figf3 ( a ) Rates of degradation of long - lived proteins in mouse fibroblasts untreated ( None ) or treated with 40 μM ATRA and maintained in the presence or absence of serum . Values are expressed as a percentage of proteolysis ; n = 3 . ( b ) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h ; n = 3 . ( c ) Immunoblot for LC3 - II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors ( PI ) . Top , representative immunoblot . Actin is shown as loading control . Bottom , ratio of amount of LC3 - II in cells treated with PI to that in untreated cells . Values are expressed as fold change in value relative to untreated cells ; n = 4 . ' I ' and ' II ' designate different forms of LC3 , as described in the text . ( d , e ) Autophagic flux in untreated and ATRA - treated cells expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged - channel images . Arrows indicate autolysosomes ( red ) . Scale bar , 2 μm . Shown in e are the number of autophagosomes ( left ) and percentage of autolysosomes ( right ) after quantification of > 50 cells . AV , autophagic vacuoles . ( d , e ) Autophagic flux in untreated and ATRA - treated cells expressing mCherry - GFP - LC3 - II and maintained in the presence or absence of serum . Shown in d are representative merged - channel images . Arrows indicate autolysosomes ( red ) . Scale bar , 2 μm . Shown in e are the number of autophagosomes ( left ) and percentage of autolysosomes ( right ) after quantification of > 50 cells . AV , autophagic vacuoles . ( f ) Mouse fibroblasts expressing the KFERQ - mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum . Left , representative images . Nuclei are labeled with DAPI . Scale bars , 10 μm . Graph shows quantification of the number of puncta per cell in > 50 cells . All values in a - c , e and f are mean ± s . e . m . , and differences with untreated ( marked with asterisk ) or with serum - supplemented cells ( marked with § ) are significant for P 0 . 01 . Full - field fluorescence images and full - length blots are shown in Supplementary Figures 3 and 21 , respectively .

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

figf3(a) Rates of degradation of long-lived proteins in mouse fibroblasts untreated (None) or treated with 40 μM ATRA and maintained in the presence or absence of serum. Values are expressed as a percentage of proteolysis; n = 3.(b) Percentage of lysosomal degradation calculated after treatment with inhibitors of lysosomal proteolysis for 12 h; n = 3.(c) Immunoblot for LC3-II of the same cells as in a and b maintained in the presence or absence of serum and protease inhibitors (PI). Top, representative immunoblot. Actin is shown as loading control. Bottom, ratio of amount of LC3-II in cells treated with PI to that in untreated cells. Values are expressed as fold change in value relative to untreated cells; n = 4. 'I' and 'II' designate different forms of LC3, as described in the text.(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.(d,e) Autophagic flux in untreated and ATRA-treated cells expressing mCherry-GFP-LC3-II and maintained in the presence or absence of serum. Shown in d are representative merged-channel images. Arrows indicate autolysosomes (red). Scale bar, 2 μm. Shown in e are the number of autophagosomes (left) and percentage of autolysosomes (right) after quantification of >50 cells. AV, autophagic vacuoles.(f) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter with or without ATRA and after photoactivation maintained in the presence or absence of serum. Left, representative images. Nuclei are labeled with DAPI. Scale bars, 10 μm. Graph shows quantification of the number of puncta per cell in >50 cells. All values in a-c, e and f are mean ± s.e.m., and differences with untreated (marked with asterisk) or with serum-supplemented cells (marked with §) are significant for P 0.01. Full-field fluorescence images and full-length blots are shown in Supplementary Figures 3 and 21, respectively.


figf5 ( a ) Mouse fibroblasts expressing the KFERQ - mCherry1 photoactivatable reporter without ( None ) or with 20 μM of the indicated compounds imaged 16 h after photoactivation . Insets show higher - magnification images . Nuclei are labeled with DAPI . Scale bars , 10 μm . ( b ) Quantification of the effect of increasing concentrations of GR1 on the same cells . Untreated cells and cells treated with 40 μM ATRA are also shown . Representative images are shown in Supplementary Figure 7 . Graph shows the average number of fluorescent puncta per cell , quantified in > 50 cells . All values are mean ± s . e . m . ( c ) Mouse fibroblasts were cotransfected with the human RARα ( hRARα ) receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units ( RLU ) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h . ( d ) Mouse fibroblasts were cotransfected with the human RARα ( hRARα ) receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h . Values are shown as RLU . ( e ) Mouse fibroblasts were cotransfected with the human RXR receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h . ( f ) Mouse fibroblasts were cotransfected with the human RXR receptor , a relevant reporter luciferase plasmid and the non - retinoid - regulated Renilla reporter to control for transfection . Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h . Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h . Values are shown as RLU . Values in c - f are mean ± s . e . m . ; n = 4 - 6 . ( g ) Left , immunoblot for LC3 - II in cells treated with 20 μM of the retinoid derivatives and protease inhibitors ( PIs ) , as labeled . Actin is shown as loading control , and full - length blots are shown in Supplementary Figure 21 . The amount of LC3 - II in untreated cells , expressed as fold change relative to control ( middle ) , and the increase in LC3 - II after PI treatment ( LC3 - II flux , right ) , expressed as fold change , were calculated from the densitometric quantification of immunoblots . Values are mean ± s . e . m . ; n = 3 .

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figf5(a) Mouse fibroblasts expressing the KFERQ-mCherry1 photoactivatable reporter without (None) or with 20 μM of the indicated compounds imaged 16 h after photoactivation. Insets show higher-magnification images. Nuclei are labeled with DAPI. Scale bars, 10 μm. (b) Quantification of the effect of increasing concentrations of GR1 on the same cells. Untreated cells and cells treated with 40 μM ATRA are also shown. Representative images are shown in Supplementary Figure 7. Graph shows the average number of fluorescent puncta per cell, quantified in >50 cells. All values are mean ± s.e.m.(c) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units (RLU) detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12h .(d) Mouse fibroblasts were cotransfected with the human RARα (hRARα) receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection.Cells transfected as in c were treated with 100 nM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU.(e) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h.(f) Mouse fibroblasts were cotransfected with the human RXR receptor, a relevant reporter luciferase plasmid and the non-retinoid-regulated Renilla reporter to control for transfection. Values show relative luciferase units detected in cells subjected to the indicated concentrations of ATRA or the three retinoid derivatives for 12 h. Cells transfected as in e were treated with 10 μM of ATRA alone or in the presence of the indicated concentrations of the three retinoid derivatives or the antagonist BMS614 for 12 h. Values are shown as RLU. Values in c-f are mean ± s.e.m.; n = 4-6.(g) Left, immunoblot for LC3-II in cells treated with 20 μM of the retinoid derivatives and protease inhibitors (PIs), as labeled. Actin is shown as loading control, and full-length blots are shown in Supplementary Figure 21. The amount of LC3-II in untreated cells, expressed as fold change relative to control (middle), and the increase in LC3-II after PI treatment (LC3-II flux, right), expressed as fold change, were calculated from the densitometric quantification of immunoblots. Values are mean ± s.e.m.; n = 3.


figf6 ( a ) Rates of degradation of long - lived proteins in control mouse fibroblasts or RARα ( − ) or LAMP - 2A ( − ) ( L - 2A ( − ) ) cells left untreated ( None ) or treated with 20 μM of the indicated compounds . Values are expressed as the fold change in proteolytic rate relative to the rate in untreated cells for each group ; n = 3 . ( b - d ) Control mouse fibroblasts or RARα ( − ) , LAMP - 2A ( − ) or LAMP - 2B ( − ) ( L - 2B ( − ) ) cells were transfected with the KFERQ - mCherry1 photoactivatable reporter with or without the indicated compounds ( 20 μM ) . In b are shown the representative fields and high - magnification images ( insets ) for GR1 . Nuclei are labeled with DAPI . Representative fields for GR2 and AR7 are shown in Supplementary Figure 13 . Scale bars , 10 μm . ( c , d ) Average number of fluorescent puncta per cell quantified in > 50 cells in at least four different fields in control cells and RAR ( − ) ( c ) or LAMP - 2B ( − ) ( d ) cells . No puncta were detected in LAMP - 2A ( − ) cells . ( e ) Mouse fibroblasts transfected with the KFERQ - mCherry1 photoactivatable reporter with the indicated concentrations of AR7 or GR1 or with both compounds to reach the same final concentration , as indicated . Top , representative fields and high - magnification images ( insets ) . Nuclei are labeled with DAPI . Bottom , quantification of the number of fluorescent puncta per field in each condition . Scale bars , 10 μm . In a , c , d and e , values are mean ± s . e . m . ; n > 50 cells . Differences with untreated samples ( marked with asterisk ) or between single and combined treatments ( § ) are significant for P 0 . 01 .

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figf6(a) Rates of degradation of long-lived proteins in control mouse fibroblasts or RARα(−) or LAMP-2A(−) (L-2A(−)) cells left untreated (None) or treated with 20 μM of the indicated compounds. Values are expressed as the fold change in proteolytic rate relative to the rate in untreated cells for each group; n = 3.(b-d) Control mouse fibroblasts or RARα(−), LAMP-2A(−) or LAMP-2B(−) (L-2B(−)) cells were transfected with the KFERQ-mCherry1 photoactivatable reporter with or without the indicated compounds (20 μM). In b are shown the representative fields and high-magnification images (insets) for GR1. Nuclei are labeled with DAPI. Representative fields for GR2 and AR7 are shown in Supplementary Figure 13. Scale bars, 10 μm. (c,d) Average number of fluorescent puncta per cell quantified in >50 cells in at least four different fields in control cells and RAR(−) (c) or LAMP-2B(−) (d) cells. No puncta were detected in LAMP-2A(−) cells.(e) Mouse fibroblasts transfected with the KFERQ-mCherry1 photoactivatable reporter with the indicated concentrations of AR7 or GR1 or with both compounds to reach the same final concentration, as indicated. Top, representative fields and high-magnification images (insets). Nuclei are labeled with DAPI. Bottom, quantification of the number of fluorescent puncta per field in each condition. Scale bars, 10 μm. In a, c, d and e, values are mean ± s.e.m.; n > 50 cells. Differences with untreated samples (marked with asterisk) or between single and combined treatments (§) are significant for P 0.01.


figf7 ( a ) Immunoblot for the indicated proteins in homogenates ( Hom ) and lysosomes ( Lys ) isolated from cells untreated ( None ) or treated for 12 h with 20 μM of the indicated compounds . ( b ) mRNA levels of LAMP - 2A in control mouse fibroblasts ( left ) or RARα ( − ) cells ( right ) treated with AR7 or paraquat ( PQ ) as in a ; n = 4 - 5 . Values are presented as fold change in mRNA relative to untreated cells . ( c ) Cellular viability of control ( left ) or LAMP - 2A ( − ) ( right ) fibroblasts exposed to 2 mM or 0 . 5 mM paraquat , respectively , and treated with the indicated compounds for 12 h before or after the paraquat treatment ; n = 3 . ( d ) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α - synuclein and left untreated ( None ) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7 ; n = 3 . In b - d , all values are mean ± s . e . m . Differences with cells that are untreated ( marked with asterisk ) or those treated only with paraquat ( § ) were significant for P 0 . 001 . Full - length blots are shown in Supplementary Figure 21 . ( e ) Immunoblot for α - synuclein ( α - syn ) in the same cells as in d . Top , higher - exposure blot to highlight oligomeric ( oligo ) species . Asterisk denotes nonspecific band . M , monomer ; MW , molecular weight .

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figf7(a) Immunoblot for the indicated proteins in homogenates (Hom) and lysosomes (Lys) isolated from cells untreated (None) or treated for 12 h with 20 μM of the indicated compounds.(b) mRNA levels of LAMP-2A in control mouse fibroblasts (left) or RARα(−) cells (right) treated with AR7 or paraquat (PQ) as in a; n = 4-5. Values are presented as fold change in mRNA relative to untreated cells.(c) Cellular viability of control (left) or LAMP-2A(−) (right) fibroblasts exposed to 2 mM or 0.5 mM paraquat, respectively, and treated with the indicated compounds for 12 h before or after the paraquat treatment; n = 3.(d) Viability of mouse fibroblasts transfected with the indicated concentrations of a plasmid encoding α-synuclein and left untreated (None) or treated with 1 mM paraquat alone or in the presence of 20 μM AR7; n = 3. In b-d, all values are mean ± s.e.m. Differences with cells that are untreated (marked with asterisk) or those treated only with paraquat (§) were significant for P 0.001. Full-length blots are shown in Supplementary Figure 21.(e) Immunoblot for α-synuclein (α-syn) in the same cells as in d. Top, higher-exposure blot to highlight oligomeric (oligo) species. Asterisk denotes nonspecific band. M, monomer; MW, molecular weight.


Figure 1B Dose response curves for viability of parental cells ( in red ) or selected clones ( in black ) upon treatment with NFR for 48h . The green dashed box highlight concentration in the physiologically relevant range . Curves are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone . Bar graph represents the EC50 ( half maximal effective concentration ) of the dose responses . Data are mean ± s . e . m . of 3 independent experiments performed in triplicate . P values were calculated using 1way ANOVA analysis of variance ( P value in red ) followed by Dunnett ' s multiple comparison tests . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 . C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real - time PCR relative to β - actin ( mean and s . e . m . from 3 independent batches of cells are shown ) . P values were calculated using 1way ANOVA analysis of variance followed by Dunnett ' s multiple comparison tests . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level . Tubulin is used as loading control . Histogram represents mean and s . e . m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates ( see also source data ) . P values were calculated using 1way ANOVA analysis of variance followed by Dunnett ' s multiple comparison tests . * * * P value ≤ 0 . 001 .

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Figure 1B Dose response curves for viability of parental cells (in red) or selected clones (in black) upon treatment with NFR for 48h. The green dashed box highlight concentration in the physiologically relevant range. Curves are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 2way ANOVA analysis of variance between parental cells and each individual clone. Bar graph represents the EC50 (half maximal effective concentration) of the dose responses. Data are mean ± s.e.m. of 3 independent experiments performed in triplicate. P values were calculated using 1way ANOVA analysis of variance (P value in red) followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01.C The parental population and the NFR resistant clones were analyzed for eEF2K mRNA expression by real-time PCR relative to β-actin (mean and s.e.m. from 3 independent batches of cells are shown). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.D The parental population and representative NFR resistant clones were analyzed by WB for eFF2K total protein level. Tubulin is used as loading control. Histogram represents mean and s.e.m of relative eEF2K level quantified from WB of 4 different batches of cells collected at different dates (see also source data). P values were calculated using 1way ANOVA analysis of variance followed by Dunnett's multiple comparison tests. *** P value ≤ 0.001.


Figure 2A Immunoblot analysis of eEF2 Thr56 phosphorylation ( P - eEF2 ) in HeLa cells treated for 6h with increasing doses of NFR , and compared with 10 μg / ml tunicamycin ( TM ) , 200 nM rapamycin ( Rapa . ) or 1h starvation ( Starv . ) . Tubulin is used as loading control . B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR , 200 nM rapamycin ( Rapa . ) , 1h starvation ( Starv . ) or 1 mM of the AMPK activator AICAR , were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 ( Thr56 ) . C Three representative NFR - resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells . For treatments , medium was replaced for 6h with medium containing DMSO ( Mock ) , increasing doses of NFR or 10 μg / ml tunicamycin ( TM ) , or for 1h with PBS for starvation ( Starv . ) . Tubulin is used as loading control . D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV - PIs as indicated or the NFR metabolite M8 ( hydroxy - tert - butylamide ) . Molecular structures of the different HIV - PIs used are indicated . Each panel is representative of at least 3 independent experiments .

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Figure 2A Immunoblot analysis of eEF2 Thr56 phosphorylation (P-eEF2) in HeLa cells treated for 6h with increasing doses of NFR, and compared with 10 μg/ml tunicamycin (TM), 200 nM rapamycin (Rapa.) or 1h starvation (Starv.). Tubulin is used as loading control.B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, 200 nM rapamycin (Rapa.), 1h starvation (Starv.) or 1 mM of the AMPK activator AICAR, were analyzed by immunoblot with antibodies directed against total or phosphorylated eEF2 (Thr56).C Three representative NFR-resistant HeLa clones were analyzed by Immunoblot for eEF2K expression level and eEF2 phosphorylation upon NFR and compared to parental HeLa cells. For treatments, medium was replaced for 6h with medium containing DMSO (Mock), increasing doses of NFR or 10 μg/ml tunicamycin (TM), or for 1h with PBS for starvation (Starv.). Tubulin is used as loading control.D Immunoblot analysis of eEF2 Thr56 phosphorylation in HeLa cells treated for 6h with increasing doses of different HIV-PIs as indicated or the NFR metabolite M8 (hydroxy-tert-butylamide). Molecular structures of the different HIV-PIs used are indicated. Each panel is representative of at least 3 independent experiments.


Figure 3B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR , Rapamycin ( Rapa ) , AICAR or starved for indicated time in PBS ( Starv . ) , were analyzed by immunoblot using indicated antibodies . Tubulin is used as loading control . C A potent mTOR inhibition does not impair NFR - mediated eEF2 phosphorylation . WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle ( Mock ) . After 30 minutes , indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2 . Tubulin is used as loading control . D NFR - mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO . AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR , Rapamycin ( Rapa ) , AICAR or starved for 1h in PBS ( Starv . ) , were analyzed by immuoblot with indicated antibodies . * anti - total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells . Tubulin is used as loading control . Each panel is representative of at least 3 independent experiments .

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

Figure 3B eEF2K WT and KO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for indicated time in PBS (Starv.), were analyzed by immunoblot using indicated antibodies. Tubulin is used as loading control.C A potent mTOR inhibition does not impair NFR-mediated eEF2 phosphorylation. WT MEFs were treated with the indicated concentrations of Rapamycin or with vehicle (Mock). After 30 minutes, indicated doses of NFR were added and cells were incubated for additional 6h and analyzed for phosphorylated S6R and eEF2. Tubulin is used as loading control.D NFR-mediated eEF2 phosphorylation is not affected in AMPKα1α2 dKO. AMPKα1α2 WT and dKO MEF treated for 6h with indicated concentration of NFR, Rapamycin (Rapa), AICAR or starved for 1h in PBS (Starv.), were analyzed by immuoblot with indicated antibodies. * anti-total AMPKα antibody give an unspecific band with a slightly higher molecular weight in dKO cells. Tubulin is used as loading control. Each panel is representative of at least 3 independent experiments.


Figure 4A - B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM . A . Quantification of newly synthesized proteins at 0 , 2 , 4 and 6h after 20 μM NFR ( left panels ) or 10 μg / ml TM ( right panels ) treatment in MEF eEF2K + / + and eEF2K - / - . Treated cells were labeled for 15 minutes with 35S - methionine and visualized by SDS PAGE and subsequent autoradiography . Autoradiographies from four different experiments ( see also source data ) were quantified and results show percentage of translation compared to untreated cells . The mean and s . e . m of four independent metabolic labeling experiments are shown . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 obtained using 2way ANOVA analysis of variance ( in red ) followed by Bonferroni posttest ( in black ) . B . eEF2K + / + , eEF2K - / - , eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR , 200 nM rapamycin , or 10 μg / ml TM , or for 30 min with 10 μg / ml CHX . 35S - methionine incorporation was measured by liquid scintillation counting . Data are shown as the percentage of translation compared to untreated cells . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest . C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM . Area under curve for Sub - polysomes ( S ) and Polysomes ( P ) used to calculate the P / S ratio were indicated ( see also raw data provided ) . Bar graph represents ratio normalized to untreated cells . OD254 nm is optical density at 254 nm . Data showed mean ± s . e . m . of P / S ratio calculated from 3 independent experiments . P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest ; * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 . D The ribosome half - transit time in MEFs eEF2K + / + and eEF2K - / - was determined as described in Materials and Methods . Incorporation of 35S - Methionine into total protein within the PMS and PRS was obtained by linear regression analysis . Presented graphs are representative of two ( CHX 1 μg / ml for 30 min and Starvation 30 min in PBS in eEF2K + / + cells ) to four ( NFR 20μM for 6h in eEF2K + / + and eEF2K - / - cells ) independent experiments . Indicated values represent the x displacement measurement ( in time ) between the PMS line at 300sec and the PRS line ( see also raw data provided ) . Histogram represents mean and s . e . m . of the ribosome half transit time from four independent experiments . P values were calculated using two tails unpaired Student ' s T - tests ; * P value ≤ 0 . 05 .

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

Figure 4A-B EEF2K contributes to the decreased translation observed with NFR without impacting translation in presence of TM. A. Quantification of newly synthesized proteins at 0, 2, 4 and 6h after 20 μM NFR (left panels) or 10 μg/ml TM (right panels) treatment in MEF eEF2K +/+ and eEF2K-/-. Treated cells were labeled for 15 minutes with 35S-methionine and visualized by SDS PAGE and subsequent autoradiography. Autoradiographies from four different experiments (see also source data) were quantified and results show percentage of translation compared to untreated cells. The mean and s.e.m of four independent metabolic labeling experiments are shown. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance (in red) followed by Bonferroni posttest (in black). B. eEF2K+/+, eEF2K-/-, eIF2αWT and eIF2αS51A MEFs were treated for 6h with indicated doses of NFR, 200 nM rapamycin, or 10 μg/ml TM, or for 30 min with 10 μg/ml CHX. 35S-methionine incorporation was measured by liquid scintillation counting. Data are shown as the percentage of translation compared to untreated cells. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001 obtained using 2way ANOVA analysis of variance followed by Bonferroni posttest.C Representative polysome profiles of eEF2K WT and KO MEF treated 6h with NFR or TM. Area under curve for Sub-polysomes (S) and Polysomes (P) used to calculate the P/S ratio were indicated (see also raw data provided). Bar graph represents ratio normalized to untreated cells. OD254 nm is optical density at 254 nm. Data showed mean ±s.e.m. of P/S ratio calculated from 3 independent experiments. P values were calculated using 1way ANOVA analysis of variance followed by Bonferroni posttest; *P value ≤ 0.05, ** P value ≤ 0.01.D The ribosome half-transit time in MEFs eEF2K+/+ and eEF2K-/- was determined as described in Materials and Methods. Incorporation of 35S-Methionine into total protein within the PMS and PRS was obtained by linear regression analysis. Presented graphs are representative of two (CHX 1 μg/ml for 30 min and Starvation 30 min in PBS in eEF2K+/+ cells) to four (NFR 20μM for 6h in eEF2K+/+ and eEF2K-/- cells) independent experiments. Indicated values represent the x displacement measurement (in time) between the PMS line at 300sec and the PRS line (see also raw data provided). Histogram represents mean and s.e.m. of the ribosome half transit time from four independent experiments. P values were calculated using two tails unpaired Student's T-tests; *P value ≤ 0.05.


Figure 5A MEF eEF2K WT , KO or KO reconstituted with human eEF2K ( Flag - eEF2K ) were analyzed for cell growth at indicated times . Fold change ( means ± s . e . m of triplicate ) of the cell number just before treatment are shown . Histogram shows percentage of growth inhibition after 72h of 10 μM NFR . P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments . * * * P value ≤ 0 . 001 . B - F Dose response curves for cell viability after 48h NFR measured using MTS assay . Curves and bar graph for EC50 are mean ± s . e . m . of 3 independent experiments performed in triplicate . For curves , p values are 2way ANOVA analysis of variance . For bar graph , p values are two tails unpaired Student ' s T - tests ( B , E ) , or 1way ANOVA with Bonferroni ( C ) or Dunnett ' s multiple comparison posttests ( D and F ) . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . ( B , C ) eEF2K - / - MEFs show a decrease susceptibly to NFR compared to eEF2K + / + controls ( B ) whereas eEF2K reconstitution restores NFR sensitivity ( C ) . B - F Dose response curves for cell viability after 48h NFR measured using MTS assay . Curves and bar graph for EC50 are mean ± s . e . m . of 3 independent experiments performed in triplicate . For curves , p values are 2way ANOVA analysis of variance . For bar graph , p values are two tails unpaired Student ' s T - tests ( B , E ) , or 1way ANOVA with Bonferroni ( C ) or Dunnett ' s multiple comparison posttests ( D and F ) . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . ( D - F ) , HeLa ( D ) , A549 ( E ) and MCF7 ( F ) clones with CRISPR - Cas9 generated eEF2K deficiency ( CrEEF2K ) show a decrease susceptibly to NFR compare to control cells ( CrLuci ) . G NFR - mediated toxicity assessed using AnnexinV / PI staining and FACS analysis after 24h treatment . Histogram shows the percentage of dead cells ( AV + / PI + ) . Data are mean ± s . e . m . of 3 independent experiments . P values are 2way ANOVA with Bonferroni posttest . * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 .

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

Figure 5A MEF eEF2K WT, KO or KO reconstituted with human eEF2K (Flag-eEF2K) were analyzed for cell growth at indicated times. Fold change (means ± s.e.m of triplicate) of the cell number just before treatment are shown. Histogram shows percentage of growth inhibition after 72h of 10 μM NFR. P values are 1way ANOVA with Bonferroni posttest calculated from 3 independent experiments. *** P value ≤ 0.001.B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (B,C) eEF2K -/- MEFs show a decrease susceptibly to NFR compared to eEF2K +/+ controls (B) whereas eEF2K reconstitution restores NFR sensitivity (C).B-F Dose response curves for cell viability after 48h NFR measured using MTS assay. Curves and bar graph for EC50 are mean ± s.e.m. of 3 independent experiments performed in triplicate. For curves, p values are 2way ANOVA analysis of variance. For bar graph, p values are two tails unpaired Student's T-tests (B, E), or 1way ANOVA with Bonferroni (C) or Dunnett's multiple comparison posttests (D and F). *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. (D-F), HeLa (D), A549 (E) and MCF7 (F) clones with CRISPR-Cas9 generated eEF2K deficiency (CrEEF2K) show a decrease susceptibly to NFR compare to control cells (CrLuci).G NFR-mediated toxicity assessed using AnnexinV/PI staining and FACS analysis after 24h treatment. Histogram shows the percentage of dead cells (AV+/PI+). Data are mean ± s.e.m. of 3 independent experiments. P values are 2way ANOVA with Bonferroni posttest. *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001.


Figure 6A Tumor volumes of eEF2K - / - and eEF2K + / + RasV12 engraft implanted subcutaneously in AGR129mice . Treatment with NFR or vehicle was initiated 6 days post - implantation . Data are mean of tumor volume ± s . e . m . ( n = 8 per group ) . P values were calculated using 2way ANOVA followed by Bonferroni posttest comparing WT tumor with vehicle or NFR treatment and KO tumors with vehicle or NFR treatment ; * P value ≤ 0 . 05 , * * P value ≤ 0 . 01 , * * * P value ≤ 0 . 001 . Experiment is representative of 2 performed in same conditions . B Immunoblots for total and phosphorylated eEF2 of tumor engraftment from ( A ) . ( NS , Non - Specific signal . ) C Bar graph shows relative phosphorylation intensities of eEF2 compared with total eEF2 determined by densitometry analysis . Mean ± s . e . m . are shown ( n = 4 ) . P values were calculated using 2way ANOVA followed by Bonferroni posttest ; * * P value ≤ 0 . 01 .

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

Figure 6A Tumor volumes of eEF2K -/- and eEF2K +/+ RasV12 engraft implanted subcutaneously in AGR129mice. Treatment with NFR or vehicle was initiated 6 days post-implantation. Data are mean of tumor volume ± s.e.m. (n = 8 per group). P values were calculated using 2way ANOVA followed by Bonferroni posttest comparing WT tumor with vehicle or NFR treatment and KO tumors with vehicle or NFR treatment; *P value ≤ 0.05, ** P value ≤ 0.01, *** P value ≤ 0.001. Experiment is representative of 2 performed in same conditions.B Immunoblots for total and phosphorylated eEF2 of tumor engraftment from (A). (NS, Non-Specific signal.)C Bar graph shows relative phosphorylation intensities of eEF2 compared with total eEF2 determined by densitometry analysis. Mean ± s.e.m. are shown (n=4). P values were calculated using 2way ANOVA followed by Bonferroni posttest ; **P value ≤ 0.01.


Figure 1 ( A ) Gating strategy used to identify ILCs and ILC subsets in humans ( hILCs ) . ( B ) Representative histogram and summary of the percentage and of the gMFI of c - Maf expression in hILC subsets ( ANOVA analysis : F ( 2 , 18 ) = 305 . 4 , * * * * p < 0 . 0001 ) . Data information : Each dot represents the expression level in one donor ( n = 7 ) . Bars represent mean ± SEM . ( C Gating strategy for cKitlow and cKithigh ILC2s ( C ) D ) expression level ( percentage and gMFI ) of c - Maf in cKitlow and cKithigh ILC2s ( D ) ( paired T tests , * * * * p < 0 . 0001 ) . Data information : Each dot represents the expression level in one donor ( n = 7 ) . Bars represent mean ± SEM .

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Figure 1(A) Gating strategy used to identify ILCs and ILC subsets in humans (hILCs).(B) Representative histogram and summary of the percentage and of the gMFI of c-Maf expression in hILC subsets (ANOVA analysis: F(2,18)=305.4, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.(C Gating strategy for cKitlow and cKithigh ILC2s (C)D) expression level (percentage and gMFI) of c-Maf in cKitlow and cKithigh ILC2s (D) (paired T tests, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.


Figure 3 ( G ) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc - sh or Maf - sh as in ( C ) . hILC2s were then stimulated for 48h with IL - 33 and the concentration of the indicated cytokines in the supernatants was measured ( Multiple Mann - Whitney tests , * p < 0 . 05 , * * p < 0 . 01 ) Bars represent mean ± SEM . Data information : Each dot represents data from one individual mouse or from one donor .

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Figure 3(G) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc-sh or Maf-sh as in (C). hILC2s were then stimulated for 48h with IL-33 and the concentration of the indicated cytokines in the supernatants was measured (Multiple Mann-Whitney tests, *p<0.05, **p<0.01) Bars represent mean ± SEM. Data information: Each dot represents data from one individual mouse or from one donor.


Figure 5 ( E ) ChIP - qPCR analysis of c - Maf occupancy on IL - 4 , IL - 5 and IL - 13 promoters in untreated mouse and human ILC2s ( IL - 2 stimulation ) and in mouse and human ILC2s treated with IL - 33 ( n = 3 biological replicates ) .

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Figure 5(E) ChIP-qPCR analysis of c-Maf occupancy on IL-4, IL-5 and IL-13 promoters in untreated mouse and human ILC2s (IL-2 stimulation) and in mouse and human ILC2s treated with IL-33 (n=3 biological replicates).


Figure 6 ( C ) Representative histograms of c - Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c - Maf + and GATA3 + ILC2s from adult PBMC or cord blood ( n = 5 different donors ; T test , * * * * p < 0 . 0001 ) . ( D ) ILC2 expression of the indicated markers . Each dot represent sample from one donor . Bars represent mean ± SEM . ( E , F ) c - Maf quantification by qPCR ( E ) and cytokine concentration ( F ) in the supernatants of adult and cord blood ILC2s unstimulated , stimulated for 3h with PMA - Ionomycin ( 3h stim ) and stimulated for an overnight with IL - 33 and then 3h with PMA - Ionomycin ( o . n . stim ) . ILC2s were sorted from 3 different donors . ( E ) Multiple T tests , unstimulated : p = 0 . 000101 ; 3h stim : p = 0 . 0036 . ( F ) Multiple T tests , IL - 13 : p = 0 . 022 ; IL - 5 : p = 0 . 015 ( * p < 0 . 05 ) . Bars represent mean ± SEM .

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

Figure 6(C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001).(D) ILC2 expression of the indicated markers. Each dot represent sample from one donor. Bars represent mean ± SEM.(E, F) c-Maf quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s unstimulated, stimulated for 3h with PMA-Ionomycin (3h stim) and stimulated for an overnight with IL-33 and then 3h with PMA-Ionomycin (o.n. stim). ILC2s were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022; IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.


Figure 1Human derived GBM tumors were processed as in ( A ) . Representative western blot were performed ( F ) and the percentage of ER - lumenal protein cytosolic localization ( G ) were quantified . Data are the average from six different tumors . N = 6 Bars and error bars indicate

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Figure 1Human derived GBM tumors were processed as in (A). Representative western blot were performed (F) and the percentage of ER-lumenal protein cytosolic localization (G) were quantified. Data are the average from six different tumors. N=6 Bars and error bars indicate mean


Figure 2Left : Representative images of cells were transfected with super - folder GFP ( sfGFP ) treated with 250ng / mL Tunicamycin ( Tm ) , 50nM Thapsigargin ( Tg ) and 250ng / mL Brefeldin - A ( BFA ) for 24 hours . Right : Quantification of the microscopy images of cells expressing ER - targeted sfGFP and the cytosolically localized mCherry . Data values are the mean ± SD of technical replicates ( n = 10 ) from 3 independent experiments ( * * * * p < 0 . 0001 ) . One - way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software . Scale bar 15μm . Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) protocols , representative western blots are showed . Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) or differential centrifugation protocols , representative western blots are showed .

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

Figure 2Left: Representative images of cells were transfected with super-folder GFP (sfGFP) treated with 250ng/mL Tunicamycin (Tm), 50nM Thapsigargin (Tg) and 250ng/mL Brefeldin-A (BFA) for 24 hours. Right: Quantification of the microscopy images of cells expressing ER-targeted sfGFP and the cytosolically localized mCherry. Data values are the mean ± SD of technical replicates (n=10) from 3 independent experiments (****p<0.0001). One-way ANOVA was applied for the statistical analysis through the GraphPad Prism 9 software. Scale bar 15μm.Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) protocols, representative western blots are showed.Subcellular protein fractionation of several ER resident proteins in HEK293T cells treated with the indicated concentrations of Tm or Tg for 16 hours using Digitonin (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) or differential centrifugation protocols, representative western blots are showed.


Figure 3Subcellular protein fractionation of several ER resident proteins in ( A ) GL261 , ( B ) U87 and ( C ) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin . ( NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer ) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261 , U87 and A549 cells as in ( A - C ) respectively . Biological triplicates , mean ± SD calculated using Prism 9 ( GraphPad ) .

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Figure 3Subcellular protein fractionation of several ER resident proteins in (A) GL261, (B) U87 and (C) A549 cells treated with the indicated concentrations of Tm or Tg using Digitonin. (NP40 represents the membrane fraction extracted with NP40 Cell Lysis Buffer) Quantification of the subcellular protein fractionation of several ER endogenous proteins in GL261, U87 and A549 cells as in (A-C) respectively. Biological triplicates, mean ± SD calculated using Prism 9 (GraphPad).


Figure 4Representative transmission electron microscopy images of the gold particles distribution ( after immunogold labelling with PDIA3 antibodies ) in A549 cells treated with DMSO or Tm . In the inserts , gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles . ( Scale bar 500 nm ) . n represents the nucleus and m represents the mitochondria . ( C ) violin plots of the gold particles distribution from the electron microscopy experiment ( immunogold labelling of PDIA3 ) as shown in Figure 4B and 4C . * P - value = 0 . 0486 . n = 8 for DMSO and n = 8 for Tm . Thick horizontal lines represent mean ± SD - lighter dashed lines . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) ,

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Figure 4Representative transmission electron microscopy images of the gold particles distribution (after immunogold labelling with PDIA3 antibodies) in A549 cells treated with DMSO or Tm. In the inserts, gold particles in the ER were surrounded by red circles and those in the rest of the cytoplasm by blue circles. (Scale bar 500 nm). n represents the nucleus and m represents the mitochondria. (C) violin plots of the gold particles distribution from the electron microscopy experiment (immunogold labelling of PDIA3) as shown in Figure 4B and 4C. *P-value=0.0486. n=8 for DMSO and n=8 for Tm. Thick horizontal lines represent mean ± SD -lighter dashed lines. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad),


Figure 5Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells . A549 cells were treated with Tunicamycin ( Tm 100ng / ml ) , Thapsigargin ( Tg 25nM ) and Brefeldin A ( BFA 0 , 1 and 0 , 25nM ) . Endogenous p53 was immunoprecipitated from the cytoplasmic fraction ( digitonin fraction ) of A549 cells . Coprecipitated endogenous AGR2 was detected by western blot . A549 were treated with Etoposide ( Eto ) for 2 hours to induce p53 pathway . Then cells have been challenged with Tm , Tg and BFA at the indicated concentrations for 16 hours . Luciferase experiments were performed after 24 hours of transfection . Graph shows the fold induction of p53 luciferase construct . Western blot experiments for phospho - p53 , pan - p53 , p21 and HSP90 were performed as control . n = 3 , biological replicates ( mean ± SD ; Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated . * p < 0 . 05 , * * * p < 0 . 001 . A549 were transfected with control siRNA ( siC ) and AGR2 - tergetd siRNA ( siAGR2 ) . After 24 hours , cells were transfected with p53 - luciferase construct . Cells were then treated as in ( C ) and luciferase experiments were performed . n = 4 , biological replicates ( mean ± SD . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated . Left : Western blot experiments for phospho - p53 , pan - p53 , and p21 in AGR2 - silenced A549 cells exposed to ER stressors : 100ng / ml Tunicamycin ( Tm ) , 25nM Thapsigargin ( Tg ) and 0 . 25 nM Brefeldin A ( BFA ) , in the presence and absence of etoposide . Right : Quantification of the western blot . n = 3 , biological replicates , mean ± SD . A549 were transfected with the indicated constructs of differently targeted nanobodies , cells were then treated with Tunicamycin ( Tm 100 ng / ml and 250 ng / ml ) , Thapsigargin ( Tg 25 nM ) and Brefeldin A ( BFA 0 , 25 nM ) for 16 hours at the indicated concentration . Western blot experiments were performed and HSP90 were used as loading control . n = 3 , biological replicates ( mean ± SD of n = 3 independent experiments ( * * * p < 0 , 001 and * * p < 0 , 01 ) . Differences were analyzed by Unpaired Student ' s t - test using Prism 9 ( GraphPad ) , except when otherwise indicated .

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

Figure 5Immunoprecipitation of p53 and AGR2 in the digitonin fraction of A549 cells. A549 cells were treated with Tunicamycin (Tm 100ng/ml), Thapsigargin (Tg 25nM) and Brefeldin A (BFA 0,1 and 0,25nM). Endogenous p53 was immunoprecipitated from the cytoplasmic fraction (digitonin fraction) of A549 cells. Coprecipitated endogenous AGR2 was detected by western blot.A549 were treated with Etoposide (Eto) for 2 hours to induce p53 pathway. Then cells have been challenged with Tm, Tg and BFA at the indicated concentrations for 16 hours. Luciferase experiments were performed after 24 hours of transfection. Graph shows the fold induction of p53 luciferase construct. Western blot experiments for phospho-p53, pan-p53, p21 and HSP90 were performed as control. n=3, biological replicates (mean ± SD; Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated. *p<0.05, ***p < 0.001.A549 were transfected with control siRNA (siC) and AGR2-tergetd siRNA (siAGR2). After 24 hours, cells were transfected with p53-luciferase construct. Cells were then treated as in (C) and luciferase experiments were performed. n=4, biological replicates (mean ± SD. Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.Left: Western blot experiments for phospho-p53, pan-p53, and p21 in AGR2-silenced A549 cells exposed to ER stressors: 100ng/ml Tunicamycin ( Tm), 25nM Thapsigargin (Tg) and 0.25 nM Brefeldin A (BFA), in the presence and absence of etoposide. Right: Quantification of the western blot. n=3, biological replicates, mean ±SD.A549 were transfected with the indicated constructs of differently targeted nanobodies, cells were then treated with Tunicamycin (Tm 100 ng/ml and 250 ng/ml), Thapsigargin (Tg 25 nM) and Brefeldin A (BFA 0,25 nM) for 16 hours at the indicated concentration. Western blot experiments were performed and HSP90 were used as loading control. n=3, biological replicates (mean ± SD of n=3 independent experiments (***p<0,001 and **p<0,01). Differences were analyzed by Unpaired Student's t-test using Prism 9 (GraphPad), except when otherwise indicated.


Figure 1 . An S . typhimurium strain devoid of its SPI - 1 TTSS effector proteins is able to induce programmed cell death in macrophages . BMDPM from wild - type ( A ) or caspase - 1 − / − ( B ) mice ( Kuida et al . , 1995 ) were infected with wild - type S . typhimurium or mutant derivative either lacking all TTSS effector proteins ( effectorless ) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system ( invG ) . 6 h after infection , cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase ( TUNEL ) staining . Values for the lactate dehydrogenase release represent the average of at least three independent measurements . Equivalent results were obtained in several repetitions of these experiments .

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Figure 1. An S. typhimurium strain devoid of its SPI-1 TTSS effector proteins is able to induce programmed cell death in macrophages. BMDPM from wild-type (A) or caspase-1−/− (B) mice (Kuida et al., 1995) were infected with wild-type S. typhimurium or mutant derivative either lacking all TTSS effector proteins (effectorless) or defective for TTSS secretion by virtue a having a mutation on an essential component of this system (invG). 6 h after infection, cytotoxicity was evaluated by lactate dehydrogenase release or by terminal deoxynucleatide transferase (TUNEL) staining. Values for the lactate dehydrogenase release represent the average of at least three independent measurements. Equivalent results were obtained in several repetitions of these experiments.


Figure 2 . Transient expression of SipB in BMDPM from caspase - 1 − / − mice results in cell death . Plasmids expressing either SipB , SipC , or SipD fused to YFP were introduced into BMDPM from caspase - 1 − / − mice as indicated in the Materials and methods section . Macrophages were then fixed , stained with DAPI ( to visualize chromatin ) , and observed under a fluorescence microscope . Cells expressing SipC or SipD do not show any signs of cytotoxicity . In contrast , cells expressing SipB show clear signs of cytotoxicity and chromatin condensation . Arrows indicate the nucleus of transfected cells .

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Figure 2. Transient expression of SipB in BMDPM from caspase-1−/−mice results in cell death. Plasmids expressing either SipB, SipC, or SipD fused to YFP were introduced into BMDPM from caspase-1−/− mice as indicated in the Materials and methods section. Macrophages were then fixed, stained with DAPI (to visualize chromatin), and observed under a fluorescence microscope. Cells expressing SipC or SipD do not show any signs of cytotoxicity. In contrast, cells expressing SipB show clear signs of cytotoxicity and chromatin condensation. Arrows indicate the nucleus of transfected cells.


Figure 3 . Transient expression of SipB in COS - 2 cells . ( A ) A plasmid expressing YFP - tagged SipB was transfected into COS - 2 cells , and its localization was examined under a confocal microscope . ( B ) SipB colocalized with mitochondrial markers in transfected cells . COS - 2 cells were transfected with a plasmid encoding either SipB - CFP ( top ) or SipB - YFP ( bottom ) . Transfected cells were stained with MitoTracker ® Red ( Molecular Probes , Inc . ) to visualize mitochondria and were examined by confocal microscopy . The SipB - induced structures were readily stained with mitochondrial markers ( top ) . Mitochondria were also seen contained within the SipB - induced structures ( bottom ) . ( C ) SipB - induced structures were labeled with the ER marker Sec - 61 . COS - 2 cells were cotransfected with plasmids encoding SipB - YFP and Sec61 - CFP , and transfected cells were examined by confocal microscopy .

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Figure 3.Transient expression of SipB in COS-2 cells. (A) A plasmid expressing YFP-tagged SipB was transfected into COS-2 cells, and its localization was examined under a confocal microscope. (B) SipB colocalized with mitochondrial markers in transfected cells. COS-2 cells were transfected with a plasmid encoding either SipB-CFP (top) or SipB-YFP (bottom). Transfected cells were stained with MitoTracker® Red (Molecular Probes, Inc.) to visualize mitochondria and were examined by confocal microscopy. The SipB-induced structures were readily stained with mitochondrial markers (top). Mitochondria were also seen contained within the SipB-induced structures (bottom).  (C) SipB-induced structures were labeled with the ER marker Sec-61. COS-2 cells were cotransfected with plasmids encoding SipB-YFP and Sec61-CFP, and transfected cells were examined by confocal microscopy.


Figure 4 . Immuno - EM of SipB - transfected COS - 2 cells . COS - 2 cells were transfected with a plasmid expressing SipB - YFP , and 24 h after transfection , cells were fixed and prepared for immuno - EM using an antibody specific for GFP and a gold - labeled anti - rabbit antibody . SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria ( A ) . Mitochondria were often seen apparently fusing to the SipB - induced membranous structure ( B , see arrows in inset on top ; C , inset to the right ) . Mitochondria were often seen contained with the SipB - induced membrane structures ( D ) . Bars , 250 nm .

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Figure 4. Immuno-EM of SipB-transfected COS-2 cells. COS-2 cells were transfected with a plasmid expressing SipB-YFP, and 24 h after transfection, cells were fixed and prepared for immuno-EM using an antibody specific for GFP and a gold-labeled anti-rabbit antibody. SipB was seen localized to structures made up of closely apposed membranes that were often in close proximity to mitochondria (A). Mitochondria were often seen apparently fusing to the SipB-induced membranous structure (B, see arrows in inset on top; C, inset to the right). Mitochondria were often seen contained with the SipB-induced membrane structures (D). Bars, 250 nm.


Figure 5 . The nonfusogenic SipB428 - 593 mutant localizes to the mitochondria , but does not form multivesicular structures and interferes with the activity of the wild - type protein . ( A ) COS - 2 cells were transfected with a plasmid expressing YFP - tagged SipB428 - 593 , stained with an antibody to the mitochondrial protein CoxIV , subunit VIc ( Molecular Probes , Inc . ) , and examined by confocal microscopy . SipB428 - 593 - YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild - type protein . ( B ) COS - 2 cells were cotransfected with plasmids expressing either SipB428 - 593 - YFP or SipB - CFP , and transfected cells were examined under a confocal microscope . Expression of SipB428 - 593 - YFP prevented wild - type SipB - CFP from forming multivesicular structures ( notice that vesicular structures are only apparent in the absence , but not in the presence , of SipB428 - 593 - YFP ; right ) .

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Figure 5. The nonfusogenic SipB428-593 mutant localizes to the mitochondria, but does not form multivesicular structures and interferes with the activity of the wild-type protein. (A) COS-2 cells were transfected with a plasmid expressing YFP-tagged SipB428-593, stained with an antibody to the mitochondrial protein CoxIV, subunit VIc (Molecular Probes, Inc.), and examined by confocal microscopy. SipB428-593-YFP localized to mitochondria and did not induce the formation of multivesicular structures seen after expression of the wild-type protein.  (B) COS-2 cells were cotransfected with plasmids expressing either SipB428-593-YFP or SipB-CFP, and transfected cells were examined under a confocal microscope. Expression of SipB428-593-YFP prevented wild-type SipB-CFP from forming multivesicular structures (notice that vesicular structures are only apparent in the absence, but not in the presence, of SipB428-593-YFP; right).


Figure 6 . The S . typhimurium SPI - 1 TTSS - secreted protein SipB localizes to the mitochondria of infected macrophages . ( A ) Bone marrow - derived macrophages were infected with either wild - type S . typhimurium or the TTSS translocation - defective sipD mutant strain with a multiplicity of infection of 50 . 3 h after infection , macrophages were collected and processed for immuno - EM using gold - labeled antibodies . SipB could be readily detected in macrophages infected with wild - type ( A and B ) , but not in macrophages infected with the sipD mutant strain ( C ) . Bars , 100 nm . ( B ) Alternatively , mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV , subunit IV , and were examined by Western immunoblot analysis using specific antibodies .

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Figure 6. The S. typhimurium SPI-1 TTSS-secreted protein SipB localizes to the mitochondria of infected macrophages. (A) Bone marrow-derived macrophages were infected with either wild-type S. typhimurium or the TTSS translocation-defective sipD mutant strain with a multiplicity of infection of 50. 3 h after infection, macrophages were collected and processed for immuno-EM using gold-labeled antibodies. SipB could be readily detected in macrophages infected with wild-type (A and B), but not in macrophages infected with the sipD mutant strain (C). Bars, 100 nm.  (B) Alternatively, mitochondria were isolated from the infected macrophages and the presence of SipB or the resident mitochondrial protein CoxIV, subunit IV, and were examined by Western immunoblot analysis using specific antibodies.


Figure 7 . S . typhimurium - infected macrophages exhibited signs of autophagocytosis . Caspase - 1 − / − BMDPM were infected with wild - type S . typhimurium or it isogenic translocation - defective sipD mutant . 6 h after infection , macrophages were either stained with MDC to label autophagosomes ( I ) or processed for EM ( II ) as described in the legend to Fig . 3 . Macrophages infected with wild - type S . typhimurium ( but not those infected with the sipD mutant ) exhibited a large number of vesicular structures that stained with MDC ( I ) Under the electron microscope ( II ) , macrophages infected with wild - type Salmonella exhibited multivesicular structures resembling autophagosomes ( A - D ) Often , electron - dense material , presumably damaged organelles , were readily observed within the multivesicular structures ( B - D ) . In contrast , macrophages infected with type III secretion - defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild - type - infected cells . Bars ( A and E ) 500 nm ; ( B , C , D and F ) 250 nm .

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Figure 7.S. typhimurium-infected macrophages exhibited signs of autophagocytosis. Caspase-1−/− BMDPM were infected with wild-type S. typhimurium or it isogenic translocation-defective sipD mutant. 6 h after infection, macrophages were either stained with MDC to label autophagosomes (I) or processed for EM (II) as described in the legend to Fig. 3. Macrophages infected with wild-type S. typhimurium (but not those infected with the sipD mutant) exhibited a large number of vesicular structures that stained with MDC (I) Under the electron microscope (II), macrophages infected with wild-type Salmonella exhibited multivesicular structures resembling autophagosomes (A-D) Often, electron-dense material, presumably damaged organelles, were readily observed within the multivesicular structures (B-D). In contrast, macrophages infected with type III secretion-defective sipD mutant did not exhibit the large number of multivesicular structures observed in wild-type-infected cells. Bars (A and E) 500 nm; (B, C, D and F) 250 nm.


Figure 8 . The S . typhimurium - induced macrophage death does not require caspase activity . BMDPM were infected with wild - type S . typhimurium or the TTSS - defective invG mutant in the presence or absence of 50 μM of the pan - caspase inhibitor Z - VAD , and cell death was measured as indicated in the legend to Fig . 1 .

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Figure 8. The S. typhimurium-induced macrophage death does not require caspase activity. BMDPM were infected with wild-type S. typhimurium or the TTSS-defective invG mutant in the presence or absence of 50 μM of the pan-caspase inhibitor Z-VAD, and cell death was measured as indicated in the legend to Fig. 1.


Figure 3a . Lipid droplet time course . Representative photomicrographs showing lipid droplet abundance in ARPE19 cells treated with DFP starting at 7 h and maximal at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in a cell treated with DFP for 24 h . Arrows highlight LD accumulation . Scale bars = 5 μm . b . Mitophagy time course . Representative photomicrographs showing time course of DFP treatment in mitophagy reporter cells ( human ARPE - 19 cells with stable expression of mito - QC ) . Mitochondrial networks are visible in yellow and mitolysosomes as red - only puncta signifying mitophagy . Nuclei are counterstained with Hoescht33342 . Mitophagy appears maximal after 24 h of DFP treatment , with little difference between control and 7 h conditions . Inset shows details of mitolysosomes within the mitochondrial network of a highly mitophagic cell following DFP treatment . Arrows highlight mitolysosomes . Scale bar = 5 μm . c . Lipid droplets accumulate at mitochondria when cells are treated with DFP . Representative photomicrographs showing lipid droplet and mitochondria in ARPE19 cells treated with DFP at 7 h and at 24 h . Neutral lipids were revealed by BODIPY 493 / 503 and mitochondria by Mitotracker . Nuclei are counterstained with Hoescht33342 . Inset shows detail of LDs in close proximity to mitochondria in cells treated with DFP for 24 h . Scale bar = 5 μm . d . Quantification of lipid droplet area per cell over time . Lipid droplet area was quantified per cell area using a pipeline generated in CellProfiler . n = 3 with at least 50 cells quantified per biological replicate for each condition . Data represent means ± SEM . e . Quantification of mitophagy over time . Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content . n = 3 with at least 50 cells quantified per biological replicate for each condition . Data represent means ± SEM . f . Quantification of lipid droplet and mitochondria proximity over time . n = 2 with at least 50 cells quantified per biological replicate for each condition . Ratios between Control and 24h DFP ; P < 0 . 05 .

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Figure 3a. Lipid droplet time course. Representative photomicrographs showing lipid droplet abundance in ARPE19 cells treated with DFP starting at 7 h and maximal at 24 h. Neutral lipids were revealed by BODIPY 493/503. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in a cell treated with DFP for 24 h. Arrows highlight LD accumulation. Scale bars =5 μm.b. Mitophagy time course. Representative photomicrographs showing time course of DFP treatment in mitophagy reporter cells (human ARPE-19 cells with stable expression of mito-QC). Mitochondrial networks are visible in yellow and mitolysosomes as red-only puncta signifying mitophagy. Nuclei are counterstained with Hoescht33342. Mitophagy appears maximal after 24 h of DFP treatment, with little difference between control and 7 h conditions. Inset shows details of mitolysosomes within the mitochondrial network of a highly mitophagic cell following DFP treatment. Arrows highlight mitolysosomes. Scale bar = 5 μm.c. Lipid droplets accumulate at mitochondria when cells are treated with DFP. Representative photomicrographs showing lipid droplet and mitochondria in ARPE19 cells treated with DFP at 7 h and at 24 h. Neutral lipids were revealed by BODIPY 493/503 and mitochondria by Mitotracker. Nuclei are counterstained with Hoescht33342. Inset shows detail of LDs in close proximity to mitochondria in cells treated with DFP for 24 h. Scale bar = 5 μm.d. Quantification of lipid droplet area per cell over time. Lipid droplet area was quantified per cell area using a pipeline generated in CellProfiler. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.e. Quantification of mitophagy over time. Mitophagy index per cell is measured as a ratio of the number of mitolysosomes per mitochondrial content. n=3 with at least 50 cells quantified per biological replicate for each condition. Data represent means ±SEM.f. Quantification of lipid droplet and mitochondria proximity over time. n=2 with at least 50 cells quantified per biological replicate for each condition. Ratios between Control and 24h DFP; P<0.05.


Figure 4d . DFP - induced Lipid droplet accumulation is dependent on DGAT1 . Representative photomicrographs of ARPE19 cells treated with DFP ( 1mM ) for 7 h followed by treatment with LD inhibitors ( 5 μM ) as indicated . Cells were fixed after 24 h of DFP treatment . Neutral lipid droplets were stained by BODIPY493 / 503 and imaged by confocal microscopy after 24 h of DFP treatment ( ' i ' denotes the presence of inhibitor e . g . DGAT1i = DGAT1 - inhibition ) . Scale bar = 5 μm . e . Quantitation of lipid droplets area per cell from experiments in 2d . Data are represented as mean ± SEM ( n = 3 with at least 80 cells analysed per condition in each biological replicate ) .

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Figure 4d. DFP-induced Lipid droplet accumulation is dependent on DGAT1. Representative photomicrographs of ARPE19 cells treated with DFP (1mM) for 7 h followed by treatment with LD inhibitors (5 μM) as indicated. Cells were fixed after 24 h of DFP treatment. Neutral lipid droplets were stained by BODIPY493/503 and imaged by confocal microscopy after 24 h of DFP treatment ('i' denotes the presence of inhibitor e.g. DGAT1i = DGAT1-inhibition). Scale bar = 5 μm.e. Quantitation of lipid droplets area per cell from experiments in 2d. Data are represented as mean ± SEM (n=3 with at least 80 cells analysed per condition in each biological replicate).


Figure 5a . ARPE - 19 ULK1 CRISPR KO cells with / without re - introduction of FLAG - ULK1 were treated with DFP for 24h as indicated . Cells were fixed and stained with BODIPY493 / 503 to visualise lipid droplets ( green ) or DAPI to visualise nuclei ( blue ) . Scale bar , 10 μm . b . Quantification from 5a , n = 3 independent experiments , statical analysis by one - way ANOVA . * = P < 0 . 05 ; * * P = < 0 . 01 . Median represented by dashed line , quartiles by dotted lines . c . Authentication of cell lines in 5a by immunoblot of protein lysates .

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Figure 5a. ARPE-19 ULK1 CRISPR KO cells with/without re-introduction of FLAG-ULK1 were treated with DFP for 24h as indicated. Cells were fixed and stained with BODIPY493/503 to visualise lipid droplets (green) or DAPI to visualise nuclei (blue). Scale bar, 10 μm.b. Quantification from 5a, n=3 independent experiments, statical analysis by one-way ANOVA. *=P<0.05; **P=<0.01. Median represented by dashed line, quartiles by dotted lines.c. Authentication of cell lines in 5a by immunoblot of protein lysates.


Figure 6f . Representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE - 19 cells treated with DFP ± DGAT1i / 2i . Samples were immunolabelled with primary antibodies to NIX ( green ) and ATPB ( red ) . Scale bar = 2 μm . g . Representative confocal photomicrograpghs of Cathepsin - reactive endolysosomes in human ARPE - 19 cells treated with with DFP ± DGAT1i / 2i . Inset shows examples of lysosomal phenotypes . Scale bar = 5 μm . h . Colocalization quantitation for experiments shown in 6g . One - way ANOVA with Bonferroni ' s post hoc test . * * * * P < 0 . 0001 . Median represented by dashed line , quartiles by dotted lines n = 2 biological replicates . i . Colocalization quantitation for ULK1 / WIPI2 immunolabelling in human ARPE - 19 cells treated with DFP ± DGAT1i / 2i . Median represented by dashed line , quartiles by dotted lines n = 2 biological replicates . j . Quantitation of lysosome positioning in DFP ± DGAT1i / 2i . One - way ANOVA with Bonferroni ' s post hoc test . * * * * = P < 0 . 0001 . Median represented by dashed line , quartiles by dotted lines n = 2 biological replicates .

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Figure 6f. Representative confocal photomicrographs showing the pattern of endogenous mitochondrial NIX in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Samples were immunolabelled with primary antibodies to NIX (green) and ATPB (red). Scale bar = 2 μm. g. Representative confocal photomicrograpghs of Cathepsin-reactive endolysosomes in human ARPE-19 cells treated with with DFP ±DGAT1i/2i. Inset shows examples of lysosomal phenotypes. Scale bar = 5 μm. h. Colocalization quantitation for experiments shown in 6g. One-way ANOVA with Bonferroni's post hoc test. ****P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. i. Colocalization quantitation for ULK1/WIPI2 immunolabelling in human ARPE-19 cells treated with DFP ±DGAT1i/2i. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates. j. Quantitation of lysosome positioning in DFP ±DGAT1i/2i. One-way ANOVA with Bonferroni's post hoc test. ****=P<0.0001. Median represented by dashed line, quartiles by dotted lines n=2 biological replicates.


Figure 7c . Representative photomicrographs of larval neuronal cell bodies expressing the matrix - QC reporter and RNAi for mdy or control driven with nSyb - GAL4 . GFP is shown in green , mCherry is shown in magenta . Mitolysosomes ( mCherry - only puncta ) are shown in greyscale . Scale bar = 5 μm . d . Quantitative analysis of mitophagy ( mitolysosome per neuronal cell body ) . Data are shown as violin plot with median ( dashed line ) and quartile range ( dotted lines ) ; n = 5 - 6 animals , with 23 - 40 cells per animal . One - way ANOVA with Šidák ' s post - test correction for multiple samples ; * * = P < 0 .

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Figure 7c. Representative photomicrographs of larval neuronal cell bodies expressing the matrix-QC reporter and RNAi for mdy or control driven with nSyb-GAL4. GFP is shown in green, mCherry is shown in magenta. Mitolysosomes (mCherry-only puncta) are shown in greyscale. Scale bar = 5 μm.d. Quantitative analysis of mitophagy (mitolysosome per neuronal cell body). Data are shown as violin plot with median (dashed line) and quartile range (dotted lines); n = 5-6 animals, with 23-40 cells per animal. One-way ANOVA with Šidák's post-test correction for multiple samples; **=P<0.01


f1a , Survival ratio after TAC ( n = 7 - 14 per group ) . b - g , Ten days after TAC . b , Gross appearance of hearts . Scale bar , 2 mm . b - g , Ten days after TAC . c , Echocardiography . Scale bars , 0 . 2 s and 5 mm . Echocardiographic parameters ( d ) ( n = 7 - 13 per group ) . LVIDd and LVIDs , end - diastolic and end - systolic left ventricular internal dimension , respectively ; LVFS , left venticular fractional shortening ; b - g , ( e ) Ten days after TAC . physiological parameters ( n = 7 - 13 per group ) . Haematoxylin and eosin - stained ( f ) and azocarmine and aniline blue ( AZAN ) - Mallory - stained ( g ) heart sections . Scale bar , 100 µm . Data are mean ± s . e . m . * P 0 . 05 versus all other groups , * * P 0 . 05 versus sham - operated controls .

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f1a, Survival ratio after TAC (n = 7-14 per group).b-g, Ten days after TAC. b, Gross appearance of hearts. Scale bar, 2 mm.b-g, Ten days after TAC. c, Echocardiography. Scale bars, 0.2 s and 5 mm. Echocardiographic parameters (d) (n = 7-13 per group). LVIDd and LVIDs, end-diastolic and end-systolic left ventricular internal dimension, respectively; LVFS, left venticular fractional shortening;b-g, (e) Ten days after TAC.physiological parameters (n = 7-13 per group).Haematoxylin and eosin-stained (f) and azocarmine and aniline blue (AZAN)-Mallory-stained (g) heart sections. Scale bar, 100 µm. Data are mean ± s.e.m. *P  0.05 versus all other groups, **P  0.05 versus sham-operated controls.


f2Mice were analysed 2 days after TAC ( a - c ) . a , Haematoxylin and eosin - stained heart sections . Scale bar , 100 µm . b , AZAN - Mallory - stained sections . Scale bar , 100 µm . Mice were analysed 2 days after TAC ( a - c ) . c , Immunohistochemical analysis using antibodies to CD45 , CD68 , Ly6G and CD3 . Scale bar , 100 µm .

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f2Mice were analysed 2 days after TAC (a-c). a, Haematoxylin and eosin-stained heart sections. Scale bar, 100 µm. b, AZAN-Mallory-stained sections. Scale bar, 100 µm.Mice were analysed 2 days after TAC (a-c). c, Immunohistochemical analysis using antibodies to CD45, CD68, Ly6G and CD3. Scale bar, 100 µm.


f3Mice were analysed 2 days after TAC ( a - e ) . a , Electron microscopic analysis . Images of mitochondria at higher magnification are shown in subsets . Scale bar , 1 µm . b , Autolysosome after incubation with anti - DNA antibody and 10 nm gold staining . Scale bar , 200 nm . Arrows indicate labelled DNA . Mice were analysed 2 days after TAC Double staining of heart sections with EdU ( green ) and anti - LAMP2a antibody ( red ) ( c ) , EdU ( green ) and anti - LC3 antibody ( red ) ( d ) or EdU and anti - TLR9 antibody ( red ) ( e ) . Arrows indicate EdU - positive and LAMP2a - , LC3 - or TLR9 - positive structures . Scale bar , 10 µm .

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f3Mice were analysed 2 days after TAC (a-e). a, Electron microscopic analysis. Images of mitochondria at higher magnification are shown in subsets. Scale bar, 1 µm. b, Autolysosome after incubation with anti-DNA antibody and 10 nm gold staining. Scale bar, 200 nm. Arrows indicate labelled DNA.Mice were analysed 2 days after TAC Double staining of heart sections with EdU (green) and anti-LAMP2a antibody (red) (c), EdU (green) and anti-LC3 antibody (red) (d) or EdU and anti-TLR9 antibody (red) (e). Arrows indicate EdU-positive and LAMP2a-, LC3- or TLR9-positive structures. Scale bar, 10 µm.


f4a , Survival ratio of TAC - operated ODN - treated mice ( n = 6 - 10 per group ) . b - d , Four days after TAC . b , Echocardiography . Scale bars , 0 . 2 s and 5 mm . b - d , Four days after TAC . c , Echocardiographic parameters . Open and closed bars represent ODN2088 control - and ODN2088 - treated groups , respectively ( n = 5 - 8 per group ) . b - d , Four days after TAC . d , Immunohistochemical analysis . Scale bar , 100 µm . TLR9 - deficient mice were analysed 10 weeks after TAC ( e , f ) . e , Scale bars , 0 . 2 s and 5 mm . f , Echocardiographic parameters ( n = 6 - 10 per group ) . Data are mean ± s . e . m . * P 0 . 05 versus all other groups .

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f4a, Survival ratio of TAC-operated ODN-treated mice (n = 6-10 per group).b-d, Four days after TAC. b, Echocardiography. Scale bars, 0.2 s and 5 mm.b-d, Four days after TAC. c, Echocardiographic parameters. Open and closed bars represent ODN2088 control- and ODN2088-treated groups, respectively (n = 5-8 per group).b-d, Four days after TAC. d, Immunohistochemical analysis. Scale bar, 100 µm.TLR9-deficient mice were analysed 10 weeks after TAC (e, f). e, Scale bars, 0.2 s and 5 mm. f, Echocardiographic parameters (n = 6-10 per group). Data are mean ± s.e.m. *P  0.05 versus all other groups.


Figure 1 ( A - D ) Immunostaining with dopamine antibody at late 3rd instar ( 96h AEL ) in control background ( domeMESO - GFP / + ) . The inset shows DomeGFP + cells ( B ' ) and the corresponding dopamine levels ( C ' ) in them . ( E - L ) Similar expression analysis of dopamine at early 3rd ( 72h AEL , E - H ) and early 2nd instar ( 48h AEL , I - L ) . ( D , H and L ) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity . ( M ) Quantifications of dopamine levels in DomeGFP + progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 14 at 48h , n = 13 at 72h and n = 16 at 96h AEL ) . Data information : DNA is stained with Hoechst in blue , intracellular Dopamine ( iDopamine ) in red , domeMESO expression in green ( representative of progenitors in the medullary zone , MZ ) . The iDopamine channel has been converted to spectral mode in panels D , H and L . All image panels show a 20µm scale bar . The inset represents a 2X zoom of region of interest ( ROIs ) marked in white dashed square . AEL indicates After Egg Laying . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome + and Dome - cells . The quantification in M shows median with whiskers indicating the maximum and minimum values , . box indicates the lower and upper quartiles . The statistical analysis applied is One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line .

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Figure 1(A-D) Immunostaining with dopamine antibody at late 3rd instar (96h AEL) in control background (domeMESO-GFP/+). The inset shows DomeGFP+ cells (B') and the corresponding dopamine levels (C') in them. (E-L) Similar expression analysis of dopamine at early 3rd (72h AEL, E-H) and early 2nd instar (48h AEL, I-L). (D, H and L) The spectral mode of iDopamine representation with blue pixels indicating low and red pixels indicating high intensity. (M) Quantifications of dopamine levels in DomeGFP+ progenitors (domeMESO-Gal4, UAS-GFP/+, n= 14 at 48h, n=13 at 72h and n=16 at 96h AEL). Data information: DNA is stained with Hoechst in blue, intracellular Dopamine (iDopamine) in red, domeMESO expression in green (representative of progenitors in the medullary zone, MZ). The iDopamine channel has been converted to spectral mode in panels D, H and L. All image panels show a 20µm scale bar. The inset represents a 2X zoom of region of interest (ROIs) marked in white dashed square. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line for segregation between Dome+ and Dome- cells. The quantification in M shows median with whiskers indicating the maximum and minimum values,. box indicates the lower and upper quartiles. The statistical analysis applied is One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.