A . Expression of netrin - 1 and DCC in lymphoma cell lines . Quantification was performed by Q - RT - PCR in 21 lymphoma cell lines . HPRT housekeeping gene was used as a standardization control . Netrin - 1 and DCC levels are indicated as follow : − , not detectable ; + to + + + + , moderate to very high expression . ABC and GC - DLBCL cell lines are highlighted respectively in blue and green , MCL ones are in grey . Expression of netrin - 1 ( in green ) by immunofluorescence using netrin - 1 antibody on OCI - Ly3 and Granta - 519 cell lines is shown on the right panel . Nuclei are counterstained in blue by Hoechst .

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A. Expression of netrin-1 and DCC in lymphoma cell lines. Quantification was performed by Q-RT-PCR in 21 lymphoma cell lines. HPRT housekeeping gene was used as a standardization control. Netrin-1 and DCC levels are indicated as follow: −, not detectable; + to ++++, moderate to very high expression. ABC and GC-DLBCL cell lines are highlighted respectively in blue and green, MCL ones are in grey. Expression of netrin-1 (in green) by immunofluorescence using netrin-1 antibody on OCI-Ly3 and Granta-519 cell lines is shown on the right panel. Nuclei are counterstained in blue by Hoechst.


B . Caspase - 3 activity in Granta - 519 cells after transfection of scrambled siRNA ( si - Ctl ) or netrin - 1 siRNA ( si - netrin - 1 ) , with or without DCC siRNA ( si - DCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 03 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test .

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B. Caspase-3 activity in Granta-519 cells after transfection of scrambled siRNA (si-Ctl) or netrin-1 siRNA (si-netrin-1), with or without DCC siRNA (si-DCC). Results are means+/-std of four independent experiments. $: p=0.03; *: p=0.02; two-sided Mann-Whitney U-test.


D . Effect of net - 1 mAb on Granta - 519 ( left panel ) and OCI - Ly3 ( right panel ) cell density . Results are means + / - std indexed to control of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 03 ; two - sided Mann - Whitney U - test .

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D. Effect of net-1 mAb on Granta-519 (left panel) and OCI-Ly3 (right panel) cell density. Results are means+/-std indexed to control of four independent experiments. $: p=0.05; *: p=0.03; two-sided Mann-Whitney U-test.


E . Effect of net - 1 mAb on induction of Granta - 519 cells apoptosis , detected by TUNEL staining . Left panel : representative images are shown . TUNEL positive cells are labelled in red . Nuclei are counterstained in blue by Hoechst staining . Right panel : quantification of one representative experiment out of three performed is exposed . Results are presented as percentage of TUNEL - positive cells per field . * : p = 0 . 04 ; two - sided Mann - Whitney U - test .

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E. Effect of net-1 mAb on induction of Granta-519 cells apoptosis, detected by TUNEL staining. Left panel: representative images are shown. TUNEL positive cells are labelled in red. Nuclei are counterstained in blue by Hoechst staining. Right panel: quantification of one representative experiment out of three performed is exposed. Results are presented as percentage of TUNEL-positive cells per field. *: p=0.04; two-sided Mann-Whitney U-test.


F . Caspase - 3 activity in Granta - 519 cells treated with net - 1 mAb antibody or with an unrelated Ig - G1 antibody ( Ctl ) , with or without silencing of DCC via siRNA ( siDCC ) . Results are means + / - std of four independent experiments . $ : p = 0 . 05 ; * : p = 0 . 02 ; two - sided Mann - Whitney U - test .

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F. Caspase-3 activity in Granta-519 cells treated with net-1 mAb antibody or with an unrelated Ig-G1 antibody (Ctl), with or without silencing of DCC via siRNA (siDCC). Results are means+/-std of four independent experiments. $: p=0.05; *: p=0.02; two-sided Mann-Whitney U-test.


G - H . Effect of netrin - 1 interfering antibody net - 1 mAb on OCI - Ly3 ( G ) and Granta - 519 ( H ) xenograft tumors growth . Results are presented as means + / - 95 % CIs of tumor growth rates from the beginning of treatment . * : p = 0 . 02 ; * * : p = 0 . 01 ; $ : p = 0 . 007 ; $ $ : p = 0 . 002 ; * * * : p = 0 . 0002 ; * * * * : p < 0 . 0001 ; two - sided Student t - test ( Granta - 519 ) or Welch t - test ( OCI - Ly3 ) .

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G-H. Effect of netrin-1 interfering antibody net-1 mAb on OCI-Ly3 (G) and Granta-519 (H) xenograft tumors growth. Results are presented as means+/-95% CIs of tumor growth rates from the beginning of treatment. *:p=0.02; **:p=0.01; $: p=0.007; $$:p=0.002; ***: p=0.0002; ****:p<0.0001; two-sided Student t-test (Granta-519) or Welch t-test (OCI-Ly3).


I . Caspase - 3 activity in tumors from control ( n = 4 ) or netrin - 1 - interfering antibody ( n = 4 ) treated mice . Tumors resection was performed at the end of the treatment . Results are means + / - std of caspase - 3 activity . * : p = 0 . 02 ; two - sided Mann - Whitney test .

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I. Caspase-3 activity in tumors from control (n=4) or netrin-1-interfering antibody (n=4) treated mice. Tumors resection was performed at the end of the treatment. Results are means +/-std of caspase-3 activity. *:p=0.02; two-sided Mann-Whitney test.


Electron microscopy showing two adjacent mitochondria ( one intact and one ruptured ) in a DM platelet . a . Lower powered view of the single DM platelet . b . The enlarged inset highlights the ruptured mitochondria ( with loss of cristae morphology ) next to the intact mitochondria . c . Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation . ( M - mitochondria )

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Electron microscopy showing two adjacent mitochondria (one intact and one ruptured) in a DM platelet. a. Lower powered view of the single DM platelet. b. The enlarged inset highlights the ruptured mitochondria (with loss of cristae morphology) next to the intact mitochondria. c. Black dash line indicates mitochondria membrane and red dash line indicate phagophore formation. (M- mitochondria)


Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I / II western .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM human platelets followed by MsrB2 and LC3I/II western.


LC3 and MsrB2 immuno - EM analysis of HC and DM platelets . 15 nm dots indicate immunogold - labeled LC3 clusters and 5nm dots indicate immunogold - labeled MsrB2 clusters . No significant clusters were found in HC ( a ) platelets . Representative areas of clusters of gold labeling in DM patients ( b - d ) are presented . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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LC3 and MsrB2 immuno-EM analysis of HC and DM platelets. 15 nm dots indicate immunogold-labeled LC3 clusters and 5nm dots indicate immunogold-labeled MsrB2 clusters. No significant clusters were found in HC (a) platelets. Representative areas of clusters of gold labeling in DM patients (b-d) are presented. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot analysis of MsrB2 and LC3 I / II in Meg - 01 cell after shMsrB2 transfection ( 72 hrs ) . Cells were then treated with H2O2 ( 1 mM for 1 hr ) alone or with NAC ( 100 μM for 30 min ) . GAPDH was used as the loading control . Quantification and analysis of individual groups . GAPDH served as the loading control . ( shMsrB2 vs . shCon ; * p = 0 . 024 , shMsrB2 / H2O2 vs . shCon / H2O2 ; * p = 0 . 0295 , shMsrB2 / H2O2 / NAC vs . shCon / H2O2 / NAC * * p = 0 . 0100 , n = 3 )

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Western blot analysis of MsrB2 and LC3 I/II in Meg-01 cell after shMsrB2 transfection (72 hrs). Cells were then treated with H2O2 (1 mM for 1 hr) alone or with NAC (100 μM for 30 min). GAPDH was used as the loading control. Quantification and analysis of individual groups. GAPDH served as the loading control. (shMsrB2 vs. shCon; *p=0.024, shMsrB2/H2O2 vs. shCon/H2O2; *p=0.0295, shMsrB2/H2O2/NAC vs. shCon/H2O2/NAC **p=0.0100, n=3)


Western blot analysis of MsrB2 , pp53 ( S15 ) and LC3 I / II in Meg - 01 cell after 25 mM HG treatment for 24 hrs . GAPDH was used as the loading control . Quantification analysis on individual groups . GAPDH served as the loading control . ( MsrB2 : shCon / HG vs . shCon ; * * p = 0 . 0017 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0189 , pp53 : shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0317 , LC3II : shCon / HG vs . shCon ; * p = 0 . 0215 , shMsrB2 / HG vs . shCon / HG ; * p = 0 . 0158 , n = 3 ) The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Western blot analysis of MsrB2, pp53(S15) and LC3 I/II in Meg-01 cell after 25 mM HG treatment for 24 hrs. GAPDH was used as the loading control. Quantification analysis on individual groups. GAPDH served as the loading control. (MsrB2: shCon/HG vs. shCon; **p=0.0017, shMsrB2/HG vs. shCon/HG; *p=0.0189, pp53: shMsrB2/HG vs. shCon/HG; *p=0.0317, LC3II: shCon/HG vs. shCon; *p=0.0215, shMsrB2/HG vs. shCon/HG; *p=0.0158, n=3) The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Levels of reactive oxygen species ( ROS ) quantified in the platelets using the fluorescent dye DCFH - DA . ( ROS ; * p = 0 . 0139 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 6 )

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Levels of reactive oxygen species (ROS) quantified in the platelets using the fluorescent dye DCFH-DA. (ROS; *p=0.0139, MsrB2 fl/fl n=5, MsrB2 -/- n=6)


C . Representative image and quantification of mitochondrial apoptosis , measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice . Results are expressed as % apoptotic cells . ( TMRE ; * p = 0 . 0118 , MsrB2 fl / fl n = 5 , MsrB2 - / - n = 5 )

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C. Representative image and quantification of mitochondrial apoptosis, measured in freshly isolated platelets from the MsrB2 knockouts and age matched floxed mice. Results are expressed as % apoptotic cells. (TMRE; *p=0.0118, MsrB2 fl/fl n=5, MsrB2 -/- n=5)


Representative images showing the intraplatelet expression of LC3 and active caspase - 3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest . Magnification : 100X . Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase - 3 .

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Representative images showing the intraplatelet expression of LC3 and active caspase-3 in CD41 stained platelets from the KO mice using fluorescent labelled antibodies against the respective proteins of interest. Magnification: 100X. Rightmost panel represents zoom in on an individual platelet showing the colocalization and expression of LC3 and active caspase-3.


Graph indicates platelets positive for active caspase - 3 signal . The y ‐ axis indicates percentage of caspase - 3 positive cells from total cells calculated from n = 10 independent images per sample . ( active caspase - 3 signal ; * * p = 0 .

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Graph indicates platelets positive for active caspase-3 signal. The y‐axis indicates percentage of caspase-3 positive cells from total cells calculated from n = 10 independent images per sample. (active caspase-3 signal ; **p=0.0003


Intracellular caspase - 3 activity measured in freshly isolated platelets using the chromophore Ac - DEVD - pNA . Experiments performed in a minimum of n = 6 samples per group . Values expressed as Mean ± SEM . ( caspase - 3 activity ; * p = 0 . 0157 )

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Intracellular caspase-3 activity measured in freshly isolated platelets using the chromophore Ac-DEVD-pNA. Experiments performed in a minimum of n=6 samples per group. Values expressed as Mean ± SEM. (caspase-3 activity ; *p=0.0157)


and H . Western blot analysis of Parkin , LC3I / II , MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice . Control peptide ( CP ) , LIF peptide ( Foroud et al ) . Quantification analysis of individual band intensity . ( Parkin ; * p = 0 . 02964 , LC3II ; * p = 0 . 03200 , MsrB2 ; * * p = 0 . 0059 , Active caspase3 ; * p = 0 . 0368 vs . chow or HFD group , each group n = 3 ) . Actin served as the loading control . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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and H. Western blot analysis of Parkin, LC3I/II, MsrB2 and active caspasee3 in platelet after cell penetration peptide injection in chow and HFD mice. Control peptide (CP), LIF peptide (Foroud et al). Quantification analysis of individual band intensity. (Parkin; *p=0.02964, LC3II; *p=0.03200, MsrB2; **p=0.0059, Active caspase3; *p=0.0368 vs. chow or HFD group, each group n=3). Actin served as the loading control. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM platelets , followed by western blot analysis of precipitated Parkin and modified Parkin .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets, followed by western blot analysis of precipitated Parkin and modified Parkin.


Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets , followed by Western blot analysis of precipitated Parkin and MsrB2 .

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Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets, followed by Western blot analysis of precipitated Parkin and MsrB2.


Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin , LC3 and MsrB2 in HC and DM platelets . Arrows indicate sites of colocalization of Parkin , LC3 and MsrB2 as determined by Volocity software ( PerkinElmer , USA ) . Graphical quantification of colocalization between Parkin , LC3 and MsrB2 signal .

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Confocal microscopy was used to corroborate the Western analysis using triple staining for Parkin, LC3 and MsrB2 in HC and DM platelets. Arrows indicate sites of colocalization of Parkin, LC3 and MsrB2 as determined by Volocity software (PerkinElmer, USA). Graphical quantification of colocalization between Parkin, LC3 and MsrB2 signal.


Signal intensity of each group was converted to fold and compared with HC values ( colocalization of parkin , LC3 and MsrB2 ; * * p = 0 . 0006 ) .

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Signal intensity of each group was converted to fold and compared with HC values (colocalization of parkin, LC3 and MsrB2; **p=0.0006).


Immunoprecipitation ( Meng et al ) of Parkin in HC and DM platelets . Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies . The detection of MetO Parkin , was performed w / o β - mercaptoethanol ( NonRe ) SDS sample buffer .

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Immunoprecipitation (Meng et al) of Parkin in HC and DM platelets. Western blot analysis of MetO Parkin using specific Parkin and MetO antibodies. The detection of MetO Parkin, was performed w/o β-mercaptoethanol (NonRe) SDS sample buffer.


In vitro assay . Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs . Western blot analysis of Parkin and MsrB2 was performed . ( red arrow , low MsrB2 expression ; blue arrow , high MsrB2 expression ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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In vitro assay. Parkin oxidation was induced with 1mM H2O2 then incubated with MsrB2 protein at 37ºC for 2hrs. Western blot analysis of Parkin and MsrB2 was performed. (red arrow, low MsrB2 expression; blue arrow, high MsrB2 expression). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot analysis of Methionine Sulfoxidation ( MetO ) , Methionine sulfide reductase B2 ( MsrB2 ) and Parkin in human Healthy Control ( HC ) and Diabetic Mellitus ( DM ) platelets . P . C . is a BSA - MetO positive control ( Cayman Chemical Co . ) . Quantitation of western blot analysis shown in Fig 1C . Graphical representation and statistical analysis of HC ( n = 3 ) and DM ( n = 12 ) individuals . ( MetO ; * * p = 0 . 0003 , MsrB2 ; * p = 0 . 0186 , Parkin ; * * p = 0 . 0003 vs . HC ) .

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Western blot analysis of Methionine Sulfoxidation (MetO), Methionine sulfide reductase B2 (MsrB2) and Parkin in human Healthy Control (HC) and Diabetic Mellitus (DM) platelets. P.C. is a BSA-MetO positive control (Cayman Chemical Co.). Quantitation of western blot analysis shown in Fig 1C. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (MetO; **p=0.0003, MsrB2; *p=0.0186, Parkin; **p=0.0003 vs. HC).


Western blot analysis of Parkin and Parkin aggregation in human Healthy Control ( HC ) ( n = 3 ) and Diabetic Mellitus ( DM ) ( n = 12 ) platelets . Graphical representation and statistical analysis of HC ( n = 3 ) and DM ( n = 12 ) individuals . ( modified parkin * p = 0 . 035 vs . HC ) . Actin served as the loading control .

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Western blot analysis of Parkin and Parkin aggregation in human Healthy Control (HC) (n=3) and Diabetic Mellitus (DM) (n=12) platelets. Graphical representation and statistical analysis of HC (n=3) and DM (n=12) individuals. (modified parkin *p=0.035 vs. HC). Actin served as the loading control.


Parkin IP was performed in HC , HC plus H2O2 , and DM patients .

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Parkin IP was performed in HC, HC plus H2O2, and DM patients.


MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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MetO sites were analyzed by mass spectrometry using the Mascot program Representative mass spectrometry analysis demonstrating identification of MetO192 peptide on Parkin. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Immunoprecipitation ( Meng et al ) of MsrB2 in HC and DM platelets . Western blot analysis of precipitated MsrB2 , Parkin and ubiquitinated MsrB2 ( UB ) .

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Immunoprecipitation (Meng et al) of MsrB2 in HC and DM platelets. Western blot analysis of precipitated MsrB2, Parkin and ubiquitinated MsrB2 (UB).


Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV ( mitochondria marker ) , ubiquitin and MsrB2 . Colocalization is demomstrated by arrows and quantitated . ( colocalization ubiquitin , MsrB2 ; * * p = 0 . 0003 vs . HC ) .

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Confocal microscopy in individual platelets in HC vs DM assessing for CoxIV (mitochondria marker), ubiquitin and MsrB2. Colocalization is demomstrated by arrows and quantitated. (colocalization ubiquitin, MsrB2; **p=0.0003 vs. HC).


MsrB2 ubiquitination assay after transient transfection of MsrB2 - GFP ( 1µg ) with RFP empty vector ( E . V . ) or RFP - Parkin ( 3µg ) in HEK293 . After transfection ( 48hrs ) , an IP was performed using GFP - Trap bead , followed by Western blot analysis using UB and MsrB2 and Parkin antibodies . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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MsrB2 ubiquitination assay after transient transfection of MsrB2-GFP (1µg) with RFP empty vector (E.V.) or RFP-Parkin (3µg) in HEK293. After transfection (48hrs), an IP was performed using GFP-Trap bead, followed by Western blot analysis using UB and MsrB2 and Parkin antibodies. The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w / o Cyclosporin A ( CsA ; 2 μM for 2 hrs . ) or w / CsA . ATPB served as the loading and negative control ( mitochondrial matrix protein ) .

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Western blot analysis of MsrB2 and Cytochrome C in isolated mitochondria after incubation w/o Cyclosporin A (CsA; 2 μM for 2 hrs.) or w/ CsA. ATPB served as the loading and negative control (mitochondrial matrix protein).


Western blot analysis of Parkin , LC3I / II , ubiquitinated MsrB2 in chow ( n = 4 ) and HFD ( n = 6 ) mouse platelets .

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Western blot analysis of Parkin, LC3I/II, ubiquitinated MsrB2 in chow (n=4) and HFD (n=6) mouse platelets.


Confocal microscopy was initially stained with Tubulin antibody ( Panel a . , Chow diet , or b . High Fat Diet ) to assess platelet architecture . This was followed by staining using ATPB ( mitochondria marker - green ) and MsrB2 antibodies ( Blue ) ( Panel c - e ) . There was loss of colocalization in the HFD mice supporting release ( blue ) from ruptured mitochondria ( green ) ( Panel d & e ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Confocal microscopy was initially stained with Tubulin antibody (Panel a., Chow diet, or b. High Fat Diet) to assess platelet architecture. This was followed by staining using ATPB (mitochondria marker - green) and MsrB2 antibodies (Blue) (Panel c-e). There was loss of colocalization in the HFD mice supporting release (blue) from ruptured mitochondria (green) (Panel d & e). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


Representative western blot analysis of MsrB2 , LC3I / II and parkin using Parkinson ' s disease ( PD ) platelets . Quantification analysis of individual band intensity . ( MsrB2 ; * p = 0 . 0255 , LC3II ; * p = 0 . 0101 vs . HC ( n = 5 ) and PD ( n = 12 ) ) . GAPDH served as the loading control .

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Representative western blot analysis of MsrB2, LC3I/II and parkin using Parkinson's disease (PD) platelets. Quantification analysis of individual band intensity. (MsrB2; *p=0.0255, LC3II; *p=0.0101 vs. HC (n=5) and PD (n=12)). GAPDH served as the loading control.


Platelet apoptosis ( Annexin V ) were measured by flow cytometry analysis in HC ( n = 9 ) and PD ( n = 13 ) platelets . ( * * p < 0 . 01 vs . HC ) . The nonparametric t test was performed for comparisons of 2 groups . Analysis was performed with Prism software ( GraphPad Software , Inc , La Jolla , CA ) . A difference of P < 0 . 05 was considered significant .

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Platelet apoptosis (Annexin V) were measured by flow cytometry analysis in HC (n=9) and PD (n=13) platelets. (**p<0.01 vs. HC). The nonparametric t test was performed for comparisons of 2 groups. Analysis was performed with Prism software (GraphPad Software, Inc, La Jolla, CA). A difference of P<0.05 was considered significant.


A - F Indirect calorimetry was performed using the Oxymax / CLAMS system in Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 5 ) mice . Whole - body oxygen consumption rate ( VO2 ) ( A - B ) , energy expenditure ( EE ) ( C - D ) and respiratory exchange ratio ( RER ) ( E - F ) were measured at 24 ° C and during a cold challenge at 6 ° C during both the light ( white rectangle ) and dark ( black rectangle ) phases over a 2 - day period .

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A-F Indirect calorimetry was performed using the Oxymax/CLAMS system in Cdk4+/+ (n=6) and Cdk4-/- (n=5) mice. Whole-body oxygen consumption rate (VO2) (A-B), energy expenditure (EE) (C-D) and respiratory exchange ratio (RER) (E-F) were measured at 24°C and during a cold challenge at 6°C during both the light (white rectangle) and dark (black rectangle) phases over a 2-day period.


CalR was used to GLM - regression plot for each group corresponding to the association between energy balance and total body mass at 24 ° C ( G ) and the association between energy balance and total body mass at 6 ° C ( H ) .

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CalR was used to GLM-regression plot for each group corresponding to the association between energy balance and total body mass at 24°C (G) and the association between energy balance and total body mass at 6°C (H).


J - K Basal rectal temperature was measured at 24 ° C ( J ) , and rectal temperature was monitored during acute cold exposure at 4 ° C for 6 hours ( K ) in Cdk4 + / + ( n = 10 ) and Cdk4 - / - ( n = 8 ) mice .

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J-K Basal rectal temperature was measured at 24°C (J), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (K) in Cdk4+/+ (n=10) and Cdk4-/- (n=8) mice.


A Gross morphology of iBAT from Cdk4 + / + and Cdk4 - / - mice .

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A Gross morphology of iBAT from Cdk4+/+ and Cdk4-/- mice.


C Hematoxylin - eosin staining and UCP1 immunohistochemical ( IHC ) staining of iBAT sections ( scale bar 100 μm ) from Cdk4 + / + and Cdk4 - / - mice .

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C Hematoxylin-eosin staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm) from Cdk4+/+ and Cdk4-/- mice.


D - E Western blot analysis of UCP1 and CDK4 protein expression ( D ) and quantified protein levels of UCP1 ( E ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as loading control .

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D-E Western blot analysis of UCP1 and CDK4 protein expression (D) and quantified protein levels of UCP1 (E) (n=4 biological replicates) in iBAT. HSP90 was used as loading control.


F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

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F Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4+/+ (n=6) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


G - H Transmission electron microscopy ( TEM ) images of iBAT ( G ) ( scale bar 5 μm ( upper panel ) and 1 μm ( lower panel , LD indicates lipid droplet and M mitochondria ) ) and quantification of lipid droplet number ( LD ) and mitochondrial volume ( Mito ) ( H ) ( Cdk4 + / + ( n = 4 ) and Cdk4 - / - ( n = 4 ) ) .

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G-H Transmission electron microscopy (TEM) images of iBAT (G) (scale bar 5 μm (upper panel) and 1 μm (lower panel, LD indicates lipid droplet and M mitochondria)) and quantification of lipid droplet number (LD) and mitochondrial volume (Mito) (H) (Cdk4+/+ (n=4) and Cdk4-/- (n=4)).


I Respirometry analysis of iBAT as measured by the Oroboros system ( Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) ) .

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I Respirometry analysis of iBAT as measured by the Oroboros system (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


A - B Rectal temperature was measured at 24 ° C ( A ) , and rectal temperature was monitored for 6 hours during acute cold exposure at 4 ° C ( B ) in Cdk4flox / flox ( n = 9 ) and Cdk4flox / flox Ucp1 - Cre mice ( n = 9 ) .

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A-B Rectal temperature was measured at 24°C (A), and rectal temperature was monitored for 6 hours during acute cold exposure at 4°C (B) in Cdk4flox/flox (n=9) and Cdk4flox/flox Ucp1-Cre mice (n=9).


C Gross morphology of iBAT from Cdk4flox / flox and Cdk4flox / flox Ucp1 - Cre mice D iBAT mass compared to body weight of Cdk4flox / flox ( n = 8 ) and Cdk4flox / flox Ucp1 - Cre ( n = 7 ) mice .

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C Gross morphology of iBAT from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice D iBAT mass compared to body weight of Cdk4flox/flox (n=8) and Cdk4flox/flox Ucp1-Cre (n=7) mice.


E Hematoxylin - eosin ( H & E ) staining and UCP1 immunohistochemical ( IHC ) staining of iBAT sections ( scale bar 100 μm ( upper panel ) and 50 μm ( lower panel ) ) from Cdk4flox / flox and Cdk4flox / flox Ucp1 - Cre mice .

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E Hematoxylin-eosin (H&E) staining and UCP1 immunohistochemical (IHC) staining of iBAT sections (scale bar 100 μm (upper panel) and 50 μm (lower panel)) from Cdk4flox/flox and Cdk4flox/flox Ucp1-Cre mice.


F - G Western blot analysis of UCP1 and CDK4 protein expression ( F ) and quantified protein levels of UCP1 ( G ) ( n = 4 biological replicates ) in iBAT . HSP90 was used as a loading control .

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F-G Western blot analysis of UCP1 and CDK4 protein expression (F) and quantified protein levels of UCP1(G) (n=4 biological replicates) in iBAT. HSP90 was used as a loading control.


H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Ucp1 - Cre ( n = 6 ) mice as assessed by RT - PCR .

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H Expression of thermogenic genes and brown adipocyte markers in iBAT of Cdk4flox/flox (n=5) and Cdk4flox/flox Ucp1-Cre (n=6) mice as assessed by RT-PCR.


A - B TH immunohistochemical staining in iBAT sections ( A ) ( scale bar 20 μm , arrows indicate TH parenchymal fibers ) and corresponding quantification of the number of TH fibers relative to 100 adipocytes ( B ) ( Cdk4 + / + ( n = 6 ) and Cdk4 - / - ( n = 6 ) ) .

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A-B TH immunohistochemical staining in iBAT sections (A) (scale bar 20 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (B) (Cdk4+/+ (n=6) and Cdk4-/- (n=6)).


C Hematoxylin - eosin staining in scWAT showing multilocular adipocytes in Cdk4 - / - ( scale bar 50 μm ) .

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C Hematoxylin-eosin staining in scWAT showing multilocular adipocytes in Cdk4-/- (scale bar 50 μm).


D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4 + / + ( n = 5 ) and Cdk4 - / - ( n = 6 ) mice as assessed by RT - PCR .

D Expression of thermogenic genes and brown adipocyte markers in scWAT of Cdk4+/+ (n=5) and Cdk4-/- (n=6) mice as assessed by RT-PCR.


E - F Acute cold test ( 4 ° C ) after treatment with the β - adrenergic antagonist propranolol ( Prop ) in Cdk4 + / + ( n = 7 ) and Cdk4 - / - ( n = 6 ) animals or with vehicle ( NaCl ) ( Cdk4 + / + ( n = 7 ) and Cdk4 - / - ( n = 6 ) ) ( E ) and corresponding quantification of the area under the curve ( AUC ) .

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E-F Acute cold test (4°C) after treatment with the β-adrenergic antagonist propranolol (Prop) in Cdk4+/+ (n=7) and Cdk4-/- (n=6) animals or with vehicle (NaCl) (Cdk4+/+ (n=7) and Cdk4-/- (n=6)) (E) and corresponding quantification of the area under the curve (AUC).


A Cdk4 mRNA expression in the dissected VMH of Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Sf1 - Cre ( n = 6 )

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A Cdk4 mRNA expression in the dissected VMH of Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre (n=6) mice


B - C Rectal temperature was measured at 24 ° C ( B ) , and rectal temperature was monitored during acute cold exposure at 4 ° C for 6 hours ( C ) in Cdk4flox / flox ( n = 15 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 15 ) .

B-C Rectal temperature was measured at 24°C (B), and rectal temperature was monitored during acute cold exposure at 4°C for 6 hours (C) in Cdk4flox/flox (n=15) and Cdk4flox/flox Sf1-Cre mice (n=15).


D Hematoxylin - eosin ( H & E ) staining of iBAT sections ( scale bar 100 μm ) from Cdk4flox / flox and Cdk4flox / flox Sf1 - Cre mice .

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D Hematoxylin-eosin (H&E) staining of iBAT sections (scale bar 100 μm) from Cdk4flox/flox and Cdk4flox/flox Sf1-Cre mice.


E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox / flox ( n = 9 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 9 ) mice as assessed by RT - PCR .

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E Expression of thermogenic genes and brown adipocyte markers expression in iBAT Cdk4flox/flox (n=9) and Cdk4flox/flox Sf1-Cre mice (n=9) mice as assessed by RT-PCR.


F - G TH immunohistochemical ( IHC ) staining in iBAT sections ( F ) ( scale bar 40 μm , arrows indicate TH parenchymal fibers ) and corresponding quantification of the number of TH fibers relative to 100 adipocytes ( G ) ( Cdk4flox / flox ( n = 5 ) and Cdk4flox / flox Sf1 - Cre mice ( n = 5 ) ) .

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F-G TH immunohistochemical (IHC) staining in iBAT sections (F) (scale bar 40 μm, arrows indicate TH parenchymal fibers) and corresponding quantification of the number of TH fibers relative to 100 adipocytes (G) (Cdk4flox/flox (n=5) and Cdk4flox/flox Sf1-Cre mice (n=5)).


H - I Western blot analysis ( H ) and quantification of p - CREBS133 protein expression following 6 hours of cold exposure ( 4 ° C ) ( I ) ( n = 3 biological replicates ) . HSP90 was used as a loading control .

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H-I Western blot analysis (H) and quantification of p-CREBS133 protein expression following 6 hours of cold exposure (4°C) (I) (n=3 biological replicates). HSP90 was used as a loading control.


A - D Confocal illustrations of frontal hypothalamic sections from Cdk4 + / + mice ( A , C , N = 3 ) and Cdk4 - / - mice ( B , D , N = 3 ) . Low magnification showing nuclei ( Hoechst , left pannel ) and c - Fos activated - cells ( white , right pannel ) in the mPOA ( A1 - 2 , B1 - 2 ) and the ARC , the VMH , the DMH , the LH ( C1 - 2 , D1 - 2 ) . High magnifications corresponding to the frame area in A1 - 2 , B1 - 2 , C2 , D2 and showing c - Fos - IR cells ( A3 - 4 , B3 - 4 , C3 - 6 , D3 - 6 ) . Scale bars ( A1 - 2 , B1 - 2 , C1 - 2 , D1 - 2 ) : 200μm , ( A3 - 4 , B3 - 4 , C3 - 4 , D3 - 4 ) : 50μm . E Quantification of c - Fos IR cells in brain sections of Cdk4 + / + and Cdk4 - / - mice ( n = 15 - 24 slices according hypothalamic nucleus and genotype ) .

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A-D Confocal illustrations of frontal hypothalamic sections from Cdk4+/+ mice (A, C, N=3) and Cdk4-/- mice (B, D, N=3). Low magnification showing nuclei (Hoechst, left pannel) and c-Fos activated-cells (white, right pannel) in the mPOA (A1-2, B1-2) and the ARC, the VMH, the DMH, the LH (C1-2, D1-2). High magnifications corresponding to the frame area in A1-2, B1-2, C2, D2 and showing c-Fos-IR cells (A3-4, B3-4, C3-6, D3-6). Scale bars (A1-2, B1-2, C1-2, D1-2): 200μm, (A3-4, B3-4, C3-4, D3-4): 50μm. E Quantification of c-Fos IR cells in brain sections of Cdk4+/+ and Cdk4-/- mice (n=15-24 slices according hypothalamic nucleus and genotype).


A , B . A fraction of OTULIN is localized at mitochondria where it suppresses M1 - linked ubiquitination . HEK293T cells were transfected with control or OTULIN siRNA . Cells were harvested 72 h ( A ) or 48 h ( B ) after transfection or 15 min after TNF treatment ( 25 ng / ml ) . Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient . 38 % ( A ) or 20 % ( B ) of the mitochondrial fractions and 2 % ( A , B ) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated . For the detection of M1 - linked ubiquitin chains , either the 1E3 ( A ) or the 1F11 / 3F5 / Y102L ( B ) antibody was used .

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A, B. A fraction of OTULIN is localized at mitochondria where it suppresses M1-linked ubiquitination. HEK293T cells were transfected with control or OTULIN siRNA. Cells were harvested 72 h (A) or 48 h (B) after transfection or 15 min after TNF treatment (25 ng/ml). Mitochondria were isolated by differential centrifugation and purified by ultracentrifugation using an OptiPrepTM density gradient. 38% (A) or 20% (B) of the mitochondrial fractions and 2% (A, B) of whole cell lysates were analyzed by immunoblotting using the antibodies indicated. For the detection of M1-linked ubiquitin chains, either the 1E3 (A) or the 1F11/3F5/Y102L (B) antibody was used.


C . TNF induces M1 - ubiquitination at mitochondria in various cell types . The indicated cell types were treated with TNF ( 25 ng / ml , 15 min ) and purified mitochondrial fractions analyzed as described in A .

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C. TNF induces M1-ubiquitination at mitochondria in various cell types. The indicated cell types were treated with TNF (25 ng/ml, 15 min) and purified mitochondrial fractions analyzed as described in A.


D . TNF - induced mitochondrial M1 - ubiquitination does not occur in HOIP - deficient cells . Wildtype ( WT ) and HOIP - KO HAP cells were treated with TNF ( 25 ng / ml , 15 min ) and analyzed as described in A .

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D. TNF-induced mitochondrial M1-ubiquitination does not occur in HOIP-deficient cells. Wildtype (WT) and HOIP-KO HAP cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.


E . Mitochondria and M1 - linked ubiquitin co - localize after TNF treatment . SH - SY5Y cells were treated with TNF ( 25 ng / ml , 15 min ) , fixed , stained with antibodies against HSP60 ( green ) and M1 - ubiquitin ( 1E3 , red ) and analyzed by SR - SIM .

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E. Mitochondria and M1-linked ubiquitin co-localize after TNF treatment. SH-SY5Y cells were treated with TNF (25 ng/ml, 15 min), fixed, stained with antibodies against HSP60 (green) and M1-ubiquitin (1E3, red) and analyzed by SR-SIM.


F . TNF induces recruitment of LUBAC components to mitochondria . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) and analyzed as described in A .

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F. TNF induces recruitment of LUBAC components to mitochondria. HEK293T cells were treated with TNF (25 ng/ml, 15 min) and analyzed as described in A.


G . TNF induces a fast and transient increase in M1 - and K63 - specific ubiquitination at mitochondria . HEK 293T cells were treated with TNF ( 25 ng / ml ) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1 - , K63 - , and K48 - specific ubiquitin antibodies .

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G. TNF induces a fast and transient increase in M1- and K63-specific ubiquitination at mitochondria. HEK 293T cells were treated with TNF (25 ng/ml) for the indicated time and the mitochondrial fractions were analyzed by immunoblotting using M1-, K63-, and K48-specific ubiquitin antibodies.


A , B . PINK1 is stabilized by TNF treatment . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) before harvesting or incubated with OTULIN - specific siRNA for 48 h ( A ) . Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies . Quantification of PINK1 - specific signals normalized to TIM23 is shown in the right panel . Data represent the mean values with standard deviations of 4 independent experiments . * p < 0 . 05 . A two - tailed non - parametric Mann - Whitney U - test was used to analyze statistical significance . In ( A ) , * unprocessed PINK1 ; * * processed PINK1 .

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A, B. PINK1 is stabilized by TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) before harvesting or incubated with OTULIN-specific siRNA for 48 h (A). Purified mitochondrial fractions were analyzed by immunoblotting using the indicated antibodies. Quantification of PINK1-specific signals normalized to TIM23 is shown in the right panel. Data represent the mean values with standard deviations of 4 independent experiments. *p < 0.05. A two-tailed non-parametric Mann-Whitney U-test was used to analyze statistical significance. In (A), *unprocessed PINK1; **processed PINK1.


C . TNF - induced PINK1 stabilization does not induce mitophagy . HeLa cells expressing mt - mKeima were treated with TNF ( 25 ng / ml ) for 30 min or 16 h . As a control , mt - mKeima - expressing Hela cells were treated with antimycin A and oligomycin ( A / O , 10 µM each ) for 1 h . The analysis was done by flow cytometry gating of lysosomal and neutral mt - mKeima .

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C. TNF-induced PINK1 stabilization does not induce mitophagy. HeLa cells expressing mt-mKeima were treated with TNF (25 ng/ml) for 30 min or 16 h. As a control, mt-mKeima -expressing Hela cells were treated with antimycin A and oligomycin (A/O, 10 µM each) for 1 h. The analysis was done by flow cytometry gating of lysosomal and neutral mt-mKeima.


D . Catalytically active HOIP ubiquitinates overexpressed PINK1 . HEK293T cells were transfected with the plasmids indicated . After 24 h , the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin .

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D. Catalytically active HOIP ubiquitinates overexpressed PINK1. HEK293T cells were transfected with the plasmids indicated. After 24 h, the cells were harvested under denaturing conditions and PINK1 was immunoprecipitated via the V5 tag followed by immunoblotting against ubiquitin.


E . Recombinant HOIP ubiquitinates PINK1 in vitro . V5 - tagged - PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1 , UBE2L3 , C - terminal HOIP and ubiquitin for in vitro ubiquitination . The samples were then analyzed by immunoblotting using V5 antibodies .

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E. Recombinant HOIP ubiquitinates PINK1 in vitro. V5-tagged-PINK1 immunoprecipitated from transiently transfected HEK293T cells via the V5 tag was incubated with recombinant mouse Ube1, UBE2L3, C-terminal HOIP and ubiquitin for in vitro ubiquitination. The samples were then analyzed by immunoblotting using V5 antibodies.


F . Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) , then endogenous PINK1 was immunoprecipitated from mitochondrial fractions . Cells mildly overexpressing PINK1 - V5 were also included ( lanes 4 , 5 , 8 ) , to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1 . As a control for the presence of M1 - linked ubiquitin chains , immunoprecipitated PINK1 was treated with recombinant OTULIN . As controls for the specificity of the immunoprecipitation , beads only ( lanes 6 - 8 ) and beads plus IgG ( lane 9 ) were included . The samples were analyzed by immunoblotting using antibodies against PINK1 , M1 - ubiquitin , and phosphorylated ubiquitin . For immunoprecipitation of overexpressed PINK1 , only 50 % of cells were used in comparison to the immunoprecipitation of endogenous PINK1 .

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F. Endogenous PINK1 is modified with M1 ubiquitin chains after TNF treatment. HEK293T cells were treated with TNF (25 ng/ml, 15 min), then endogenous PINK1 was immunoprecipitated from mitochondrial fractions. Cells mildly overexpressing PINK1-V5 were also included (lanes 4, 5, 8), to make sure that the immunoreactive bands seen for endogenous PINK1 indeed correspond to PINK1. As a control for the presence of M1-linked ubiquitin chains, immunoprecipitated PINK1 was treated with recombinant OTULIN. As controls for the specificity of the immunoprecipitation, beads only (lanes 6 - 8) and beads plus IgG (lane 9) were included. The samples were analyzed by immunoblotting using antibodies against PINK1, M1-ubiquitin, and phosphorylated ubiquitin. For immunoprecipitation of overexpressed PINK1, only 50% of cells were used in comparison to the immunoprecipitation of endogenous PINK1.


A . PINK1 antagonizes OTULIN activity in cells . HEK293T cells expressing HOIP and HOIL - 1L , were transfected with PINK1 , WT OTULIN or the inactive OTULIN mutant W96A , as indicated . The cells were lysed 24 h later under denaturing conditions , and lysates were subjected to affinity purification using the Strep - tagged UBAN domain of NEMO to enrich proteins modified with M1 - linked ubiquitin . Proteins affinity - purified by Strep - Tactin beads were immunoblotted against ubiquitin .

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A. PINK1 antagonizes OTULIN activity in cells. HEK293T cells expressing HOIP and HOIL-1L, were transfected with PINK1, WT OTULIN or the inactive OTULIN mutant W96A, as indicated. The cells were lysed 24 h later under denaturing conditions, and lysates were subjected to affinity purification using the Strep-tagged UBAN domain of NEMO to enrich proteins modified with M1-linked ubiquitin. Proteins affinity-purified by Strep-Tactin beads were immunoblotted against ubiquitin.


B . Catalytically active PINK1 increases p - S65 - ubiquitin at mitochondria . HEK293T cells were transfected with wildtype PINK1 or kinase - dead ( K / D ) PINK1 . 48 h after transfection , the cells were treated with TNF ( 25 ng / ml , 15 min ) and lysed . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p - S65 - ubiquitin , PINK1 , and VDAC ( loading control ) .

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B. Catalytically active PINK1 increases p-S65-ubiquitin at mitochondria. HEK293T cells were transfected with wildtype PINK1 or kinase-dead (K/D) PINK1. 48 h after transfection, the cells were treated with TNF (25 ng/ml, 15 min) and lysed. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against p-S65-ubiquitin, PINK1, and VDAC (loading control).


C . The TNF - induced increase in p - S65 - ubiquitin is abolished in PINK1 - deficient cells . HEK293T cells were transfected with control or PINK1 - specific siRNA and treated with TNF ( 25 ng / ml , 15 min ) or CCCP ( 10 µM , 90 min ) 48 h after transfection . The whole cell lysates were analyzed by immunoblotting using the indicated antibodies .

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C. The TNF-induced increase in p-S65-ubiquitin is abolished in PINK1-deficient cells. HEK293T cells were transfected with control or PINK1-specific siRNA and treated with TNF (25 ng/ml, 15 min) or CCCP (10 µM, 90 min) 48 h after transfection. The whole cell lysates were analyzed by immunoblotting using the indicated antibodies.


D . TNF - induced M1 ubiquitination is reduced in PINK1 - deficient cells . WT and PINK1 - KO MEFs were treated with TNF ( 25 ng / ml , 15 min ) and then harvested . Purified mitochondrial fractions were subjected to affinity purification using the Strep - tagged UBAN domain , as described in A , followed by immunoblotting using M1 - ubiquitin - specifc antibodies , Strep - Tactin conjugated to horse radish peroxidase ( to control the UBAN pulldown efficiency ) , and TIM23 ( input control ) . Quantification of the M1 - ubiquitin - positive signal intensities is shown in the lower panel . Data represent the mean values with standard deviations of 3 independent experiments ; n . s . not significant , * * * p < 0 . 001 . A two - way ANOVA test was used to analyze statistical significance .

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D. TNF-induced M1 ubiquitination is reduced in PINK1-deficient cells. WT and PINK1-KO MEFs were treated with TNF (25 ng/ml, 15 min) and then harvested. Purified mitochondrial fractions were subjected to affinity purification using the Strep-tagged UBAN domain, as described in A, followed by immunoblotting using M1-ubiquitin-specifc antibodies, Strep-Tactin conjugated to horse radish peroxidase (to control the UBAN pulldown efficiency), and TIM23 (input control). Quantification of the M1-ubiquitin-positive signal intensities is shown in the lower panel. Data represent the mean values with standard deviations of 3 independent experiments; n.s. not significant, ***p < 0.001. A two-way ANOVA test was used to analyze statistical significance.


A . STS - induced mitochondrial Bax recruitment is reduced by TNF . HeLa cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) and then harvested . Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1 - ubiquitin . The input was immunoblotted for TIM23 ( upper panel ) . Bax - specific signal intensities were quantified and normalized to TIM23 - specific signals ( lower panel ) .

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A. STS-induced mitochondrial Bax recruitment is reduced by TNF. HeLa cells were treated with STS (1 μM, 1 h) with or without a 15 min pretreatment with TNF (25 ng/ml) and then harvested. Purified mitochondrial fractions were analyzed by immunoblotting using antibodies against Bax and M1-ubiquitin. The input was immunoblotted for TIM23 (upper panel). Bax-specific signal intensities were quantified and normalized to TIM23-specific signals (lower panel).


B . STS - induced cytochrome C release is decreased by TNF . HeLa cells were treated as described in A . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . GAPDH was used as a reference . Quantification of 5 biological replicates is shown in the lower panel . Signal intensities were quantified and normalized to that of GAPDH . * p < 0 . 05 . Kolmogorow - Smirnov normality test , paired t - test , two - tailed , n = 5 .

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B. STS-induced cytochrome C release is decreased by TNF. HeLa cells were treated as described in A. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. GAPDH was used as a reference. Quantification of 5 biological replicates is shown in the lower panel. Signal intensities were quantified and normalized to that of GAPDH. *p < 0.05. Kolmogorow-Smirnov normality test, paired t-test, two-tailed, n=5.


C , D . TNF treatment prevents damage of mitochondrial cristae under pro - apoptotic conditions . C . SH - SY5Y cells were treated as described in A , fixed , embedded and the mitochondrial ultrastructure was imaged by electron microscopy . Scale bar : 400 nm . D . Cristae abundance ( average cristae number per mitochondrial area ) and cristae length ( average cristae length per mitochondrium ) were analyzed by Imaris 9 . 8 . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 01 . Cristae abundance : Shapiro - Wilk normality test , One - way ANOVA followed by Tukey ' s multiple comparison test , n = 27 . Cristae length : Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 24 - 27 . Bars represent mean ±

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C, D. TNF treatment prevents damage of mitochondrial cristae under pro-apoptotic conditions. C. SH-SY5Y cells were treated as described in A, fixed, embedded and the mitochondrial ultrastructure was imaged by electron microscopy. Scale bar: 400 nm. D. Cristae abundance (average cristae number per mitochondrial area) and cristae length (average cristae length per mitochondrium) were analyzed by Imaris 9.8. *p < 0.05, **p < 0.01, ***p < 0.01. Cristae abundance: Shapiro-Wilk normality test, One-way ANOVA followed by Tukey's multiple comparison test, n = 27. Cristae length: Kruskal-Wallis test followed by Dunn's multiple comparison test, n=24-27. Bars represent mean ± SEM


E . The fast anti - apoptotic effect of TNF is not affected by the NF - κΒ inhibitor IκΒα . SH - SY5Y cells were transiently transfected with the NF - κΒ super - repressor IᴋBα - 2S or luciferase as a control . 24 h later , the cells were treated with STS ( 5 µM , 2 h ) with or without a 15 min pretreatment with TNF ( 25 ng / ml ) . Cells were fixed and stained by antibodies against active caspase - 3 . Signal intensities were quantified by immunocytochemistry and fluorescence microscopy . Expression of IᴋBα - 2S was tested by immunoblotting using antibodies against the HA tag . Data represent the standard deviation of three independent experiments ; at least 300 cells were counted per experiment . * p < 0 . 05 , * * p < 0 . 01 . Student ' s t - test , two - tailed , n = 3 .

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E. The fast anti-apoptotic effect of TNF is not affected by the NF-κΒ inhibitor IκΒα. SH-SY5Y cells were transiently transfected with the NF-κΒ super-repressor IᴋBα-2S or luciferase as a control. 24 h later, the cells were treated with STS (5 µM, 2 h) with or without a 15 min pretreatment with TNF (25 ng/ml). Cells were fixed and stained by antibodies against active caspase-3. Signal intensities were quantified by immunocytochemistry and fluorescence microscopy. Expression of IᴋBα-2S was tested by immunoblotting using antibodies against the HA tag. Data represent the standard deviation of three independent experiments; at least 300 cells were counted per experiment. *p < 0.05, **p < 0.01. Student's t-test, two-tailed, n=3.


F . The protective effect of TNF is dependent on HOIP expression . HeLa cells were transiently transfected with control or HOIP siRNA . 48 h after transfection , cells were treated with STS ( 1 μM , 1 h ) with or without a 15 min TNF pretreatment ( 25 ng / ml ) and then harvested . The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies . HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP . GAPDH and actin were immunoblotted as input controls .

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F. The protective effect of TNF is dependent on HOIP expression. HeLa cells were transiently transfected with control or HOIP siRNA. 48 h after transfection, cells were treated with STS (1 μM, 1 h) with or without a 15 min TNF pretreatment (25 ng/ml) and then harvested. The cytosolic fractions were analyzed by immunoblotting using cytochrome c antibodies. HOIP silencing efficiency was analyzed in whole cell lysates using antibodies against HOIP. GAPDH and actin were immunoblotted as input controls.


G . The protective effect of TNF is dependent on PINK1 expression . HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F . The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies . GAPDH was immunoblotted as input control .

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G. The protective effect of TNF is dependent on PINK1 expression. HeLa cells were transiently transfected with control or PINK1 siRNA and treated as described in F. The cytosolic fractions were analyzed by immunoblotting using cytochrome c and PINK1 antibodies. GAPDH was immunoblotted as input control.


A . NF - κB signaling components are recruited to mitochondria upon TNF treatment . A . HEK293T cells were treated with TNF ( 25 ng / ml , 15 min ) , harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated ; p - p65 : phospho - S536 - p65 ; p - TBK1 : phospho - S172 - TBK1 .

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A. NF-κB signaling components are recruited to mitochondria upon TNF treatment. A. HEK293T cells were treated with TNF (25 ng/ml, 15 min), harvested and purified mitochondrial fractions were analyzed by immunoblotting using the antibodies indicated; p-p65: phospho-S536-p65; p-TBK1: phospho-S172-TBK1.


B . Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment . Representative immunofluorescence images of SH - SY5Y cells using the proximity ligation assay ( PLA ) between phospho - S536 - p65 ( rabbit ) and TOM20 ( mouse ) couple antibodies . One set of cells were treated with TNF ( 25 ng / ml , 15 min ) before fixation . Nuclei were stained with DAPI , mitochondria were stained with MitoTrackerTM Red CMXRos ( red ) , and the PLA amplification reaction was visualized by green foci . As a positive control , fixed cells were incubated with primary TOM20 ( mouse ) and TOM70 ( rabbit ) antibodies and subjected to the PLA assay . As negative controls , fixed cells were incubated with either TOM20 or p - p65 antibodies prior to the PLA assay .

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B. Phosphorylated p65 is in close proximity to TOM20 in response to TNF treatment. Representative immunofluorescence images of SH-SY5Y cells using the proximity ligation assay (PLA) between phospho-S536-p65 (rabbit) and TOM20 (mouse) couple antibodies. One set of cells were treated with TNF (25 ng/ml, 15 min) before fixation. Nuclei were stained with DAPI, mitochondria were stained with MitoTrackerTM Red CMXRos (red), and the PLA amplification reaction was visualized by green foci. As a positive control, fixed cells were incubated with primary TOM20 (mouse) and TOM70 (rabbit) antibodies and subjected to the PLA assay. As negative controls, fixed cells were incubated with either TOM20 or p-p65 antibodies prior to the PLA assay.


C . Quantification of the phospho - S536 - p65 / TOM20 PLA foci per cells . Data represent the number of PLA foci per cell ( mean ± SD ) ; at least 84 cells in total were analyzed per condition . * * * * p < 0 . 0001 . A two - tailed non - parametric Mann - Whitney test was used to analyze statistical significance .

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C. Quantification of the phospho-S536-p65/TOM20 PLA foci per cells. Data represent the number of PLA foci per cell (mean ±SD); at least 84 cells in total were analyzed per condition. ****p < 0.0001. A two-tailed non-parametric Mann-Whitney test was used to analyze statistical significance.


A . Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression . SH - SY5Y cells were transfected with control or PINK1 - specific siRNAs . For rescue experiments , cells were co - transfected with wildtype PINK1 , PINK1Δ77 , or kinase - dead PINK1 - K / D . Two days after transfection the cells were treated with TNF ( 25 ng / ml , 25 min ) , fixed and stained with p65 antibodies . The fraction of cells showing nuclear translocation of p65 was determined for each condition . Data represent the mean ± SEM of 3 independent experiments . The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition .

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A. Nuclear translocation of p65 is impaired in cells silenced for PINK1 expression. SH-SY5Y cells were transfected with control or PINK1-specific siRNAs. For rescue experiments, cells were co-transfected with wildtype PINK1, PINK1Δ77, or kinase-dead PINK1-K/D. Two days after transfection the cells were treated with TNF (25 ng/ml, 25 min), fixed and stained with p65 antibodies. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3 independent experiments. The experiment was performed in triplicates and more than 300 cells were quantified per experiment and condition.


B . PINK1 phosphorylates ubiquitinated NEMO . HEK293T cells were co - transfected with V5 - tagged wildtype PINK1 or kinase - dead PINK1 - K / D and HA - tagged NEMO . One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using p - S65 - ubiquitin antibodies . The input was immunoblotted for NEMO , PINK1 , p - S65 - ubiquitin , and GAPDH .

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B. PINK1 phosphorylates ubiquitinated NEMO. HEK293T cells were co-transfected with V5-tagged wildtype PINK1 or kinase-dead PINK1-K/D and HA-tagged NEMO. One day after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using p-S65-ubiquitin antibodies. The input was immunoblotted for NEMO, PINK1, p-S65-ubiquitin, and GAPDH.


C . Linear ubiquitination of NEMO is reduced in PINK1 - deficient cells . HEK293T cells were co - transfected with HA - NEMO and control or PINK1 - specific siRNAs . 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA . Precipitated proteins were then detected by immunoblotting using M1 - ubiquitin antibodies . The input was immunoblotted for NEMO and actin . PINK1 silencing efficiency was determined by real - time RT - PCR . Bars represent mean ± SD with three technical replicates .

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C. Linear ubiquitination of NEMO is reduced in PINK1-deficient cells. HEK293T cells were co-transfected with HA-NEMO and control or PINK1-specific siRNAs. 48 h after transfection the cells were lysed and subjected to immunoprecipitation using antibodies against HA. Precipitated proteins were then detected by immunoblotting using M1-ubiquitin antibodies. The input was immunoblotted for NEMO and actin. PINK1 silencing efficiency was determined by real-time RT-PCR. Bars represent mean± SD with three technical replicates.


A . TNF induces the movement of peripheral mitochondria towards the nucleus . Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF ( 25 ng / ml ) . The motility of mitochondria was analyzed using Imaris 9 . 8 spot function . The graphical representation of the top 25 % peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment . Exemplified dataset shows the median distance with the upper and lower quartile ( shaded area ) of n = 105 - 187 mitochondrial spots per time point .

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A. TNF induces the movement of peripheral mitochondria towards the nucleus. Primary macrophages were stained by MitotrackerTM Green to visualize mitochondria and by Hoechst 33342 to visualize the nucleus and monitored every 30 s for 15 min after treatment with TNF (25 ng/ml). The motility of mitochondria was analyzed using Imaris 9.8 spot function. The graphical representation of the top 25% peripheral mitochondrial indicates a decrease of the average mitochondrial distance to the nucleus after TNF treatment. Exemplified dataset shows the median distance with the upper and lower quartile (shaded area) of n=105-187 mitochondrial spots per time point.


B . Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1 / 2 expression . HeLa cells were transfected with control or Miro1 and Miro2 siRNAs . 48 h later , cells were treated with TNF ( 25 ng / ml , 15 min ) , fixed and stained using antibodies against p65 and tubulin and DAPI . Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4 . 2 . 1 . Quantification is based on 3 biological replicates . Kruskal - Wallis test followed by Dunn ' s multiple comparison test , n = 79 - 157 , * p < 0 . 05 .

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B. Nuclear p65 translocation upon TNF treatment is reduced cells silenced for Miro1/2 expression. HeLa cells were transfected with control or Miro1 and Miro2 siRNAs. 48 h later, cells were treated with TNF (25 ng/ml, 15 min), fixed and stained using antibodies against p65 and tubulin and DAPI. Images were segmented in a cytoplasmic and nuclear compartment and the fluorescence signal intensity ratio of p65 was measured using CellProfiler 4.2.1. Quantification is based on 3 biological replicates. Kruskal-Wallis test followed by Dunn's multiple comparison test, n=79-157, *p < 0.05.


E , F , G . TNF increases mitochondria - nucleus contact sites . Representative immunofluorescence images ( E , F ) and quantification ( G ) of mitochondria - nucleus contact sites in HeLa cells transfected with the SPLICSNU - MT and treated with 25 ng / ml TNF or PBS for 15 min . TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation . Nuclei were stained with Hoechst33342 ( ThermoFisher , 1 μg / ml ) . Unpaired two - tailed t - test , n = 43 ( control ) or 51 ( TNF ) ,

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E, F, G. TNF increases mitochondria-nucleus contact sites. Representative immunofluorescence images (E, F) and quantification (G) of mitochondria-nucleus contact sites in HeLa cells transfected with the SPLICSNU-MT and treated with 25 ng/ml TNF or PBS for 15 min. TOM20 antibodies were used to stain mitochondria and p65 antibodies to assess p65 nuclear translocation. Nuclei were stained with Hoechst33342 (ThermoFisher, 1 μg/ml). Unpaired two-tailed t-test, n=43 (control) or 51 (TNF),


H , I , J . TNF increases mitochondria - nucleus contact sites in a HOIP - dependent manner . Representative immunofluorescence images ( H , I ) and quantification ( J ) of mitochondria - nucleus contact sites in control or HOIP - silenced ( HOIP siRNA ) HeLa cells transfected with the SPLICSNU - MT probe treated with 25 ng / ml TNF or PBS for 15 minutes . Mitochondria and nuclei were stained as described in E and F .

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H, I, J. TNF increases mitochondria-nucleus contact sites in a HOIP-dependent manner. Representative immunofluorescence images (H, I) and quantification (J) of mitochondria-nucleus contact sites in control or HOIP-silenced (HOIP siRNA) HeLa cells transfected with the SPLICSNU-MT probe treated with 25 ng/ml TNF or PBS for 15 minutes. Mitochondria and nuclei were stained as described in E and F.


( C ) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 ( ACT2 ) as a loading control .

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(C) Northern analysis of EVD RNA isoforms using a probe for the GAG region or for ACTIN2 (ACT2) as a loading control.


( D ) qPCR quantification of shGAG and flGAG - POL normalized to ACT2 and to GLYCERALDEHYDE - 3 - PHOSPHATE DEHYDROGENASE C SUBUNIT ( GAPC ) levels . qPCR was performed on n = 3 biological replicates ; bars : standard error . ( * * ) = p - value < 0 . 01 ( two - sided t - test between indicated values ) .

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(D) qPCR quantification of shGAG and flGAG-POL normalized to ACT2 and to GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE C SUBUNIT (GAPC) levels. qPCR was performed on n=3 biological replicates; bars: standard error. (**) = p-value < 0.01 (two-sided t-test between indicated values).


( E ) Western analysis of GAG and RDR6 with Coomassie ( coom . ) staining as a loading control . Arrow indicates cognate RDR6 protein band .

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(E) Western analysis of GAG and RDR6 with Coomassie (coom.) staining as a loading control. Arrow indicates cognate RDR6 protein band.


( F - G ) sRNA - seq profiles from EVD de - repressed in the ddm1 ( F ) or ddm1 dcl1 ( G ) backgrounds . Different siRNA size categories are stacked . Nomenclature as in ( B ) .

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(F-G) sRNA-seq profiles from EVD de-repressed in the ddm1 (F) or ddm1 dcl1 (G) backgrounds. Different siRNA size categories are stacked. Nomenclature as in (B).


( B ) Expression levels of shGFP ( spliced ) and GFP - EVDint / ter - GUS ( unspliced ) transcripts , relative to ACT2 and AT4G26410 ( RHIP1 ) , in the WT or rdr6 background . qPCR was performed on three biological replicates and error bars represent the standard error on . ( * ) = p - value < 0 . 05 ( two - sided t - test against corresponding controls ) .

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(B) Expression levels of shGFP (spliced) and GFP-EVDint/ter-GUS (unspliced) transcripts, relative to ACT2 and AT4G26410 (RHIP1), in the WT or rdr6 background. qPCR was performed on three biological replicates and error bars represent the standard error on. (*) = p-value < 0.05 (two-sided t-test against corresponding controls).


( C ) sRNA - seq profile mapped on the genomic 35S : GFP - EVDint / ter - GUS locus . ( RPM ) Reads per million . Positions indicated in nucleotides ( nt ) from the start of the 35S sequence . Dashed vertical lines : shGFP and GFP - GUS 3 ' ends .

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(C) sRNA-seq profile mapped on the genomic 35S:GFP-EVDint/ter-GUS locus. (RPM) Reads per million. Positions indicated in nucleotides (nt) from the start of the 35S sequence. Dashed vertical lines: shGFP and GFP-GUS 3' ends.


( D ) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2 . qPCR was performed on three biological replicates and error bars represent the standard error . ( ns . ) = non - significant , ( * ) = p - value < 0 . 05 , ( * * ) = p - value < 0 . 01 , ( two - sided t - test between indicated samples / targets ) .

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(D) Relative expression levels of spliced and unspliced transcripts in the three EVD constructs relative to ACT2. qPCR was performed on three biological replicates and error bars represent the standard error. (ns.) = non-significant, (*) = p-value < 0.05, (**) = p-value < 0.01, (two-sided t-test between indicated samples/targets).


( E ) High and low molecular - weight RNA analysis of EVD GAG ( GAG - ex1 ) and EVD intron ( GAG - in ) in two independent T1 bulks from each indicated line . The filled arrows on the right hand - side or with an asterisk on the blots correspond to the transcripts depicted in ( A - C ) . ACT2 : loading control for mRNAs ; tasiR255 , miR173 and U6 : loading controls for sRNAs . Hybridizations for GAG - ex2 and POL probes are found in Supp . Fig . 5A .

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(E) High and low molecular-weight RNA analysis of EVD GAG (GAG-ex1) and EVD intron (GAG-in) in two independent T1 bulks from each indicated line. The filled arrows on the right hand-side or with an asterisk on the blots correspond to the transcripts depicted in (A-C). ACT2: loading control for mRNAs; tasiR255, miR173 and U6: loading controls for sRNAs. Hybridizations for GAG-ex2 and POL probes are found in Supp. Fig.5A.


( B ) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in ( A ) .

(B) Boxplots of RNA expression levels of TEs in ddm1 from the quartiles in (A).


( A ) Comparison of RNA isoforms and sRNA patterns generated by 35S : GFP - GUS and 35S : GFP - EVDint / ter - GUS . High and low molecular - weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background . mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig . 2A . EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs , respectively .

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(A) Comparison of RNA isoforms and sRNA patterns generated by 35S:GFP-GUS and 35S:GFP-EVDint/ter-GUS. High and low molecular-weight RNA analysis using a GFP or GUS probe in two independent transgenic lines from each construct in the WT or rdr6 background. mRNA isoforms are indicated with arrows and correspond to the transcripts depicted in Fig.2A. EtBr staining of the agarose gel and miR171 probe serve as loading control for mRNAs and sRNAs, respectively.


( B ) . Western blot analysis of the translation products from GFP and GFP - GUS transcripts . Coomassie ( coom . ) staining as a loading control . Black arrow : GFP - GUS fusion protein ; white arrow : GFP protein .

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(B). Western blot analysis of the translation products from GFP and GFP-GUS transcripts. Coomassie (coom.) staining as a loading control. Black arrow: GFP-GUS fusion protein; white arrow: GFP protein.


( C ) Nucleo - cytosolic distribution of 35S : EVD and 35S : GFP - EVDint / ter - GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR . RNA extracted from Total , nuclear ( Nucl ) and cytoplasmic ( Cyto ) fractions was reverse transcribed with random hexamers and oligo ( dT ) . snoRNA U5 is shown as a nuclear - only RNA control . ( D ) Same as in ( C ) but using exclusively oligo ( dT ) to reverse transcribe poly ( A ) + RNAs .

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(C) Nucleo-cytosolic distribution of 35S:EVD and 35S:GFP-EVDint/ter-GUS RNA isoforms in rdr6 relative to that of ACT2 analyzed by qPCR. RNA extracted from Total, nuclear (Nucl) and cytoplasmic (Cyto) fractions was reverse transcribed with random hexamers and oligo(dT). snoRNA U5 is shown as a nuclear-only RNA control. (D) Same as in (C) but using exclusively oligo(dT) to reverse transcribe poly(A)+ RNAs.


( B ) RIBO - seq coverage profiles from 35S : EVD in WT or rdr6 . RPM : Reads per million . ( C ) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P - sites to calculate codon coverage . The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence .

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(B) RIBO-seq coverage profiles from 35S:EVD in WT or rdr6. RPM: Reads per million. (C) Ribosomal footprints on shGAG in rdr6 displaying codon occupancy at P-sites to calculate codon coverage. The coverage observed at each codon position was divided by the expected mean coverage along the entire GAG coding sequence.


C . Autoradiograph of hTrmt13 - RNA complexes in the MDA - MB - 231 cells immunoprecipitated with an antibody against hTrmt13 ( right lane ) and IgG as a negative control ( left lane ) . * RNA residing in the regions between marked lines was recovered for sequencing .

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C. Autoradiograph of hTrmt13-RNA complexes in the MDA-MB-231 cells immunoprecipitated with an antibody against hTrmt13 (right lane) and IgG as a negative control (left lane). * RNA residing in the regions between marked lines was recovered for sequencing.


H . Mass chromatograms of the nucleosides , Um ( Q1 / Q3 = 259 . 1 / 113 . 1 ) , Cm ( Q1 / Q3 = 258 . 1 / 112 . 1 ) , and G ( Q1 / Q3 = 284 . 1 / 152 . 2 ) of HctRNAGly ( GCC ) , HctRNAGly ( CCC ) , HctRNAGly ( UCC ) , and HctRNAPros isolated from WT and hTrmt13 KO cells . Um , 2 ′ - O - methyluridine , Cm , 2 ′ - O - methylcytidine . Q1 / Q3 : the mass of the precursor ion and the mass of the product ion . These endogenous tRNAs were purified separately from total cellular RNAs using sequence - specific probes .

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H. Mass chromatograms of the nucleosides, Um (Q1/Q3 = 259.1/113.1), Cm (Q1/Q3 = 258.1/112.1), and G (Q1/Q3 = 284.1/152.2) of HctRNAGly(GCC), HctRNAGly(CCC), HctRNAGly(UCC), and HctRNAPros isolated from WT and hTrmt13 KO cells. Um, 2′-O-methyluridine, Cm, 2′-O-methylcytidine. Q1/Q3: the mass of the precursor ion and the mass of the product ion. These endogenous tRNAs were purified separately from total cellular RNAs using sequence-specific probes.


I . Quantification of the Um / G and Cm / G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells . Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch . Statistical analysis was performed using t - tests . Error bars represent mean ± SD for three independent experiments .

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I. Quantification of the Um/G and Cm/G ratios in endogenous tRNAs isolated from WT and hTrmt13 KO cells. Quantification was performed by comparison with the standard curve obtained from pure nucleoside standards running in the same batch. Statistical analysis was performed using t-tests. Error bars represent mean ± SD for three independent experiments.


A . De novo protein synthesis measured by puromycin incorporation in shSCR , hTrmt13 knockdown cells ( sh - 1 and sh - 2 ) , and sh - 1 with stable expression of wild type hTrmt13 ( sh - 1 + hTrmt13 ) or enzymatically - inactive hTrmt13 ( sh - 1 + E463A ) . Puromycin - labeled proteins were detected by immunoblotting . Representative images ( left ) and statistical analysis ( right ) are shown . Knockdown and overexpression efficiency of hTrmt13 in MDA - MB - 231 verified by Western blots . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

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A. De novo protein synthesis measured by puromycin incorporation in shSCR, hTrmt13 knockdown cells (sh-1 and sh-2), and sh-1 with stable expression of wild type hTrmt13 (sh-1+hTrmt13) or enzymatically-inactive hTrmt13 (sh-1+E463A). Puromycin-labeled proteins were detected by immunoblotting. Representative images (left) and statistical analysis (right) are shown. Knockdown and overexpression efficiency of hTrmt13 in MDA-MB-231 verified by Western blots. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.


C . The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot . The samples were also deacylated ( DA ) by incubating with alkaline buffer ( pH 9 . 0 ) to obtain fully uncharged tRNA control .

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C. The in vivo aminoacylation level of hTrmt13 substrate tRNAs assayed by Northern blot. The samples were also deacylated (DA) by incubating with alkaline buffer (pH 9.0) to obtain fully uncharged tRNA control.


D . RT - qPCR using a stem - loop primer to quantify the tRFs levels in shSCR , sh - 1 , and sh - 2 . Data information : Statistical analysis was performed using t - tests , and error bars represent mean ± SD for three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. RT-qPCR using a stem-loop primer to quantify the tRFs levels in shSCR, sh-1, and sh-2. Data information: Statistical analysis was performed using t-tests, and error bars represent mean ± SD for three independent experiments.