) followed by IB using anti-SUMO2/3 antibody.


USP36 interacts with Ubc9 in cells . H1299 cell transfected with the indicated plasmids were subjected to co - IP with anti - Flag antibody followed by IB .

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

USP36 interacts with Ubc9 in cells. H1299 cell transfected with the indicated plasmids were subjected to co-IP with anti-Flag antibody followed by IB.


USP36 SUMOylates PARP1 in vitro . Recombinant T7 - PARP1 protein ( 0 . 1 μM ) was incubated with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the presence of USP361 - 800 ( 50 nM ) and / or ATP ( 2 . 5 mM ) at 30ºC for 5 hours and then assayed by IB .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

USP36 SUMOylates PARP1 in vitro. Recombinant T7-PARP1 protein (0.1 μM) was incubated with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the presence of USP361-800 (50 nM) and/or ATP (2.5 mM) at 30ºC for 5 hours and then assayed by IB.


USP36 SUMOylates Nhp2 and Nop58 in vitro . In vitro SUMOylation assays were performed by incubating recombinant Nhp2 ( F , G ) or Nop58 ( H ) ( 0 . 1 μM ) with SUMO E1 ( 30 nM ) , Ubc9 ( 50 nM ) , SUMO2 ( 4 μM ) in the absence or presence of USP361 - 800 ( 50 nM , 1x ; 100 nM , 2x ) and / or ATP ( 2 . 5 mM ) at 37ºC for two hours or the indicated times . The reactions were assayed by IB using anti - Nhp2 and anti - Nop58 antibodies , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

USP36 SUMOylates Nhp2 and Nop58 in vitro. In vitro SUMOylation assays were performed by incubating recombinant Nhp2 (F, G) or Nop58 (H) (0.1 μM) with SUMO E1 (30 nM), Ubc9 (50 nM), SUMO2 (4 μM) in the absence or presence of USP361-800 (50 nM, 1x; 100 nM, 2x) and/or ATP (2.5 mM) at 37ºC for two hours or the indicated times. The reactions were assayed by IB using anti-Nhp2 and anti-Nop58 antibodies, respectively.


USP36 does not change the levels of endogenous snoRNP proteins . HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB .

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

USP36 does not change the levels of endogenous snoRNP proteins. HeLa cells infected with scrambled or the indicated USP36 shRNA lentiviruses were assayed by IB.


USP36 promotes the binding of Nhp2 to snoRNAs . H1299 cells transfected with Flag - Nhp2 and V5 - USP36 individually or together were assayed by RNA - IP with anti - Flag or control mouse IgG , followed by RT - qPCR detection of the indicated box H / ACA snoRNAs ( C ) . Shown is one representative experiment of three independent experiments . Data were presented as mean ± SD , n = 3 technical replicates . * P < 0 . 05 ; * * P < 0 . 01 , compared to Nhp2 alone ( Student ' s t - test ) .

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

USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).


( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


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 .

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

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

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.


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

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.


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

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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 ) .

O O O O O O O O O O O O O O O O O O O O O O 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) 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 .

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

(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 .

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

(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 .

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

(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


( 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 .

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

(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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(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.


( 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 .

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

(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 .

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(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 ) .

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(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 .

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(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.


( 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 .

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(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 .

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(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 ) .

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(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 .

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(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 .

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(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 ) .

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(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).


Overexpression 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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Overexpression 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.


( 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 .

(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 .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 ) .

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(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 ) .

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(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 ) .

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(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).


( 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 .

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(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 .

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(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 .

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(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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(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.


( A ) Quantitative PCR analysis was performed in triplicates using specific oligonucleotides ( see Supplementary Table 1 ) ( * P0 . 01 ) .

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(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 .

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(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 .

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(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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(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.


Representative 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 .

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Representative 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.