( C ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment . Expression of GFP - XopL was verified with an anti - GFP antibody , while expression of GUS - HA was confirmed with an anti - HA antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( D ) GFP - ATG8g - labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA . Puncta were calculated from z - stacks ( 15 ) of n = 12 individuals using ImageJ . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * * P < 0 . 01 , * * * P < 0 . 001 ) was determined by one way ANOVA . The experiment was repeated twice with similar results .

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(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.


( E ) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ , Xcv ΔxopQ ΔxopL or mock infected N . benthamiana plants at 2dpi . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( F ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co - infiltrated with Agrobacteria containing the respective constructs . RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post - infiltration using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values ( * * * P < 0 . 001 ) was revealed by Student ' s t - test . The experiment was repeated 3 times with similar results .

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

(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.


( A ) Interaction of XopL with SH3P2 in yeast two - hybrid assays . XopL fused to the GAL4 DNA - binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain ( AD ) in yeast strain Y190 . Cells were grown on selective media before a LacZ filter assay was performed . pSV40 / p53 served as positive control , while the empty AD or BD vector served as negative control . NtSH3P2 = Nicotiana tabacum SH3P2 . - LT = yeast growth on medium without Leu and Trp , - HLT = yeast growth on medium lacking His , Leu , and Trp , indicating expression of the HIS3 reporter gene . LacZ , activity of the lacZ reporter gene .

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(A) Interaction of XopL with SH3P2 in yeast two-hybrid assays. XopL fused to the GAL4 DNA-binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control, while the empty AD or BD vector served as negative control. NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast growth on medium without Leu and Trp, -HLT = yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.


( B ) Coimmunoprecipitation of GFP - XopL with AtSH3P2 - HA . GFP - XopL or GFP were transiently coexpressed with AtSH3P2 - HA in leaves of N . benthamiana . After 48 h , total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis using either anti - GFP or anti - HA antibodies . GFP blot was split for visualization purpose . AtSH3P2 = Arabidopsis thaliana SH3P2 . Two repetitions with similar results have been conducted .

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(B) Coimmunoprecipitation of GFP-XopL with AtSH3P2-HA. GFP-XopL or GFP were transiently coexpressed with AtSH3P2-HA in leaves of N. benthamiana. After 48 h, total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis using either anti-GFP or anti-HA antibodies. GFP blot was split for visualization purpose. AtSH3P2 = Arabidopsis thaliana SH3P2. Two repetitions with similar results have been conducted.


( C ) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay . Yellow fluorescent protein ( YFP ) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173 - XopL in combination with AtSH3P2 - VenusC155 . A positive control showing the dimerization of fructose - 1 , 6 - bisphosphatase ( FBPase ) within the cytosol . White arrows indicate reconstitution of YFP fluorescence . The red structures indicate autofluorescence of chloroplasts . The combination of VenusN173 - XopL with FBPase - VenusC155 or VenusN173 - FBPase with AtSH3P2 - VenusC155 do not induce YFP fluorescence and serve as negative controls . Bars = 20 μm .

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(C) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay. Yellow fluorescent protein (YFP) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173-XopL in combination with AtSH3P2-VenusC155. A positive control showing the dimerization of fructose-1,6-bisphosphatase (FBPase) within the cytosol. White arrows indicate reconstitution of YFP fluorescence. The red structures indicate autofluorescence of chloroplasts. The combination of VenusN173-XopL with FBPase-VenusC155 or VenusN173-FBPase with AtSH3P2-VenusC155 do not induce YFP fluorescence and serve as negative controls. Bars = 20 μm.


( D ) Colocalization analysis of GFP - XopL with SH3P2 - RFP , RFP - ATG8e and RFP - ATG8g in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( scale bars = 20 μm and 10 μm , lower panel ) . White arrows indicate colocalization of GFP and RFP signals . The experiment was repeated twice with similar results .

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(D) Colocalization analysis of GFP-XopL with SH3P2-RFP, RFP-ATG8e and RFP-ATG8g in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (scale bars = 20 μm and 10 μm, lower panel). White arrows indicate colocalization of GFP and RFP signals. The experiment was repeated twice with similar results.


( E ) Total proteins were extracted 48 hpi with A . tumefaciens harboring the respective GFP - XopL , HA - XopL and SH3P2 - GFP expression constructs . SH3P2 - GFP protein levels ( lower band ) were detected using an anti - GFP antibody . Expression of the XopL was verified using an anti - HA or anti - GFP antibody . Expression of GUS - HA served as a control . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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(E) Total proteins were extracted 48 hpi with A. tumefaciens harboring the respective GFP-XopL, HA-XopL and SH3P2-GFP expression constructs. SH3P2-GFP protein levels (lower band) were detected using an anti-GFP antibody. Expression of the XopL was verified using an anti-HA or anti-GFP antibody. Expression of GUS-HA served as a control. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( F ) Growth of Xcv and Xcv ΔxopL strains in roq1 N . benthamiana plants silenced for SH3P2 ( pTRV2 - SH3P2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 and bacteria were quantified at 3 and 6 dpi . Experimental repeats are indicated by data points in red ( n = 8 ) and yellow ( n = 6 ) data points . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Different letters indicate statistically significant differences ( P < 0 . 05 ) as determined by one - way ANOVA .

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(F) Growth of Xcv and Xcv ΔxopL strains in roq1 N. benthamiana plants silenced for SH3P2 (pTRV2-SH3P2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2 and bacteria were quantified at 3 and 6 dpi. Experimental repeats are indicated by data points in red (n=8) and yellow (n=6) data points. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA.


( A ) SH3P2 - GFP was transiently coexpressed together with GUS - HA and GFP - XopL in N . benthamiana using agroinfiltration . At 42 hpi , 200 μM MG132 was infiltrated into A . tumefaciens - inoculated leaves , and leaf material was collected 48 hpi . Expression of SH3P2 - GFP ( lower band ) and GFP - XopL ( upper band ) was detected using an anti - GFP antibody . GUS - HA expression was confirmed with an anti - HA antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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

(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( B ) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL . GST - XopL , and MBP - SH3P2 were tested using the Arabidopsis His - AtUBA1 and His - AtUBC8 . Lanes 2 to 4 are negative controls . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . Asterisks indicate bands that are not ubiquitinated an present in controls . The experiment was repeated twice with similar results .

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(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.


( C ) SH3P2 - GFP was transiently coexpressed together with GFP , GFP - XopL and GFP - XopL ΔE3 in N . benthamiana using agroinfiltration . GFP protein levels were detected with an anti - GFP antibody . Ponceau S staining serves as a loading control . The experiment was repeated three times with similar results .

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(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( D ) Immunoblot analysis of ATG8 protein levels in N . benthamiana plants transiently expressing GFP - XopL , GFP - XopL ΔE3 or GFP control at 2dpi . Expression of binary constructs was verified with an anti - GFP antibody . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( E ) RLUC - ATG8a constructs were coexpressed with internal control FLUC in N . benthamiana . GFP - XopL , GFP - XopL ΔE3 or GFP control were co - infiltrated together with RLUC / FLUC mixture . Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual - luciferase system . Values represent the ratio of RLUC - ATG8a and FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Statistical significance ( * P < 0 . 5 , * * P < 0 . 01 ) was revealed by Student ' s t - test . The experiment was repeated 3 times .

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(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.


( A ) GFP - XopL was coexpressed with GFP or AIMp - RFP . Proteins were separated by SDS - PAGE and detected by immunoblotting using the indicated antibodies . GFP blot was split for visualization purpose . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

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(A) GFP-XopL was coexpressed with GFP or AIMp-RFP. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( B ) Colocalization of RFP - XopL with GFP - Joka2 in N . benthamiana leaves . Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells ( bars = 20 μm ) . The experiment was repeated twice with similar results .

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(B) Colocalization of RFP-XopL with GFP-Joka2 in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (bars = 20 μm). The experiment was repeated twice with similar results.


( C ) FRET FLIM measurements of GFP - Joka2 and RFP - XopL in N . benthamiana leaves . The freeRFP construct served as a negative control and RFP - ATG8E ( n = 9 ) as a positive control . Scattered points show individual data points , color indicates biological repeats . The lifetime ( in ns ) of GFP - Joka2 ( donor , n = 41 ) was significantly reduced in the presence of RFP - XopL ( n = 40 ) but not in the presence of freeRFP ( n = 35 ) . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Wilcoxon rank sum test , with significantly different groups denoted by different letters . The experiment was repeated three times with similar results .

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(C) FRET FLIM measurements of GFP-Joka2 and RFP-XopL in N. benthamiana leaves. The freeRFP construct served as a negative control and RFP-ATG8E (n = 9) as a positive control. Scattered points show individual data points, color indicates biological repeats. The lifetime (in ns) of GFP-Joka2 (donor, n = 41) was significantly reduced in the presence of RFP-XopL (n = 40) but not in the presence of freeRFP (n = 35). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Wilcoxon rank sum test, with significantly different groups denoted by different letters. The experiment was repeated three times with similar results.


( D ) Immunoprecipitation ( IP ) of GFP - XopL reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP or GFP - XopL were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(D) Immunoprecipitation (IP) of GFP-XopL reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP or GFP-XopL were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( E ) Immunoprecipitation ( IP ) of GFP - XopL and GFP - XopL ΔE3 reveals association with NBR1 . Immunoblots of input and IP samples from N . benthamiana plants transiently expressing GFP , GFP - XopL and GFP - XopL ΔE3 were probed with anti - GFP and anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(E) Immunoprecipitation (IP) of GFP-XopL and GFP-XopL ΔE3 reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP, GFP-XopL and GFP-XopL ΔE3 were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( F ) GFP - XopL was transiently expressed in pTRV2 , pTRV2 - Joka2 and N . benthamiana WT plants . Expression of binary constructs was verified with an anti - GFP antibody . Joka2 silencing was verified using an anti - NBR1 antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated twice with similar results .

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(F) GFP-XopL was transiently expressed in pTRV2, pTRV2-Joka2 and N. benthamiana WT plants. Expression of binary constructs was verified with an anti-GFP antibody. Joka2 silencing was verified using an anti-NBR1 antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( G ) Growth of Xcv ΔxopQ in N . benthamiana plants silenced for Joka2 ( pTRV2 - Joka2 ) compared to control plants ( pTRV2 ) . Leaves were dip - inoculated with a bacteria suspension at OD600 = 0 . 2 . and bacteria were quantified at 3 and 6 dpi . Red , blue , and green data points represent repeats of the experiments . Middle horizontal bars of boxplots represent the median , the bottom and top represent the 25th and 75th percentiles , whiskers extend to at most 1 . 5 times the interquartile range . Significant differences were calculated using Student ' s t - test and are indicated by : * * , P < 0 . 01 . The experiment was repeated three times with similar trends .

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(G) Growth of Xcv ΔxopQ in N. benthamiana plants silenced for Joka2 (pTRV2-Joka2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2. and bacteria were quantified at 3 and 6 dpi. Red, blue, and green data points represent repeats of the experiments. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Student's t-test and are indicated by: **, P < 0.01. The experiment was repeated three times with similar trends.


( H ) GFP - XopL , GFP - XopL ΔE3 were transiently expressed in N . benthamiana . RFP - AIMp was co - infiltrated to stabilize both XopL variants . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a negative control . RFP - AIMp expression was verified by an anti - RFP antibody . GFP blot was split for visualization purpose . Asterisk indicates the GFP - XopL full - length protein . The experiment was repeated three times with similar results .

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(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp was co-infiltrated to stabilize both XopL variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a negative control. RFP-AIMp expression was verified by an anti-RFP antibody. GFP blot was split for visualization purpose. Asterisk indicates the GFP-XopL full-length protein. The experiment was repeated three times with similar results.


( I ) GFP - XopL was transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with the ubiquitin pan selector , followed by immunoblot analysis of the precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a control . Asterisk indicates the GFP - XopL full - length protein . GFP blot was split for visualization purpose . The experiment was repeated two times with similar results .

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(I) GFP-XopL was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a control. Asterisk indicates the GFP-XopL full-length protein. GFP blot was split for visualization purpose. The experiment was repeated two times with similar results.


( J ) Immunoblot analysis GFP - XopL and GFP - XopLK191A at 1 and 2 dpi using an anti - GFP antibody . Ponceau Staining ( PS ) served as a loading control . The experiment was repeated three times with similar results .

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(J) Immunoblot analysis GFP-XopL and GFP-XopLK191A at 1 and 2 dpi using an anti-GFP antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( K ) HA - XopL and GFP - Joka2 and its variants were transiently expressed in N . benthamiana . Samples were taken 48 hpi , and total proteins ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot analysis of the precipitates using either anti - GFP , anti - ubiquitin and anti - NBR1 antibodies . GFP served as a control .

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(K) HA-XopL and GFP-Joka2 and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP, anti-ubiquitin and anti-NBR1 antibodies. GFP served as a control.


a , The 2 - bp deletion identified in exon 9b of patient 1 . b , The T - to - A nonsense mutation in exon 4 of patient 2 ( stop codon is underlined ) . c , Upper panel , intronic mutation in patient 3 ; lower panel , sequencing of the RT - PCR product in patient 3 , showing exon - 6 skipping . d , Upper panel , The G - to - A point mutation at the splice - donor site in intron 5 of patient 6 ; lower panel , sequencing of the RT - PCR product in patient 6 , showing a 6 - bp insertion at the junction between exons 5 and 6 . The inserted sequence ( underlined ) is derived from the first six nucleotides of intron 5 ( underlined ) , which creates a stop codon ( dotted ) . Most probably , nucleotides 7 and 8 , GT ( double underlined ) , in intron 5 are alternatively recognized as a splice - donor site . e , Upper panel , the 10 - bp deletion in patient 9 . This mutation deletes four nucleotides from intron 5 and six from exon 6 . Lower panel , sequence of the RT - PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5 ′ end of exon 6 . The ' AG ' ( double underlined ) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice - acceptor site .

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a, The 2-bp deletion identified in exon 9b of patient 1. b, The T-to-A nonsense mutation in exon 4 of patient 2 (stop codon is underlined). c, Upper panel, intronic mutation in patient 3; lower panel, sequencing of the RT-PCR product in patient 3, showing exon-6 skipping. d, Upper panel, The G-to-A point mutation at the splice-donor site in intron 5 of patient 6; lower panel, sequencing of the RT-PCR product in patient 6, showing a 6-bp insertion at the junction between exons 5 and 6. The inserted sequence (underlined) is derived from the first six nucleotides of intron 5 (underlined), which creates a stop codon (dotted). Most probably, nucleotides 7 and 8, GT (double underlined), in intron 5 are alternatively recognized as a splice-donor site. e, Upper panel, the 10-bp deletion in patient 9. This mutation deletes four nucleotides from intron 5 and six from exon 6. Lower panel, sequence of the RT-PCR product in patient 9 reveals the deletion of the 10 nucleotides at the 5′ end of exon 6. The 'AG' (double underlined) in exon 6 immediately after the genomic DNA deletion appears to be recognized as an alternative splice-acceptor site.


Figure 2 Representation of the mutant LAMP - 2b . ' Normal ' represents the LAMP - 2b amino - acid sequence . Because patient 1 , who developed full DD symptoms , has a mutation in exon 9b , the LAMP - 2b isoform is most probably responsible for DD . Residue 265 , one of the putative disulphide - bond - creating cysteines , and two glycosylation sites are lost by exon - 6 skipping in patient 3 , which probably results in a significant change in the secondary structure . Black boxes represent the loops created between two cystein residues flanking each of the four loop regions ; Y depicts a glycosylation site ; hatched boxes show altered amino acids caused by frameshift mutations ; dashes represent missing amino acids due to exon - 6 skipping in patient 3 . All of the other mutant proteins lack the transmembrane domain ( grey bar ) because of nonsense or frameshift mutations . Thus , those molecules cannot function as lysosomal ' membrane ' proteins .

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Figure 2 Representation of the mutant LAMP-2b. 'Normal' represents the LAMP-2b amino-acid sequence. Because patient 1, who developed full DD symptoms, has a mutation in exon 9b, the LAMP-2b isoform is most probably responsible for DD. Residue 265, one of the putative disulphide-bond-creating cysteines, and two glycosylation sites are lost by exon-6 skipping in patient 3, which probably results in a significant change in the secondary structure. Black boxes represent the loops created between two cystein residues flanking each of the four loop regions; Y depicts a glycosylation site; hatched boxes show altered amino acids caused by frameshift mutations; dashes represent missing amino acids due to exon-6 skipping in patient 3. All of the other mutant proteins lack the transmembrane domain (grey bar) because of nonsense or frameshift mutations. Thus, those molecules cannot function as lysosomal 'membrane' proteins.


a , Skeletal muscle . Muscle extracts from patients 1 - 6 and 10 and a control were blotted and labelled with antibodies against LAMP - 2 ( top panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein , which is probably the LAMP - 2a isoform . In support of this idea , LAMP - 2b is the principal LAMP - 2 species in skeletal muscle6 . Numbers denote patients as in Table 1 ; C , control . b , Heart . Protein was extracted from cardiac muscle of patient 4 and a control , blotted onto a nitrocellulose membrane , and labelled with the antibodies against LAMP - 2 ( too panel ) and β - actin ( bottom panel ) . LAMP - 2 is deficient also in cardiac muscle . C , control ; 4 , patient 4 .

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a, Skeletal muscle. Muscle extracts from patients 1-6 and 10 and a control were blotted and labelled with antibodies against LAMP-2 (top panel) and β-actin (bottom panel). LAMP-2 is deficient in all muscles examined except the sample from patient 1 that showed a trace amount of protein, which is probably the LAMP-2a isoform. In support of this idea, LAMP-2b is the principal LAMP-2 species in skeletal muscle6. Numbers denote patients as in Table 1; C, control. b, Heart. Protein was extracted from cardiac muscle of patient 4 and a control, blotted onto a nitrocellulose membrane, and labelled with the antibodies against LAMP-2 (too panel) and β-actin (bottom panel). LAMP-2 is deficient also in cardiac muscle. C, control; 4, patient 4.


Serial transverse sections of the biopsiedmuscle from six DD patients , and samples from a XMEA patient and a normal control , were immunostained with antibodies against LAMP - 2 and limp - I . LAMP - 2 is absent in all DD muscles ( patient 6 shown here ) , whereas it is present in the XMEA muscle . The antibody against limp - I highlighted vacuoles in both DD and XMEA muscles . LAMP - 1 antibody faintly stained both DD and XMEA muscles , suggesting that the expression level of LAMP - 1 might be lower in skeletal muscle than LAMP - 2 or limp - I ( data not shown ) . The muscle from the patient without a lamp - 2 mutation also showed LAMP - 2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP - 2 deficiency ( data not shown ) . The immunohistochemical stains of LAMP - 2 , limp - I , and LAMP - 1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres . H & amp ; amp ; E , haematoxylin and eosin , original magnification × 150 .

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

Serial transverse sections of the biopsiedmuscle from six DD patients, and samples from a XMEA patient and a normal control, were immunostained with antibodies against LAMP-2 and limp-I. LAMP-2 is absent in all DD muscles (patient 6 shown here), whereas it is present in the XMEA muscle. The antibody against limp-I highlighted vacuoles in both DD and XMEA muscles. LAMP-1 antibody faintly stained both DD and XMEA muscles, suggesting that the expression level of LAMP-1 might be lower in skeletal muscle than LAMP-2 or limp-I (data not shown). The muscle from the patient without a lamp-2 mutation also showed LAMP-2 deficiency on immunohistochemistry similar to genetically proven DD patients with primary LAMP-2 deficiency (data not shown). The immunohistochemical stains of LAMP-2, limp-I, and LAMP-1 in normal muscle shows numerous tiny particles and accentuates type 1 fibres. H&amp;E, haematoxylin and eosin, original magnification ×150.


Representative confocal 3D images of LysoTracker ( red ) , DAPI ( blue ) , and BODIPY ( green ) stained ATM subsets obtained from eWAT of HFD ( 16 weeks ) treated mice ( n = 8 mice from two independent experiments ) . The merged figures are the result of the overlap of all three fluorochromes . Scale bar , 10 μm .

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Representative confocal 3D images of LysoTracker (red), DAPI (blue), and BODIPY (green) stained ATM subsets obtained from eWAT of HFD (16 weeks) treated mice (n = 8 mice from two independent experiments). The merged figures are the result of the overlap of all three fluorochromes. Scale bar, 10 μm.


Mito Stress assay of sorted ATM subsets ( red , MHCIIlow ; green , MHCIIhi ; and blue , CD11c + ) from 16 - week - HFD obese mice showing overall oxygen consumption rate ( OCR ) , maximal respiration , spare capacity respiration , basal respiration , ATP production , and non - mitochondrial respiration . OCR data shown are representative of four independent experiments and expressed as mean ± SD ( n = 4 MHCIIlow ; n = 7 MHCIIhi ; n = 5 CD11c + ) , with statistical significance determined using one - way ANOVA test ( * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns : no significant difference ) .

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Mito Stress assay of sorted ATM subsets (red, MHCIIlow; green, MHCIIhi; and blue, CD11c+) from 16-week-HFD obese mice showing overall oxygen consumption rate (OCR), maximal respiration, spare capacity respiration, basal respiration, ATP production, and non-mitochondrial respiration. OCR data shown are representative of four independent experiments and expressed as mean ± SD (n= 4 MHCIIlow; n= 7 MHCIIhi; n=5 CD11c+), with statistical significance determined using one-way ANOVA test (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference).


Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169 - DTR ND - ( A ) or HFD - treated ( 16 weeks ) ( B ) mice . A representative analysis of three independent experiments is shown .

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Representative flow cytometric showing the specific depletion efficiency of ATM subsets from eWAT of CD169-DTR ND- (A) or HFD-treated (16 weeks) (B) mice. A representative analysis of three independent experiments is shown.


eWAT was collected from lean ( C ) and obese ( HFD 16 weeks ) ( D ) WT or CD169 - DTR mice after 12 days DT - mediated ATM depletion . Representative pictures of dissected eWAT are shown on the left , the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right , with statistical significance calculated using the Student ' s t - test ( * * P < 0 . 01 and * * * * P < 0 . 0001 ) . The zoomed - in area shows clear , oily lipid accumulation in the absence of ATMs ( lower left part ) .

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

eWAT was collected from lean (C) and obese (HFD 16 weeks) (D) WT or CD169-DTR mice after 12 days DT-mediated ATM depletion. Representative pictures of dissected eWAT are shown on the left, the corresponding eWAT weights expressed as mean ± SD of more than 12 samples are shown in the right, with statistical significance calculated using the Student's t-test (**P < 0.01 and ****P < 0.0001). The zoomed-in area shows clear, oily lipid accumulation in the absence of ATMs (lower left part).


Adipocyte hypertrophy is visible in the absence of ATMs . Representative images of hematoxylin and eosin staining ( H & E ) ( E ) and corresponding adipocyte diameters ( F ) of lean and obese ( HFD 16 weeks ) eWAT from WT or CD169 - DTR mice treated for 7 or 12 days with DT . Scale bar , 50 μm . Each group comprised 3 - 5 mice . Statistical significance was determined using one - way ANOVA test . * * P < 0 . 01 ; * * * * P < 0 . 0001 ; ns : no significant difference . The box and whisker plots show the median value and 10 - 90 percentiles of adipocyte diameters .

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Adipocyte hypertrophy is visible in the absence of ATMs. Representative images of hematoxylin and eosin staining (H&E) (E) and corresponding adipocyte diameters (F) of lean and obese (HFD 16 weeks) eWAT from WT or CD169-DTR mice treated for 7 or 12 days with DT. Scale bar, 50 μm. Each group comprised 3-5 mice. Statistical significance was determined using one-way ANOVA test. **P < 0.01; ****P < 0.0001; ns: no significant difference. The box and whisker plots show the median value and 10-90 percentiles of adipocyte diameters.


qPCR showing the relative expression of genes for adipogenesis ( Pparγ ) , lipolysis ( Lpl ) , and lipogenesis ( Dgat1 ) , plus other lipid receptor / transporters ( Cd36 , Abca1 ) or chaperones ( Fabp4 ) in obese ( HFD 16 weeks ) eWAT of CD169 - DTR ( red bars ) mice and DT - injected WT controls ( blue bars ) ( upper panel ) and obese eWAT of anti - CSF1R antibody - injected mice ( green bars ) mice and isotype control - injected WT controls ( white bars ) ( lower panel ) . Mice were under DT or anti - CSF1R antibody treatment for 12 days . ( n = 5 pooled mice per group , mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , ns : no significant difference .

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qPCR showing the relative expression of genes for adipogenesis (Pparγ), lipolysis (Lpl), and lipogenesis (Dgat1), plus other lipid receptor/transporters (Cd36, Abca1) or chaperones (Fabp4) in obese (HFD 16 weeks) eWAT of CD169-DTR (red bars) mice and DT-injected WT controls (blue bars) (upper panel) and obese eWAT of anti-CSF1R antibody-injected mice (green bars) mice and isotype control-injected WT controls (white bars) (lower panel). Mice were under DT or anti-CSF1R antibody treatment for 12 days. (n = 5 pooled mice per group, mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significant difference.


Violin plots depicting the expression of representative marker genes for each CD45 - cell cluster : ASC1 ( Gsn , Pdgfrb , Eln ) ( red ) , ASC2 ( Pi16 , Pdgfra , Cebpd ) ( yellow ) , stromal cells ( Col1a1 , Col1a2 , Myl9 ) ( green ) , VEC1 / VEC2 ( Pecam - 1 , Cldn5 , Aqp1 ) ( purple and blue ) , and mesothelial - like cells ( MLCs ) ( Upk3b , Msln , Acta2 ) ( orange ) .

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Violin plots depicting the expression of representative marker genes for each CD45- cell cluster: ASC1 (Gsn, Pdgfrb, Eln) (red), ASC2 (Pi16, Pdgfra, Cebpd) (yellow), stromal cells (Col1a1, Col1a2, Myl9) (green), VEC1/VEC2 (Pecam-1, Cldn5, Aqp1) (purple and blue), and mesothelial-like cells (MLCs) (Upk3b, Msln, Acta2) (orange).


Representative 3D fluorescence imaging of lean and obese ( HFD 16 weeks ) eWAT samples stained with anti - MHCII ( green in lean samples ) , anti - CD11c ( green in obese samples ) , anti - CD31 ( blue ) , anti - CD169 ( pink ) , and DAPI ( white ) . The two images at the bottom show the images merged for the lean and obese conditions without DAPI . Scale bar : 100 μm ( lean ) and 70 μm ( obese ) .

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Representative 3D fluorescence imaging of lean and obese (HFD 16 weeks) eWAT samples stained with anti-MHCII (green in lean samples), anti-CD11c (green in obese samples), anti-CD31 (blue), anti-CD169 (pink), and DAPI (white). The two images at the bottom show the images merged for the lean and obese conditions without DAPI. Scale bar: 100 μm (lean) and 70 μm (obese).


Higher magnification of specific eWAT areas . Scale bar : 30 μm ( lean ) and 50 μm ( obese ) . ( left panels ) Distance of different ATM subsets to blood vessels . Distances ( in μm ) were calculated using the Imaris software , as described in the Materials and Methods section . The box and whisker plots show the median value and 10 - 90 percentiles of distance values . The right panels show the distribution of ATM subsets to blood vessels within a certain distance ( ≤ 1 μm , 1 - 10 μm , ≥ 10 μm ) . C : CD169 + MHCIIlow and CD169 + MHChi ATMs in lean eWAT . D : CD169 + CD11c - and CD169 + CD11c + ATMs in obese ( HFD 16 weeks ) eWAT . Four mice from two independent experiments were analyzed ( n = 4 - 5 ; mean ± SD ) . Statistical significance was determined using an unpaired Student ' s t - test . * P < 0 . 05 , * * * * P < 0 . 0001 , bars with no asterisks showed no significant difference .

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

Higher magnification of specific eWAT areas. Scale bar: 30 μm (lean) and 50 μm (obese). (left panels) Distance of different ATM subsets to blood vessels. Distances (in μm) were calculated using the Imaris software, as described in the Materials and Methods section. The box and whisker plots show the median value and 10-90 percentiles of distance values. The right panels show the distribution of ATM subsets to blood vessels within a certain distance (≤1 μm, 1-10 μm, ≥10 μm). C: CD169+MHCIIlow and CD169+MHChi ATMs in lean eWAT. D: CD169+CD11c- and CD169+CD11c+ ATMs in obese (HFD 16 weeks) eWAT. Four mice from two independent experiments were analyzed (n = 4-5; mean ± SD). Statistical significance was determined using an unpaired Student's t-test. *P < 0.05, ****P < 0.0001, bars with no asterisks showed no significant difference.


Model construction based on 3D reconstructed images showing the relative distance of CD11c + ATMs from traced large blood vessels . Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing ( white filaments ) . CD11c + ATMs are close ( < 10µm ) to the traced large blood vessels ( red ellipsoids ) or far ( > 10 µm ) ( grey ellipsoids ) from the traced blood vessel . Blood vessels are shown in green . Scale bar : 100 μm . The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤ 10 or > 10 μm to blood vessels quantified from 3 biological replicates from 1 experiment , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 .

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

Model construction based on 3D reconstructed images showing the relative distance of CD11c+ ATMs from traced large blood vessels. Blood vessels larger than 10µm diameter in the image were marked out on the image by tracing (white filaments). CD11c+ ATMs are close (< 10µm) to the traced large blood vessels (red ellipsoids) or far (>10 µm) (grey ellipsoids) from the traced blood vessel. Blood vessels are shown in green. Scale bar: 100 μm. The graph to the right shows the percentage of CD11chi ATMs showing a proximity of ≤10 or >10 μm to blood vessels quantified from 3 biological replicates from 1 experiment, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Two representative images of eWAT dissected from Evans blue - injected obese ( HFD 16 weeks ) WT and CD169 - DTR mice treated for 7 days with DT . Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm ( the absorption values were normalized to the tissue weight in grams ) . A chart showing the normalized mean optical density ± SD ( n = 5 - 7 mice per group ) . Statistical significance was determined using an unpaired Student ' s t - test . * * * P < 0 . 001 .

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

Two representative images of eWAT dissected from Evans blue-injected obese (HFD 16 weeks) WT and CD169-DTR mice treated for 7 days with DT. Evans blue dye was extracted from eWAT using formamide and the absorption of the extract was measured at 620 nm (the absorption values were normalized to the tissue weight in grams). A chart showing the normalized mean optical density ± SD (n = 5-7 mice per group). Statistical significance was determined using an unpaired Student's t-test. ***P < 0.001.


Mmp12 ( H ) and Vegfα ( F ) are highly expressed in MHCIIhi and CD11c + ATMs purified from obese ( HFD 16 weeks ) eWAT . qPCR analysis showed the relative expression in all three ATM subsets ( n = 3 ) , data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one - way ANOVA test . * P < 0 . 05 , * * P < 0 . 01 . qPCR analysis showing the relative expression of Vegfα in obese ( HFD 16 weeks ) eWAT of WT ( n = 3 ) or CD169 - DTR mice ( n = 5 ) treated for 7 days with DT , data was expressed as mean ± SD . Statistical significance was determined using an unpaired Student ' s t - test . * * * * P < 0 . 0001 .

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

Mmp12 (H) and Vegfα (F) are highly expressed in MHCIIhi and CD11c+ ATMs purified from obese (HFD 16 weeks) eWAT. qPCR analysis showed the relative expression in all three ATM subsets (n=3), data was expressed as mean ± SD . Statistical significance between the three ATM subpopulations was determined using one-way ANOVA test. *P < 0.05, **P < 0.01. qPCR analysis showing the relative expression of Vegfα in obese (HFD 16 weeks) eWAT of WT (n=3) or CD169-DTR mice (n=5) treated for 7 days with DT, data was expressed as mean ± SD. Statistical significance was determined using an unpaired Student's t-test. ****P < 0.0001.


Elastin staining showing more pronounced ECM accumulation in eWAT of CD169 - DTR ( HFD 16 weeks ) when compared with ECM accumulation in the WT obese ( HFD 16 weeks ) control ( DAPI , blue ; elastin , red ; CD31 , green ) . The fourth image in each row shows the three channels merged . Scale bar : 50 μm . Picrosirius red - staining depicts excessive collagen deposits in obese ( HFD 16 weeks ) eWAT of CD169 - DTR mice when compared with collagen deposits in WT controls . Scale bar : 250 μm . Upper panels : brightfield microscopy ; lower panels : fluorescence microscopy .

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

Elastin staining showing more pronounced ECM accumulation in eWAT of CD169-DTR (HFD 16 weeks) when compared with ECM accumulation in the WT obese (HFD 16 weeks) control (DAPI, blue; elastin, red; CD31, green). The fourth image in each row shows the three channels merged. Scale bar: 50 μm. Picrosirius red-staining depicts excessive collagen deposits in obese (HFD 16 weeks) eWAT of CD169-DTR mice when compared with collagen deposits in WT controls. Scale bar: 250 μm. Upper panels: brightfield microscopy; lower panels: fluorescence microscopy.


Violin plots showing the pro - inflammatory cytokine profile ( Il - 6 , Tnf - α , and Il - 1β ) of all 23 cell clusters obtained from obese ( HFD 16 weeks ) WT and CD169 - DTR eWAT . Cluster 0 : ASC1 ; Cluster 1 : Stromal cells ; Cluster 2 : VEC1 ; Cluster 3 : VEC2 ; Cluster 4 : NK1 ; Cluster 5 : MAC / Mono1 ; Cluster 6 : ASC2 ; Cluster 7 : B ; Cluster 8 : cDC1 ; Cluster 9 : mono - derived cDC ; Cluster 10 : Th2 / ILC2 / Treg ; Cluster 11 : MAC / Mono3 ; Cluster 12 : Th17 ; Cluster 13 : VEC2 ; Cluster 14 : CD8 ; Cluster 15 : MLCs ; Cluster 16 : CD11b + DC ; Cluster 17 : Neutrophils ; Cluster 18 : cDC2 ; Cluster 19 : cDC ; Cluster 20 : NKT ; Cluster 21 : NK2 ; Cluster 22 : Mast cells / Basophils . ASC : adipocyte stem cell ; VEC : vascular endothelial cell ; MLC : mesothelial - like cell ; NK : natural killer ; MAC : macrophage ; Mono : monocyte ; DC : dendritic cell ; NKT : natural killer T cells .

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

Violin plots showing the pro-inflammatory cytokine profile (Il-6, Tnf-α, and Il-1β) of all 23 cell clusters obtained from obese (HFD 16 weeks) WT and CD169-DTR eWAT. Cluster 0: ASC1; Cluster 1: Stromal cells; Cluster 2: VEC1; Cluster 3: VEC2; Cluster 4: NK1; Cluster 5: MAC/Mono1; Cluster 6: ASC2; Cluster 7: B; Cluster 8: cDC1; Cluster 9: mono-derived cDC; Cluster 10: Th2/ILC2/Treg; Cluster 11: MAC/Mono3; Cluster 12: Th17; Cluster 13: VEC2; Cluster 14: CD8; Cluster 15: MLCs; Cluster 16: CD11b+ DC; Cluster 17: Neutrophils; Cluster 18: cDC2; Cluster 19: cDC; Cluster 20: NKT; Cluster 21: NK2; Cluster 22: Mast cells/Basophils. ASC: adipocyte stem cell; VEC: vascular endothelial cell; MLC: mesothelial-like cell; NK: natural killer; MAC: macrophage; Mono: monocyte; DC: dendritic cell; NKT: natural killer T cells.


( a - b ) eIF4A knockdown in either S2 ( a ) or Kc167 ( b ) cells blunts the inactivation of TORC1 upon amino acid removal . Cells were treated with dsRNA targeting GFP as a negative control , or three independent , non - overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes . Representative of 3 biological replicates .

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

(a-b) eIF4A knockdown in either S2 (a) or Kc167 (b) cells blunts the inactivation of TORC1 upon amino acid removal. Cells were treated with dsRNA targeting GFP as a negative control, or three independent, non-overlapping dsRNAs targeting eIF4A for 5 days and then incubated with complete medium or medium lacking only amino acids for 30 minutes. Representative of 3 biological replicates.


( d ) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids , caused by removal of amino acid subsets . After 5 days of knockdown , S2 cells were treated for 30 min with either complete Schneider ' s medium ( + aa ) , Schneider ' s medium lacking all amino acids ( - aa ) or various subsets of amino acids , as indicated ( where EAA " essential amino acids " = H , I , L , K , M , T , W , V ) . Error bars : std dev . n = 3 biological replicates .

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

(d) Impaired inactivation of TORC1 in response to eIF4A knockdown is most apparent upon partial depletion of amino acids, caused by removal of amino acid subsets. After 5 days of knockdown, S2 cells were treated for 30 min with either complete Schneider's medium (+aa), Schneider's medium lacking all amino acids (-aa) or various subsets of amino acids, as indicated (where EAA "essential amino acids" = H,I,L,K,M,T,W,V). Error bars: std dev. n=3 biological replicates.


( e ) Time course of amino - acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying ( see drop in S6K and tubulin levels ) . Representative of two biological replicates .

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

(e) Time course of amino-acid removal reveals that eIF4A knockdown Kc167 cells maintain elevated S6K phosphorylation up to the maximum possible timepoint of 60 minutes when the cells start dying (see drop in S6K and tubulin levels). Representative of two biological replicates.


( f ) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids . Control ( w1118 ) or eIF4A1006 / 1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS / 1 % agarose + 2 % sucrose for 1h prior to lysis and immunoblot analysis . Two biological replicates are shown . Representative of 3 biological replicates .

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

(f) eIF4A mutant larvae have impaired TORC1 inactivation upon shifting to food lacking amino acids. Control (w1118) or eIF4A1006/1013 1st instar larvae were transferred from standard food to plates containing either standard fly food or PBS/1% agarose+2%sucrose for 1h prior to lysis and immunoblot analysis. Two biological replicates are shown. Representative of 3 biological replicates.


( a - a ' ) Although knockdown of eIF4A or eIF3 - S2 equally blunt translation of EGFP from an inducible plasmid ( a ) , only knockdown of eIF4A but not eIF3 - S2 impairs TORC1 inactivation upon amino acid removal ( a ' ) . ( a ) S2 cells , treated with indicated dsRNAs for 5 days , on day 3 transfected with an inducible EGFP plasmid ( pMT - EGFP ) and on day 4 induced for 18h . Non - targeting LacZ dsRNA used as a negative control . ( a ' ) S2 cells treated with indicated dsRNAs for 5 days , then incubated with medium lacking amino acids for indicated time prior to lysis . Representative of two biological replicates .

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

(a-a') Although knockdown of eIF4A or eIF3-S2 equally blunt translation of EGFP from an inducible plasmid (a), only knockdown of eIF4A but not eIF3-S2 impairs TORC1 inactivation upon amino acid removal (a'). (a) S2 cells, treated with indicated dsRNAs for 5 days, on day 3 transfected with an inducible EGFP plasmid (pMT-EGFP) and on day 4 induced for 18h. Non-targeting LacZ dsRNA used as a negative control. (a') S2 cells treated with indicated dsRNAs for 5 days, then incubated with medium lacking amino acids for indicated time prior to lysis. Representative of two biological replicates.


( b ) Knockdown of eIF4A , but not other translation initiation factors , blunts TORC1 inactivation upon amino acid withdrawal . Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider ' s medium or Schneider ' s medium lacking the indicated amino acids for 30 minutes prior to lysis .

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

(b) Knockdown of eIF4A, but not other translation initiation factors, blunts TORC1 inactivation upon amino acid withdrawal. Kc167 cells treated with indicated dsRNAs for 4 days and then incubated with complete Schneider's medium or Schneider's medium lacking the indicated amino acids for 30 minutes prior to lysis.


( c ) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells . Kc167 cells treated with either eIF4A dsRNA for 4 days , or cycloheximide ( 50 µg / mL , " CHX " ) for 5 min prior to , as well as during removal of amino acids ( - LIVASTQP ) . CHX treated cells still inactivate TORC1 ( compare lanes 8 to 7 ) whereas eIF4A knockdown cells do not ( lanes 14 vs 13 ) . Quantifications of two biological replicates are shown . CHX and eIF4A samples were normalized to their respective control conditions .

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

(c) Inhibition of translation with cycloheximide does not block inactivation of TORC1 upon amino acid withdrawal in Kc167 cells. Kc167 cells treated with either eIF4A dsRNA for 4 days, or cycloheximide (50 µg/mL, "CHX") for 5 min prior to, as well as during removal of amino acids (-LIVASTQP). CHX treated cells still inactivate TORC1 (compare lanes 8 to 7) whereas eIF4A knockdown cells do not (lanes 14 vs 13). Quantifications of two biological replicates are shown. CHX and eIF4A samples were normalized to their respective control conditions.


( d ) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min . Quantification of total intracellular amino acids , analyzed in a blinded fashion . Levels of individual amino acids shown in Figure S2D . For CHX samples , cycloheximide ( 50µg / mL ) was added 5 minutes prior to , and during treatment with medium containing or lacking amino acids . Statistical significance tested by ANOVA2 using Scheffé ' s multiple comparisons method ( p < 0 . 05 ) . Error bars : std . dev . n = 5 .

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

(d) Knockdown of eIF4A in Kc167 cells does not prevent a drop in intracellular amino acids levels when amino acids are removed from the medium for 30 min. Quantification of total intracellular amino acids, analyzed in a blinded fashion. Levels of individual amino acids shown in Figure S2D. For CHX samples, cycloheximide (50µg/mL) was added 5 minutes prior to, and during treatment with medium containing or lacking amino acids. Statistical significance tested by ANOVA2 using Scheffé's multiple comparisons method (p<0.05). Error bars: std. dev. n=5.


( a ) Knockdown of eIF4F components ( eIF - 4E and eIF - 4G ) partially phenocopy the eIF4A knockdown , leading to elevated TORC1 activity upon amino acid removal ( 30 min ) . Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal ( main Figures 1c , 2a ' and 2b ) . Error bars : std . dev . n = 3 biological replicates .

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

(a) Knockdown of eIF4F components (eIF-4E and eIF-4G) partially phenocopy the eIF4A knockdown, leading to elevated TORC1 activity upon amino acid removal (30 min). Knockdown of all other tested translation initiation factors do not cause elevated TORC1 upon amino acid removal (main Figures 1c, 2a' and 2b). Error bars: std. dev. n=3 biological replicates.


( b ) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown , explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity ( panel a ) are a bit milder than the eIF4A knockdown . Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click - it OPP reagent 30min before fixation and fluorescent labeling . Quantification of OPP fluorescence per cell ( nuclear count ) for two independent experiments is displayed ( 3 independent images per condition ) , normalized to the no dsRNA condition . Scale bars : 25µm .

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

(b) Quantification of de novo protein synthesis rates by OPP incorporation reveals that eIF4E and eIF4G knockdowns deplete eIF4F function less efficiently than the eIF4A knockdown, explaining why the effects of eIF4E and eIF4G knockdowns on TORC1 activity (panel a) are a bit milder than the eIF4A knockdown. Kc167 cells treated with dsRNA for 4 days then incubated with 20μM Click-it OPP reagent 30min before fixation and fluorescent labeling. Quantification of OPP fluorescence per cell (nuclear count) for two independent experiments is displayed (3 independent images per condition), normalized to the no dsRNA condition. Scale bars: 25µm.


( a ) Binding of eIF4G to the Rag GTPases detected by co - immunoprecipitation from Kc167 cells in absence or presence of the chemical cross - linker DSP . Representative of > 3 biological replicates .

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

(a) Binding of eIF4G to the Rag GTPases detected by co-immunoprecipitation from Kc167 cells in absence or presence of the chemical cross-linker DSP. Representative of >3 biological replicates.


( b ) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC , detected by expressing and immunoprecipitating either RagA or RagC alone . Representative of two biological replicates .

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

(b) Binding between eIF4G and the Rag GTPase complex is mainly mediated by RagC, detected by expressing and immunoprecipitating either RagA or RagC alone. Representative of two biological replicates.


( c ) Interaction between eIF4A and Raptor detected via co - immunoprecipitation of epitope - tagged proteins . Proteins were cross - linked with DSP prior to lysis and immunoprecipitation . Representative of 3 biological replicates .

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

(c) Interaction between eIF4A and Raptor detected via co-immunoprecipitation of epitope-tagged proteins. Proteins were cross-linked with DSP prior to lysis and immunoprecipitation. Representative of 3 biological replicates.


( d - d ' ' ) eIF4A and Raptor form a complex , detected by proximity ligation assay ( PLA ) . Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167 . ( d ) Representative images . Cells outlined in white . ( d ' ) Quantification of number of PLA spots per cell . ( n > 200 . * * * p < 0 . 0001 , ANOVA1 , Dunnett ' s multiple comparisons test , was performed on log - transformed data , box = + / - 1 quartile , whiskers = max / min ) . ( d ' ' ) Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns .

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(d-d'') eIF4A and Raptor form a complex, detected by proximity ligation assay (PLA). Specificity of the PLA signal was controlled by knocking down either eIF4A or Raptor in Kc167. (d) Representative images. Cells outlined in white. (d') Quantification of number of PLA spots per cell. (n>200. ***p<0.0001, ANOVA1, Dunnett's multiple comparisons test, was performed on log-transformed data, box = +/- 1 quartile, whiskers = max/min). (d'') Immunoblotting to detect efficiency of eIF4A and Raptor knockdowns.


( e - f ) The eIF4A - Raptor interaction does not take place on lysosomes ( e ) or the endoplasmic reticulum ( ER ) ( f ) . Kc167 cells were either incubated with 100 µg / mL Dextran for 1 hour , washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes ( e ) , or transfected to express an ER - resident GFP ( f ) . Amino acid removal was for 45 min . Pearson ' s correlation coefficients for eIF4A - Raptor PLA and Dextran in panel ( e ) are 0 . 09 and 0 . 08 for + aa and - LIVASTQP respectively ( calculated for all cells in the acquired fields , which were chosen randomly , n = 40 ) . Scale bars : 5µm .

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(e-f) The eIF4A-Raptor interaction does not take place on lysosomes (e) or the endoplasmic reticulum (ER) (f). Kc167 cells were either incubated with 100 µg/mL Dextran for 1 hour, washed and incubated for 14 hours in normal growth medium to label late endosomes and lysosomes (e), or transfected to express an ER-resident GFP (f). Amino acid removal was for 45 min. Pearson's correlation coefficients for eIF4A-Raptor PLA and Dextran in panel (e) are 0.09 and 0.08 for +aa and -LIVASTQP respectively (calculated for all cells in the acquired fields, which were chosen randomly, n=40). Scale bars: 5µm.


( a ) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner , indicating they act in the same , and not parallel pathways . Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells ( lanes 1 - 2 and 5 - 6 ) , eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells ( lanes 3 - 4 and 7 - 8 ) . Error bars : std dev . n = 3 biological replicates .

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(a) Knockdown of eIF4A and TSC2 do not elevate TORC1 activity in an additive manner, indicating they act in the same, and not parallel pathways. Whereas knockdown of eIF4A increases TORC1 activity in control Kc167 cells (lanes 1-2 and 5-6), eIF4A knockdown cannot further increase TORC1 activity in TSC2 knockdown cells (lanes 3-4 and 7-8). Error bars: std dev. n=3 biological replicates.


( b ) Activation of TSC2 with the p90RSK inhibitor BI - D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells , consistent with TSC2 acting downstream of eIF4A . BI - D1870 leads to inhibition of TORC1 ( lanes 1 - 4 ) in a TSC2 - dependent manner ( see also Appendix Figure S5A ) . Representative of two biological replicates .

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(b) Activation of TSC2 with the p90RSK inhibitor BI-D1870 rescues the elevated TORC1 activity caused by eIF4A knockdown in Kc167 cells, consistent with TSC2 acting downstream of eIF4A. BI-D1870 leads to inhibition of TORC1 (lanes 1-4) in a TSC2-dependent manner (see also Appendix Figure S5A). Representative of two biological replicates.


( c ) FLAG - eIF4A and TSC2 - V5 co - immunoprecipitate in Kc167 cells in the absence of chemical cross - linker . Cells were transfected with FLAG - eIF4A and TSC2 - V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti - FLAG immunoprecipitation . The experiment was performed in absence of the chemical cross - linker DSP . Representative of 3 biological replicates .

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(c) FLAG-eIF4A and TSC2-V5 co-immunoprecipitate in Kc167 cells in the absence of chemical cross-linker. Cells were transfected with FLAG-eIF4A and TSC2-V5 expression vectors and treated with media containing or lacking amino acids for indicated timepoints prior to lysis and anti-FLAG immunoprecipitation. The experiment was performed in absence of the chemical cross-linker DSP. Representative of 3 biological replicates.


( d ) Expression of TSC2 - insensitive ( S15H or Q63L ) , but not wild - type ( WT ) Rheb causes TORC1 activity to remain high upon amino acid removal . Kc167 cells were transfected to express either wild - type or mutant Rheb and then incubated with Schneider ' s medium either containing or lacking amino acids for 30 minutes . Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K , or phosphorylation of an HA - tagged S6K that was co - transfected with the Rheb constructs to assay specifically the transfected cells . Representative of two biological replicates .

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(d) Expression of TSC2-insensitive (S15H or Q63L), but not wild-type (WT) Rheb causes TORC1 activity to remain high upon amino acid removal. Kc167 cells were transfected to express either wild-type or mutant Rheb and then incubated with Schneider's medium either containing or lacking amino acids for 30 minutes. Elevated TORC1 activity can be observed either by looking at phosphorylation of endogenous S6K, or phosphorylation of an HA-tagged S6K that was co-transfected with the Rheb constructs to assay specifically the transfected cells. Representative of two biological replicates.


( a ) Mass spectrometry analysis of proteins co - immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins . eIF4A immunoprecipitations were performed in triplicate , from cells treated with medium either containing ( + aa ) or lacking amino acids ( - aa ) , and average values are shown . Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts . Raw peptide counts shown in parentheses . ( a ' ) Schematic diagram of eIF4G and NAT1 primary protein structures . Binding sites for other initiation factors are shown .

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(a) Mass spectrometry analysis of proteins co-immunoprecipitating with eIF4A reveals NAT1 as one of the top interacting proteins. eIF4A immunoprecipitations were performed in triplicate, from cells treated with medium either containing (+aa) or lacking amino acids (-aa), and average values are shown. Peptide counts for each protein in each replicate were normalized to eIF4A peptide counts. Raw peptide counts shown in parentheses.(a') Schematic diagram of eIF4G and NAT1 primary protein structures. Binding sites for other initiation factors are shown.


( b ) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1 - independent fashion . Co - immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells , or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times . Representative of 3 biological replicates .

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(b) Binding between eIF4A and NAT1 is regulated by amino acid availability in a TORC1-independent fashion. Co-immunoprecipitation of tagged eIF4A and NAT1 in control Kc167 cells, or cells treated with medium lacking amino acids or supplemented with 20 nM rapamycin for indicated times. Representative of 3 biological replicates.


( c ) Knockdown of NAT1 using 4 independent , non - overlapping dsRNAs leads to reduced TORC1 activity . Kc167 cells , treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(c) Knockdown of NAT1 using 4 independent, non-overlapping dsRNAs leads to reduced TORC1 activity. Kc167 cells, treated with indicated dsRNAs for 4 days and then incubated with medium containing or lacking amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( d ) eIF4A is epistatic to NAT1 for TORC1 regulation . In the absence of amino acids , eIF4A knockdown cells have elevated TORC1 activity , NAT1 knockdown cells have reduced TORC1 activity , and eIF4A NAT1 double - knockdown cells have elevated TORC1 activity compared to control cells . Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min . Error bars : std . dev . n = 3 biological replicates .

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(d) eIF4A is epistatic to NAT1 for TORC1 regulation. In the absence of amino acids, eIF4A knockdown cells have elevated TORC1 activity, NAT1 knockdown cells have reduced TORC1 activity, and eIF4A NAT1 double-knockdown cells have elevated TORC1 activity compared to control cells. Cells were treated with indicated dsRNAs for 4 days and then treated with medium containing or lacking the indicated amino acids for 30 min. Error bars: std. dev. n=3 biological replicates.


( e ) TSC2 is epistatic to NAT1 for TORC1 regulation . As in ( d ) , knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown .

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(e) TSC2 is epistatic to NAT1 for TORC1 regulation. As in (d), knockdown of TSC2 rescues the reduced TORC1 activity caused by NAT1 knockdown.


A and B ) Confocal microscopy images showing mitochondrial fragmentation in HBV - and HBx - expressing cells , respectively . Huh7 cells were transiently transfected with HBV ( A ) or the HBx - flag construct ( B ) . At 2 days post - transfection , cells prestained with MitoTracker ( Mito , white ) were immunostained with anti - HBsAg ( A , green ) and anti - flag ( B , green ) antibodies , respectively . In the zoomed images , typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown .

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A and B) Confocal microscopy images showing mitochondrial fragmentation in HBV- and HBx-expressing cells, respectively. Huh7 cells were transiently transfected with HBV (A) or the HBx-flag construct (B). At 2 days post-transfection, cells prestained with MitoTracker (Mito, white) were immunostained with anti-HBsAg (A, green) and anti-flag (B, green) antibodies, respectively. In the zoomed images, typical tubular mitochondria in untransfected cells and fragmented mitochondria in transfected cells are shown.


C ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins . β - actin was used as an internal loading control . Mitochondrial fraction ( bottom panel ) isolated from HepAD38 cells ( see the purity in Figure 2B ) was analyzed by Western blotting with phospho - Drp1 ( Ser616 ) antibody .

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C) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct were analyzed by Western blotting with antibodies against the indicated proteins. β-actin was used as an internal loading control. Mitochondrial fraction (bottom panel) isolated from HepAD38 cells (see the purity in Figure 2B) was analyzed by Western blotting with phospho-Drp1 (Ser616) antibody.


( D - F ) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV - and HBx - expressing cells , respectively . Huh7 cells transfected with HBV ( D and E ) or HBx - flag construct ( F ) were immunostained with antibodies specific to TOM20 ( red ) , Drp1 ( D , green ) , and phospho - Drp1 ( Ser616 ) ( E and F , green ) , respectively . HBV and HBx gene expression ( white ) was verified using anti - HBsAg ( D and E ) and anti - flag ( F ) antibodies , respectively . In the zoomed images , the yellow color indicates the merge of Drp1 or phospho - Drp1 ( Ser616 ) with mitochondria . ( A , B , D , E , and F ) Transfected ( + ) and untransfected ( − ) cells are marked . Nuclei were stained with DAPI ( blue ) .

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(D-F) Confocal immunofluorescence showing mitochondrial translocation of Drp1 in HBV- and HBx-expressing cells, respectively. Huh7 cells transfected with HBV (D and E) or HBx-flag construct (F) were immunostained with antibodies specific to TOM20 (red), Drp1 (D, green), and phospho-Drp1 (Ser616) (E and F, green), respectively. HBV and HBx gene expression (white) was verified using anti-HBsAg (D and E) and anti-flag (F) antibodies, respectively. In the zoomed images, the yellow color indicates the merge of Drp1 or phospho-Drp1 (Ser616) with mitochondria. (A, B, D, E, and F) Transfected (+) and untransfected (−) cells are marked. Nuclei were stained with DAPI (blue).


( A ) Huh7 cells transfected with HBV construct were prestained with MitoTracker ( Mito , red ) and subsequently , immunostained with anti - Parkin ( green ) and anti - HBsAg ( white ) antibodies . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria . Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV - expressing cells .

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(A) Huh7 cells transfected with HBV construct were prestained with MitoTracker (Mito, red) and subsequently, immunostained with anti-Parkin (green) and anti-HBsAg (white) antibodies. Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, yellow color indicates the accumulation of endogenous Parkin recruited to the mitochondria. Quantification of fluorescence intensity of Parkin aggregates on the mitochondria in HBV-expressing cells.


( B ) The cytosolic ( Cyto ) and mitochondrial ( Mito ) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h . Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins . Fractions : WCL , whole cell lysates ; Cyto , purified cytoplasm ; Mito , purified mitochondria . Organelle markers : VDAC1 , mitochondria ; GAPDH , cytoplasm .

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(B) The cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated from HepAD38 cells grown in the absence or presence of tetracycline for 48 h. Cellular fractions were analyzed by Western blotting with antibodies specific for the indicated proteins. Fractions: WCL, whole cell lysates; Cyto, purified cytoplasm; Mito, purified mitochondria. Organelle markers: VDAC1, mitochondria; GAPDH, cytoplasm.


( C ) Parkin protein in whole cell lysates of HBV - expressing cells was immunoprecipitated by anti - Parkin antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal rabbit IgG was used as a control for immunoprecipitation ( IP ) . Western blot analysis for β - actin indicates equivalent amount of cell lysates for IP ( C and D ) .

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(C) Parkin protein in whole cell lysates of HBV-expressing cells was immunoprecipitated by anti-Parkin antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal rabbit IgG was used as a control for immunoprecipitation (IP). Western blot analysis for β-actin indicates equivalent amount of cell lysates for IP (C and D).


( D ) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non - targeting ( NT ) and Parkin ( P ) siRNA , respectively , for 48 h was immunoprecipitated by anti - Mfn2 antibody , followed by immunoblotting ( IB ) with anti - ubiquitin ( Ub ) antibody . Normal mouse IgG was used as a control for immunoprecipitation ( IP ) . The protein expression was analyzed by Western blotting with antibodies specific to Mfn2 , Parkin , and β - actin proteins . (

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(D) Mfn2 protein in whole cell lysates extracted from HepAD38 cells transfected with non-targeting (NT) and Parkin (P) siRNA, respectively, for 48 h was immunoprecipitated by anti-Mfn2 antibody, followed by immunoblotting (IB) with anti-ubiquitin (Ub) antibody. Normal mouse IgG was used as a control for immunoprecipitation (IP). The protein expression was analyzed by Western blotting with antibodies specific to Mfn2, Parkin, and β-actin proteins. (


( E ) Intracellular mRNA levels of Parkin , PINK1 , and LC3B were analyzed by real - time qRT - PCR . GAPDH was used to normalize changes in Parkin , PINK1 , and LC3B gene expression .

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(E) Intracellular mRNA levels of Parkin, PINK1, and LC3B were analyzed by real-time qRT-PCR. GAPDH was used to normalize changes in Parkin, PINK1, and LC3B gene expression.


( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading cont ( F ) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins . β - actin was used as a loading control ( F and G ) . The relative intensity of ATF4 , Parkin , PINK1 , and LC3B protein expression normalized to β - actin was analyzed by ImageJ software .

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(F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading cont(F) The protein expression was analyzed by Western blotting with antibodies specific for the indicated proteins. β-actin was used as a loading control (F and G). The relative intensity of ATF4, Parkin, PINK1, and LC3B protein expression normalized to β-actin was analyzed by ImageJ software.


. ( G ) Whole cell lysates extracted from Huh7 cells with empty vector ( Mock ) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins .

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. (G) Whole cell lysates extracted from Huh7 cells with empty vector (Mock) and those with HBV construct for 48 h were analyzed by Western blotting with antibodies specific to the indicated proteins.


( A ) Huh7 cells transiently expressing GFP - LC3 protein were transfected with HBV construct in the absence or presence of 3 - MA ( 10 mM ) and BafA1 ( 100 nM ) , respectively , for 8 h before fixation . At 2 days post - transfection , cells were immunostained with antibodies against HBsAg ( white ) , TOM20 ( blue ) , and Parkin ( red ) . Nuclei are demarcated with white dots . Transfected ( + ) and untransfected ( − ) cells are marked . In the zoomed images , the arrows ( white puncta ) indicate GFP - LC3 puncta ( green ) colocalized with TOM20 and Parkin .

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(A) Huh7 cells transiently expressing GFP-LC3 protein were transfected with HBV construct in the absence or presence of 3-MA (10 mM) and BafA1 (100 nM), respectively, for 8 h before fixation. At 2 days post-transfection, cells were immunostained with antibodies against HBsAg (white), TOM20 (blue), and Parkin (red). Nuclei are demarcated with white dots. Transfected (+) and untransfected (−) cells are marked. In the zoomed images, the arrows (white puncta) indicate GFP-LC3 puncta (green) colocalized with TOM20 and Parkin.