Figure 3Expression data ( RNAseq ) for significantly differentially expressed genes ( q - value < 0 . 05 , FDR - adjusted p - value , n = 2 biological replicates for each condition ) . Scatter plot shows log2 ( fold change ) of gene expression data comparing endpoint to initial populations for Exp . 1 and Exp . 2 ( grey dots ) with the location of the gene in the reference genome as the x - axis . Those genes that are associated with AraC transcription units are highlighted ( red dots for Exp . 1 and blue dots for Exp . 2 ) . Above the plot , the transcription units are labeled green if AraC activates expression ( in the presence of arabinose ) or red if AraC represses expression of those genes . Growth rate analysis of various weaned ( starting point of static phase ) and optimized ( endpoint of static phase ) strains with or without fucK or araB genes knocked out . Strains were grown in triplicate ( n = 3 ) on M9 minimal media with D - arabinose as the sole carbon source . The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation . The p - values reported were calculated using a two - sided Welch ' s t - test .

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

Figure 3Expression data (RNAseq) for significantly differentially expressed genes (q-value < 0.05, FDR-adjusted p-value, n=2 biological replicates for each condition). Scatter plot shows log2(fold change) of gene expression data comparing endpoint to initial populations for Exp. 1 and Exp. 2 (grey dots) with the location of the gene in the reference genome as the x-axis. Those genes that are associated with AraC transcription units are highlighted (red dots for Exp. 1 and blue dots for Exp. 2). Above the plot, the transcription units are labeled green if AraC activates expression (in the presence of arabinose) or red if AraC represses expression of those genes.Growth rate analysis of various weaned (starting point of static phase) and optimized (endpoint of static phase) strains with or without fucK or araB genes knocked out. Strains were grown in triplicate (n=3) on M9 minimal media with D-arabinose as the sole carbon source. The colored bars represent the calculated mean growth rate and the error bars represent the standard deviation. The p-values reported were calculated using a two-sided Welch's t-test.


Figure 1 ( A ) Cell lysates prepared from HeLa cells were solubilized with Triton X - 100 and subjected to immunoprecipitation using preimmune sera ( lane 1 ) or anti - Beclin antibodies ( lanes 2 and 3 ) . The immunoprecipitates were incubated with phosphatidylinositol , [ γ - 32P ] ATP and 60 μM cold ATP in the presence of Mn2 + ( lanes 1 and 2 ) or Mg2 + ( lane 3 ) for 5 min at 30 ° C . ( B ) Immunoprecipitates of anti ‐ Beclin antibodies prepared as described in ( A ) were assayed for PI 3 ‐ kinase activity in the presence of wortmannin at concentrations of 0 nM ( lane 1 ) , 1 nM ( lane 2 ) , 10 nM ( lane 3 ) , 100 nM ( lane 4 ) . The labeled lipids were extracted and separated by thin layer chromatography , followed by detection by autoradiography using a bioimaging analyzer BAS2000 ( Fuji Film ) . ( C ) Cell lysates prepared from HeLa cells were solubilized with Triton X ‐ 100 and incubated with protein A ‐ immobilized anti ‐ Beclin ( lane 2 ) or anti ‐ PtdIns 3 ‐ kinase antibodies ( lane 4 ) . As controls , preimmune sera of the respective antibodies ( lanes 1 and 3 ) were used . Retained proteins were separated by SDS - PAGE and detected by immunoblotting with anti ‐ PtdIns 3 ‐ kinase ( upper panels ) and anti ‐ Beclin ( lower panels ) antibodies .

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 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 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

Figure 1(A) Cell lysates prepared from HeLa cells were solubilized with Triton X-100 and subjected to immunoprecipitation using preimmune sera (lane 1) or anti-Beclin antibodies (lanes 2 and 3). The immunoprecipitates were incubated with phosphatidylinositol, [γ-32P]ATP and 60 μM cold ATP in the presence of Mn2+ (lanes 1 and 2) or Mg2+ (lane 3) for 5 min at 30°C.(B) Immunoprecipitates of anti‐Beclin antibodies prepared as described in (A) were assayed for PI 3‐kinase activity in the presence of wortmannin at concentrations of 0 nM (lane 1), 1 nM (lane 2), 10 nM (lane 3), 100 nM (lane 4). The labeled lipids were extracted and separated by thin layer chromatography, followed by detection by autoradiography using a bioimaging analyzer BAS2000 (Fuji Film).(C) Cell lysates prepared from HeLa cells were solubilized with Triton X‐100 and incubated with protein A‐immobilized anti‐Beclin (lane 2) or anti‐PtdIns 3‐kinase antibodies (lane 4). As controls, preimmune sera of the respective antibodies (lanes 1 and 3) were used. Retained proteins were separated by SDS-PAGE and detected by immunoblotting with anti‐PtdIns 3‐kinase (upper panels) and anti‐Beclin (lower panels) antibodies.


Figure 2 ( A ) Total cell lysates were treated with DSP ( lanes 3 , 4 , 7 and 8 ) or its solvent , dimethylsulfoxide ( lanes 1 , 2 , 5 and 6 ) , at 4 ° C for 2 h . Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A ‐ immobilized anti ‐ PtdIns 3 ‐ kinase ( PtdIns 3 ‐ K ) ( lanes 1 - 4 ) or Beclin ( lanes 5 - 8 ) antibodies . Immunoprecipitates were eluted with 1 × SDS sample buffer ( 62 . 5 mM Tris - HCl pH 6 . 8 , 2 % SDS , 10 % glycerol , a trace amount of Bromophenol blue ) . After treatment with 5 % 2 ‐ mercaptoethanol , proteins were separated by SDS - PAGE , followed by detection by immunoblotting with anti ‐ Beclin ( upper panels ) and anti ‐ PtdIns 3 ‐ kinase ( lower panels ) antibodies . ( B ) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h . The membrane ( lanes 1 and 3 ) and supernatant ( lanes 2 and 4 ) fractions were subjected to SDS - PAGE , followed by immunoblotting with anti ‐ PtdIns 3 ‐ kinase ( lanes 1 and 2 ) and anti ‐ Beclin ( lanes 3 and 4 ) antibodies . ( C ) Membrane ( lanes 1 - 4 ; 100 000 g , 30 min , pellet ) and soluble fractions ( lanes 5 - 8 ; 100 000 g , 30 min , supernatant ) prepared from HeLa cells were treated with DSP ( lanes 3 , 4 , 7 and 8 ) or dimethylsulfoxide ( lanes 1 , 2 , 5 and 6 ) . Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A - immobilized anti - PtdIns 3 - kinase antibodies . Immunoprecipitates were separated by SDS - PAGE under a reducing condition , followed by detection by immunoblotting with Beclin antibodies . F and E indicate flow - through and elute fracions , respectively .

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

Figure 2(A) Total cell lysates were treated with DSP (lanes 3, 4, 7 and 8) or its solvent, dimethylsulfoxide (lanes 1, 2, 5 and 6), at 4°C for 2 h. Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A‐immobilized anti‐PtdIns 3‐kinase (PtdIns 3‐K) (lanes 1-4) or Beclin (lanes 5-8) antibodies. Immunoprecipitates were eluted with 1× SDS sample buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, a trace amount of Bromophenol blue). After treatment with 5% 2‐mercaptoethanol, proteins were separated by SDS-PAGE, followed by detection by immunoblotting with anti‐Beclin (upper panels) and anti‐PtdIns 3‐kinase (lower panels) antibodies.(B) Cell lysates prepared from HeLa cells were subjected to subcellular fractionation by centrifugation at 100 000 g for 1 h. The membrane (lanes 1 and 3) and supernatant (lanes 2 and 4) fractions were subjected to SDS-PAGE, followed by immunoblotting with anti‐PtdIns 3‐kinase (lanes 1 and 2) and anti‐Beclin (lanes 3 and 4) antibodies.(C) Membrane (lanes 1-4; 100 000 g, 30 min, pellet) and soluble fractions (lanes 5-8; 100 000 g, 30 min, supernatant) prepared from HeLa cells were treated with DSP (lanes 3, 4, 7 and 8) or dimethylsulfoxide (lanes 1, 2, 5 and 6). Proteins were solubilized with SDS and subjected to immunoprecipitation using protein A-immobilized anti-PtdIns 3-kinase antibodies. Immunoprecipitates were separated by SDS-PAGE under a reducing condition, followed by detection by immunoblotting with Beclin antibodies. F and E indicate flow-through and elute fracions, respectively.


Figure 3Beclin localizes to the TGN . HeLa ( A , D , E and F ) , BNL CL . 2 ( B ) and H ‐ 4 ‐ II ‐ E cells ( C ) were grown on cover slips . Cells were permeabilized with digitonin before ( C , D and E ) or after ( A , B and F ) fixation with 3 % paraformaldehyde . Cells were double ‐ labeled with anti ‐ Beclin ( A - F , left panels ) and either anti ‐ syntaxin 6 ( A and B , middle panels ) , anti ‐ TGN38 ( C , middle panel ) , anti ‐ EEA1 ( D , middle panel ) , anti ‐ LBPA ( E , middle panel ) or anti ‐ TfR ( F , middle panel ) antibodies . In the merged images ( right panels ) , yellow indicates co ‐ localization . Bar , 10 μm .

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Figure 3Beclin localizes to the TGN. HeLa (A, D, E and F), BNL CL.2 (B) and H‐4‐II‐E cells (C) were grown on cover slips. Cells were permeabilized with digitonin before (C, D and E) or after (A, B and F) fixation with 3% paraformaldehyde. Cells were double‐labeled with anti‐Beclin (A-F, left panels) and either anti‐syntaxin 6 (A and B, middle panels), anti‐TGN38 (C, middle panel), anti‐EEA1 (D, middle panel), anti‐LBPA (E, middle panel) or anti‐TfR (F, middle panel) antibodies. In the merged images (right panels), yellow indicates co‐localization. Bar, 10 μm.


Figure 4Immunofluorescence localization of PtdIns 3 ‐ kinase . HeLa ( A and C ) and H ‐ 4 ‐ II ‐ E cells ( B ) were permeabilized , fixed , and then subjected to immunostaining . Cells were double ‐ labeled with anti ‐ PtdIns 3 ‐ kinase ( A - C , left panels ) and either anti ‐ syntaxin 6 ( A , middle panel ) , anti ‐ TGN38 ( B , middle panel ) or anti ‐ LBPA ( C , middle panel ) antibodies . Bar , 10 μm .

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Figure 4Immunofluorescence localization of PtdIns 3‐kinase. HeLa (A and C) and H‐4‐II‐E cells (B) were permeabilized, fixed, and then subjected to immunostaining. Cells were double‐labeled with anti‐PtdIns 3‐kinase (A-C, left panels) and either anti‐syntaxin 6 (A, middle panel), anti‐TGN38 (B, middle panel) or anti‐LBPA (C, middle panel) antibodies. Bar, 10 μm.


Figure 1Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH . Shown are maximum projections of 50 µm z - stacks covering an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions of the CV ( orange ) and PV ( blue ) areas are shown as magnifications on the left and right , respectively . Quantification of BC diameter within 18 zones along the CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) in livers from untreated animals or mice at indicated timepoints post PH . The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in ( a ) . The zones directly adjacent to the CV and PV were excluded from the analysis ( ~ 1 cell layer ) . Mean ± s . e . m , n = 3 - 6 mice per timepoint . BC diameter of untreated mice vs . 0 . 8 d , p = 1 . 62 * 10 - 11 ; untreated mice vs . 1 . 5 d , p = 1 . 18 * 10 - 12 ; untreated mice vs . 2 . 0 d , p = 5 . 69 * 10 - 11 ; untreated mice vs . 3 . 0 d , p = 6 . 92 * 10 - 12 ; untreated mice vs . 5 . 0 d , p = 1 . 65 * 10 - 7 . EM images of BC on liver tissue sections of untreated mice ( left ) and 1 . 8 d post sham OP ( middle ) or PH ( right ) . Quantification of BC perimeter ( d ) and total BC membrane length ( e ) from EM images as representatively shown in ( c ) . Box - whisker plot with median , 25 - 75 quartiles and minimum / maximum error bars , n = 5 - 6 mice per condition . In ( d ) , BC perimeter of untreated vs . sham condition , p = 0 . 94 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 01 . In ( e ) , BC membrane length of untreated vs . sham condition , p = 0 . 86 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 03 . IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL . Shown are maximum projections of 50 µm z - stacks in the CV ( left , orange ) and PV ( right , blue ) region . Images in ( a ) and ( f ) are background - subtracted . Scale bars , 50 µm ( a ) , 1 µm ( c ) , 5 µm ( f ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 1Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH. Shown are maximum projections of 50 µm z-stacks covering an entire CV-PV axis (CV, left; PV, right). Indicated regions of the CV (orange) and PV (blue) areas are shown as magnifications on the left and right, respectively.Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) in livers from untreated animals or mice at indicated timepoints post PH. The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=3-6 mice per timepoint. BC diameter of untreated mice vs. 0.8 d, p=1.62*10-11; untreated mice vs. 1.5 d, p=1.18*10-12; untreated mice vs. 2.0 d, p=5.69*10-11; untreated mice vs. 3.0 d, p=6.92*10-12; untreated mice vs. 5.0 d, p=1.65*10-7.EM images of BC on liver tissue sections of untreated mice (left) and 1.8 d post sham OP (middle) or PH (right).Quantification of BC perimeter (d) and total BC membrane length (e) from EM images as representatively shown in (c). Box-whisker plot with median, 25-75 quartiles and minimum/maximum error bars, n=5-6 mice per condition. In (d), BC perimeter of untreated vs. sham condition, p=0.94 (n.s.); Untreated vs. PH condition, p=0.01. In (e), BC membrane length of untreated vs. sham condition, p=0.86 (n.s.); Untreated vs. PH condition, p=0.03.IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL. Shown are maximum projections of 50 µm z-stacks in the CV (left, orange) and PV (right, blue) region. Images in (a) and (f) are background-subtracted. Scale bars, 50 µm (a), 1 µm (c), 5 µm (f).


Figure 2Fluorescence stainings for F - actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm Quantification of apical F - actin intensity from images as representatively shown in ( a , as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) . Data is normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 ( b ) mice per timepoint . Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm Quantification of pMLC ( d ) intensity from images as representatively shown in ( a , c ) as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) . Data is normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 2 - 5 ( d ) mice per timepoint .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2Fluorescence stainings for F-actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of apical F-actin intensity from images as representatively shown in (a, as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) mice per timepoint.Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=2-5 (d) mice per timepoint.


Figure 3Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH . Images show an entire CV - PV axis ( CV , left ; PV , right ) . Note , the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct . Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH . Images show an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions ( orange squares ) are shown as magnifications in ( b ) . Note , the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct . Quantification of the mean nuclear YAP ( green ) and PCNA ( magenta ) intensity from images of liver tissue sections at indicated timepoints post PH ( solid line ) and sham OP ( dashed line ) as representatively shown in ( a ) and Appendix Fig . S4A . Data was normalized to untreated animals ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . Nuclear YAP intensity of PH vs . sham time course , p = 2 . 97 * 10 - 5 . Nuclear PCNA intensity of PH vs . sham time course , p = 7 . 51 * 10 - 6 . Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in ( a ) and Appendix Fig . S4A , at indicated time points post PH ( solid line ) or sham OP ( dashed line ) in 10 zones within the CV - PV axis ( zone 1 , CV area ; zone 10 , PV area ) . Data is normalized to untreated animals ( not shown ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . Nuclear YAP intensity of sham vs . PH mice at 0 . 8d , p = 5 . 35 * 10 - 5 ; 1 . 5d , p = 3 . 95 * 10 - 5 ; 2d , p = 5 . 99 * 10 - 5 ; 3d , p = 2 . 16 * 10 - 5 ; 5d , p = 0 . 01 . Fluorescence stainings for YAP , CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH . Arrows indicate BC . Note the enrichment of apical YAP at 0 . 5 and 1 . 5d post PH . Fractionation of liver tissue at 2d post PH or sham ( Sh ) OP and untreated ( UT ) mice for nuclear ( Nuc ) , cytoplasmic ( Cyto ) and membrane ( Mem ) fraction . TRF2 , transferrin receptor2 ( membrane marker ) ; Histone H3 ( nuclear marker ) ; GAPDH , Glyceraldehyde 3 - phosphate dehydrogenase ( cytoplasmic marker ) . Quantification of YAP in the nuclear , cytoplasmic and membrane fraction from immunoblots as representatively shown in ( f ) . YAP signal is normalized to the fraction marker ( GAPDH , cytoplasm ; Histon H3 , nuclei ; TRF2 , membrane ) and to the untreated condition ( not shown ) . n = 3 ( sham ) or 4 ( PH ) ; data is shown as mean + / - s . e . m . Differences between sham and PH conditions for cytoplasmic , nuclear and membrane fractions are not significant .

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

Figure 3Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.Quantification of the mean nuclear YAP (green) and PCNA (magenta) intensity from images of liver tissue sections at indicated timepoints post PH (solid line) and sham OP (dashed line) as representatively shown in (a) and Appendix Fig. S4A. Data was normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of PH vs. sham time course, p=2.97*10-5. Nuclear PCNA intensity of PH vs. sham time course, p=7.51*10-6.Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in (a) and Appendix Fig. S4A, at indicated time points post PH (solid line) or sham OP (dashed line) in 10 zones within the CV-PV axis (zone 1, CV area; zone 10, PV area). Data is normalized to untreated animals (not shown). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of sham vs. PH mice at 0.8d, p=5.35*10-5; 1.5d, p=3.95*10-5; 2d, p=5.99*10-5; 3d, p=2.16*10-5; 5d, p=0.01.Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.Fractionation of liver tissue at 2d post PH or sham (Sh) OP and untreated (UT) mice for nuclear (Nuc), cytoplasmic (Cyto) and membrane (Mem) fraction. TRF2, transferrin receptor2 (membrane marker); Histone H3 (nuclear marker); GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (cytoplasmic marker). Quantification of YAP in the nuclear, cytoplasmic and membrane fraction from immunoblots as representatively shown in (f). YAP signal is normalized to the fraction marker (GAPDH, cytoplasm; Histon H3, nuclei; TRF2, membrane) and to the untreated condition (not shown). n = 3 (sham) or 4 (PH); data is shown as mean +/- s.e.m. Differences between sham and PH conditions for cytoplasmic, nuclear and membrane fractions are not significant.


Figure 4EM image of immunogold - labelled YAP on liver tissue sections at 1 . 8 d post PH . Image ( A ) shows a BC in between two hepatocytes . Indicated regions ( black squares ) of the apical ( a ' ) and the basolateral ( a ' ' ) membrane of the hepatocyte are shown as magnification . Arrows indicate gold particles . Correlative light and electron microcopy of F - actin ( B ) and YAP ( C ) on a liver tissue section at 1 . 8 d post PH . YAP was detected by immunogold - labelling , F - actin by fluorescence staining with phalloidin . Indicated BC regions with high ( c ' , c ' ' ) or low ( c ' ' ' ) F - actin levels are shown as magnifications . Arrows indicate gold particles .

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 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

Figure 4EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.


Figure 5Fluorescence stainings for YAP ( green ) , F - actin ( magenta ) and with the nuclear marker DAPI ( blue ) of primary hepatocyte cultures treated with DMSO ( control ) , Y27 , deoxycholic acid ( DCA ) or DCA + Y27 for ~ 18 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets . Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO ( control ) , Y27 , DCA or DCA + Y27 for ~ 18 h as representatively shown in ( A ) . Data is normalized to untreated cells ( not shown ) . Mean ± s . e . m . , n = 7 ; DCA vs . DMSO p = 8 . 98 * 10 - 5 ; DCA + Y27 vs . DMSO , p = 0 . 05 ; Y27 vs . DMSO , p = 0 . 55 ( n . s . ) ; DCA + Y27 vs . DCA p = 0 . 05Western blot of pMLC and GAPDH ( loading control ) of primary hepatocyte culture lysates . Cells were untreated or incubated for 18 h with the indicated compoundsFluorescence stainings of primary hepatocytes for YAP ( green ) , F - actin ( magenta ) and with the nuclear marker DAPI ( blue ) . Cells were treated with DMSO ( control ) , Y27 , SMIFH2 or Cytochalasin D ( CytoD ) for 6 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets . Note the dilation of canaliculi ( asterisks ) and fragmentation of F - actin ( arrows ) upon CytoD treatment . Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO , Saline , Y27 , Fasudil , SMIFH2 , CK666 , CytoD or Latrunculin A ( LatA ) for 6 h . Saline serves as control for Fasudil , DMSO serves as control for all other conditions . Inhibitors affecting similar actin processes are displayed in the same grey level . Saline , Fasudil , CK666 and LatA conditions are not shown in ( d ) . Data is normalized to untreated cells ( not shown ) . Mean ± s . e . m . , n = 3 - 5 ; DMSO vs . Y27 , p = 0 . 002 ; DMSO vs . CytoD , p = 5 . 11 * 10 - 7 ; DMSO vs . LatA , p = 0 . 01 ; DMSO vs . CK666 , p = 0 . 15 ( n . s . ) ; DMSO vs . SMIFH2 , p = 0 . 11 ( n . s . ) ; Saline vs . Fasudil , p = 0 . 92 . Western blot of pMLC and GAPDH ( loading control ) in untreated or actin inhibitor - treated primary hepatocyte culture lysates . Inhibitor treatments are the same as in ( E ) . Scale bars , 20 µm ( A , D ) .

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

Figure 5Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compoundsFluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).


Figure 6Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH , treated with saline ( control ) or the Rho kinase inhibitor Fasudil for 1 h . Shown are maximum projections of 50 µm stacks in the PV area . Quantification of BC diameter within 18 zones along the CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) from mice at 2 d post sham OP ( green ) or PH ( red ) , treated with saline ( control , solid line ) or Fasudil ( dashed line ) . Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in ( a ) . The zones directly adjacent to the CV and PV were excluded from the analysis ( ~ 1 cell layer ) . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . BC diameter of sham operated mice treated with saline vs . Fasudil , p = 6 . 59 * 10 - 8 ; BC diameter of PH mice treated with saline vs . Fasudil , p = 2 . 31 * 10 - 14 . Fluorescence stainings for YAP , PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH , treated with saline ( control ) or Fasudil for 1 h . Indicated regions ( dashed rectangle ) are shown as magnifications in insets . Quantification of the mean nuclear YAP intensity within 10 zones along the CV - PV axis , ( zone 1 , peri - central ; zone 10 , peri - portal ) from IF images as representatively shown in ( c ) . Data was normalized to untreated animals . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . Nuclear YAP intensity of sham operated mice treated with saline vs . Fasudil , p = 0 . 13 ( n . s . ) ; nuclear YAP intensity of PH mice treated with saline vs . Fasudil , p = 0 . 0004 .

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 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

Figure 6Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area. Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.


Figure 4A Pancreatic sections ( 20X magnification ) from 10 - months old WT and Glo1KD mice stained with hematoxylin and eosin ( H & E ) , anti - insulin , anti - glucagon and anti - F4 / 80 antibodies , are representative of sections from 7 mice each group . H , I and J Insulin and glucagon IS , calculated as the intensity score multiplied with percentage of positive cells ( H , I ) and the percentage of islets area stained with F4 / 80 ( J ) ( n = 7 mice for each genotype ) . Data information : box plots show the minimum and maximum values ( ends of the whiskers ) , interquartile range ( length of the box ) , median ( line through the box ) and mean ( + ) values . * p ≤ 0 . 05 , * * p ≤ 0 . 01 ( Mann - Whitney test ) .

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Figure 4A Pancreatic sections (20X magnification) from 10-months old WT and Glo1KD mice stained with hematoxylin and eosin (H&E), anti-insulin, anti-glucagon and anti-F4/80 antibodies, are representative of sections from 7 mice each group.H, I and J Insulin and glucagon IS, calculated as the intensity score multiplied with percentage of positive cells (H, I) and the percentage of islets area stained with F4/80 (J) (n=7 mice for each genotype). Data information: box plots show the minimum and maximum values (ends of the whiskers), interquartile range (length of the box), median (line through the box) and mean (+) values. *p≤0.05, **p≤0.01 (Mann-Whitney test).


Figure 7C Whole cell protein lysates of INS - 1 832 / 13 β - cells analysed by Western Blot with anti - p53 and anti - p21 antibodies ; anti - vinculin antibody was used as loading control ( n = 9 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± SEM . In C and D , blots show representative image of replicate experiments ; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney testD Nuclear protein lysates of INS - 1 832 / 13 β - cells analysed by Western Blot with anti - HMGB1 antibody ; anti - histone H3 antibody was used as loading control ( n = 5 biological replicates for each treatment group ) . Data information : Data in bar graphs are presented as mean ± SEM . In C and D , blots show representative image of replicate experiments ; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 : MGO vs CTR ; # p ≤ 0 . 05 , # # p ≤ 0 . 01 , # # # p ≤ 0 . 001 : D / Q vs MGO ( Mann - Whitney

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 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

Figure 7C Whole cell protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-p53 and anti-p21 antibodies; anti-vinculin antibody was used as loading control (n=9 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney testD Nuclear protein lysates of INS-1 832/13 β-cells analysed by Western Blot with anti-HMGB1 antibody; anti-histone H3 antibody was used as loading control (n=5 biological replicates for each treatment group). Data information: Data in bar graphs are presented as mean ± SEM. In C and D, blots show representative image of replicate experiments; protein levels were quantified by the densitometric analysis of independent experiments and showed in the bar graphs as the percentage over not treated control cells. *p≤0.05, **p≤0.01, ***p≤0.001: MGO vs CTR; #p≤0.05, ##p≤0.01, ###p≤0.001: D/Q vs MGO (Mann-Whitney test


Figure 1A Left : Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non - apoptotic cells labeled with Bax ( green ) and Tom22 ( red ) . Bax translocates to the mitochondria in apoptotic cells . Right : Individual display of the two color channels as shown in the overlay image . The asterisk denotes the apoptotic cell . Scale bars : 10 µm . B , C Dual color image of an apoptotic U2OS cell decorated with antibodies against Bax ( green ) and Tom20 ( red ) . The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction - limited confocal mode . ( B ) and ( C ) display the same data set . The small images are magnifications of the areas indicated by the rectangles . In ( B ) the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters . In ( C ) the color table was adjusted such that the dimmer Bax - rings are visible and the clusters are saturated . The arrows point to Bax - rings . Scale bars : 1 µm . D Optical sections of an apoptotic mitochondrion labelled for Bax ( green ) and Tom22 ( red ) . Shown are three optical sections and a maximum intensity projection of the entire image stack . Scale bar : 0 . 5µm . E Bax - rings on apoptotic mitochondria of various mammalian cell types . Shown are apoptotic mitochondria labelled for Bax ( green ) and Tom22 ( red ) in HT1080 , SH - SY5Y , HeLa and CV - 1 cells , as indicated . Scale bars : 0 . 5 µm . F Quantification of the diameter of Bax - rings in apoptotic U2OS cells . Only the long ring - axis was measured . Bax - rings smaller than 200 nm exist , but were not considered .

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 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 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 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

Figure 1A Left: Overlay image of a confocal recording of an apoptotic U2OS cell surrounded by non-apoptotic cells labeled with Bax (green) and Tom22 (red). Bax translocates to the mitochondria in apoptotic cells. Right: Individual display of the two color channels as shown in the overlay image. The asterisk denotes the apoptotic cell. Scale bars: 10 µm.B, C Dual color image of an apoptotic U2OS cell decorated with antibodies against Bax (green) and Tom20 (red). The Bax signal was recorded in the STED mode and the Tom20 signal in the diffraction-limited confocal mode. (B) and (C) display the same data set. The small images are magnifications of the areas indicated by the rectangles. In (B) the color table was adjusted to the brightest Bax signal in the image resulting in a clear display of the clusters. In (C) the color table was adjusted such that the dimmer Bax-rings are visible and the clusters are saturated. The arrows point to Bax-rings. Scale bars: 1 µm.D Optical sections of an apoptotic mitochondrion labelled for Bax (green) and Tom22 (red). Shown are three optical sections and a maximum intensity projection of the entire image stack. Scale bar: 0.5µm.E Bax-rings on apoptotic mitochondria of various mammalian cell types. Shown are apoptotic mitochondria labelled for Bax (green) and Tom22 (red) in HT1080, SH-SY5Y, HeLa and CV-1 cells, as indicated. Scale bars: 0.5 µm.F Quantification of the diameter of Bax-rings in apoptotic U2OS cells. Only the long ring-axis was measured. Bax-rings smaller than 200 nm exist, but were not considered.


Figure 2Apoptotic mitochondria were decorated with antibodies against Bax ( green ) and Tom22 ( red ) . The Bax signal was recorded in the STED mode . Shown are the individual color channels as well as an overlay ( from top to bottom ) of several apoptotic mitochondria . We used Lagrange interpolation to double the number of pixels . Note that throughout this manuscript except for contrast stretching no image manipulation was applied . Scale bar : 0 . 5 µm .

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

Figure 2Apoptotic mitochondria were decorated with antibodies against Bax (green) and Tom22 (red). The Bax signal was recorded in the STED mode. Shown are the individual color channels as well as an overlay (from top to bottom) of several apoptotic mitochondria. We used Lagrange interpolation to double the number of pixels. Note that throughout this manuscript except for contrast stretching no image manipulation was applied. Scale bar: 0.5 µm.


Figure 3A , B Three - color images of mitochondria of unchallenged ( A ) and apoptotic ( B ) U2OS cells . From left to right : Tom22 , Bax ( recorded in the STED - mode ) , cytochrome c and an overlay of all three channels . The arrows denote Bax - rings . Scale bars : 2 µm .

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

Figure 3A, B Three-color images of mitochondria of unchallenged (A) and apoptotic (B) U2OS cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows denote Bax-rings. Scale bars: 2 µm.


Figure 4A In apoptotic Drp1 knockdown U2OS cells , cytochrome c release is delayed . Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 ( red ) , cytochrome c ( blue ) and Bax ( green ) . Large image : Overlay of all three channels . Small images : Magnifications of the area indicated by the rectangle in the large image . Shown are the individual channels , as indicated . The arrows point to mitochondria that had not released cytochrome c ; the asterisks denote mitochondria that had released cytochrome c . Scale bars : 10 µm ( overview ) and 2 µm ( magnifications ) . B In Drp1 knockdown cells Bax - ring formation and cytochrome c release is not correlated . Three - color images of apoptotic mitochondria of Drp1 knockdown cells . From left to right : Tom22 , Bax ( recorded in the STED - mode ) , cytochrome c and an overlay of all three channels . The arrows point to Bax - rings . Scale bar : 1 µm .

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 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

Figure 4A In apoptotic Drp1 knockdown U2OS cells, cytochrome c release is delayed. Confocal overview image of an apoptotic Drp1 knockdown cell labelled with antibodies against Tom20 (red), cytochrome c (blue) and Bax (green). Large image: Overlay of all three channels. Small images: Magnifications of the area indicated by the rectangle in the large image. Shown are the individual channels, as indicated. The arrows point to mitochondria that had not released cytochrome c; the asterisks denote mitochondria that had released cytochrome c. Scale bars: 10 µm (overview) and 2 µm (magnifications).B In Drp1 knockdown cells Bax-ring formation and cytochrome c release is not correlated. Three-color images of apoptotic mitochondria of Drp1 knockdown cells. From left to right: Tom22, Bax (recorded in the STED-mode), cytochrome c and an overlay of all three channels. The arrows point to Bax-rings. Scale bar: 1 µm.


Figure 5Drp1 knockdown cells were treated with DMSO ( control ) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax , cytochrome c , and Mic27 or Mic60 ( see Appendix Figure S4 ) . Three - color images were taken , whereby Mic27 or Mic60 were recorded in the STED mode . Relying on the Bax and the cytochrome c signals , we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release . The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity , which is a sensitive measure for the distribution of the labelled protein . The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean .

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

Figure 5Drp1 knockdown cells were treated with DMSO (control) or with actinomycin D for 14 h to induce apoptosis and were decorated with antisera against Bax, cytochrome c, and Mic27 or Mic60 (see Appendix Figure S4). Three-color images were taken, whereby Mic27 or Mic60 were recorded in the STED mode. Relying on the Bax and the cytochrome c signals, we discriminated between healthy and apoptotic mitochondria before and after cytochrome c release. The distributions of Mic27 or Mic60 were analyzed by determining the normalized variance of the fluorescence intensity, which is a sensitive measure for the distribution of the labelled protein. The numbers within the columns represent the numbers of images analyzed and the error bars represent the standard error of the mean.


Figure 6Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell . Top and middle row : Tom22 and Bax ( recorded in STED ) channel , respectively . Bottom row : overlay ( Tom22 : red ; Bax : green ) . Note the absence of Tom22 signal in the area enclosed by the Bax - ring . Displayed are raw data . Scale bar : 0 . 5 µm .

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

Figure 6Shown are seven optical sections through a mitochondrion of an apoptotic U2OS cell. Top and middle row: Tom22 and Bax (recorded in STED) channel, respectively. Bottom row: overlay (Tom22: red; Bax: green). Note the absence of Tom22 signal in the area enclosed by the Bax-ring. Displayed are raw data. Scale bar: 0.5 µm.


Figure 1Antibody response in plasma and saliva at different time points ( T0 , T1 , T2 ) from BNT162b2 vaccination in naïve ( plasma : n = 64 ; saliva : n = 66 ) and SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received the first vaccine dose at day 0 ( T0 ) , and the 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after the 2nd dose . Plasma and saliva were tested for IgG to full - length spike and its receptor binding domain ( RBD ) . For plasma samples the titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted line : LoD ( spike ) = 12 , LoD ( RBD ) = 13 . 8 . For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS - CoV - 2 specific Ig by total IgG concentration of each sample . The normalization was applied only to values higher than LoD . The adjusted values are expressed in AU . The box plots show the interquartile range , the horizontal lines show the median values , and the whiskers indicate the minimum - to - maximum range .

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

Figure 1Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (plasma: n=64; saliva: n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgG to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=12, LoD (RBD)=13.8. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgG concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU. The box plots show the interquartile range, the horizontal lines show the median values, and the whiskers indicate the minimum-to-maximum range.


Figure 2Antibody response in plasma and saliva at different time points ( T0 , T1 , T2 ) from BNT162b2 vaccination in naïve ( n = 66 ) and SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received the first vaccine dose at day 0 ( T0 ) , and the 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after the 2nd dose . Plasma and saliva were tested for IgA to full - length spike and its receptor binding domain ( RBD ) . For plasma samples the titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted line : LoD ( spike ) = 54 . 22 , LoD ( RBD ) = 54 . 08 . For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS - CoV - 2 specific Ig by total IgA concentration of each sample . The normalization was applied only to values higher than LoD .

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

Figure 2Antibody response in plasma and saliva at different time points (T0, T1, T2) from BNT162b2 vaccination in naïve (n=66) and SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike)=54.22, LoD (RBD)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD.


Figure 3The antibody response of vaccinated naïve ( plasma : n = 64 ; saliva n = 66 ) or SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects at 7 - 10 days after the second dose ( T2 ) was compared to that obtained in COVID - 19 patients ( plasma n = 28 ; saliva n = 26 ) . As a control , SARS - CoV - 2 specific antibody response was analyzed in non - vaccinated subjects ( n = 19 , Control ) . Plasma and saliva were tested for IgA to full - length spike and its receptor binding domain ( RBD ) . For plasma samples the titers of antigen specific Ig are expressed in IU / ml . LoD is indicated by a dotted line : LoD ( spike IgG ) = 12 , LoD ( RBD IgG ) = 13 . 8 , LoD ( spike IgA ) = 54 . 22 , LoD ( RBD IgA ) = 54 . 08 . For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS - CoV - 2 specific Ig by total IgA or total IgG concentrations of each sample . The normalization was applied only to values higher than LoD . The adjusted values are expressed in AU .

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

Figure 3The antibody response of vaccinated naïve (plasma: n=64; saliva n=66) or SARS-CoV-2-Exp (plasma: n= 21; saliva: n=18) subjects at 7-10 days after the second dose (T2) was compared to that obtained in COVID-19 patients (plasma n=28; saliva n=26). As a control, SARS-CoV-2 specific antibody response was analyzed in non-vaccinated subjects (n=19, Control). Plasma and saliva were tested for IgA to full-length spike and its receptor binding domain (RBD). For plasma samples the titers of antigen specific Ig are expressed in IU/ml. LoD is indicated by a dotted line: LoD (spike IgG)=12, LoD (RBD IgG)=13.8, LoD (spike IgA)=54.22, LoD (RBD IgA)=54.08. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA or total IgG concentrations of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU.


Figure 4A , IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva at different time point ( T0 , T1 , T2 ) of vaccinated naïve ( plasma : n = 64 ; saliva : n = 63 ( IgA1 ) , n = 55 ( IgA2 ) ) or SARS - CoV - 2 - Exp ( plasma : n = 21 ; saliva : n = 18 ) subjects . Subjects received the first vaccine dose at day 0 ( T0 ) , and the 2nd dose at day 21 ( T1 ) ( indicated with an arrow ) . T2 corresponds to 7 - 10 days after the 2nd dose . For plasma samples the titers of antigen specific IgA1 and IgA2 are expressed in IU / ml and AU , respectively ( see Methods ) . LoD is indicated by a dotted line : LoD ( IgA1 ) = 74 . 64 ; LoD ( IgA2 ) = 0 . 009 . For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS - CoV - 2 specific Ig by total IgA concentration of each sample . The normalization was applied only to values higher than LoD . The adjusted values are expressed in AU . B , IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva of vaccinated naïve and SARS - CoV - 2 - Exp subjects 7 - 10 days after second dose ( T2 ) and the antibody response was compared to that obtained in COVID - 19 patients ( plasma : n = 28 ; saliva : n = 26 ) and in non - vaccinated subjects ( n = 19 , Control ) .

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 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 4A, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva at different time point (T0,T1, T2) of vaccinated naïve (plasma: n=64; saliva: n=63 (IgA1), n=55 (IgA2)) or SARS-CoV-2-Exp (plasma: n=21; saliva: n=18) subjects. Subjects received the first vaccine dose at day 0 (T0), and the 2nd dose at day 21 (T1) (indicated with an arrow). T2 corresponds to 7-10 days after the 2nd dose. For plasma samples the titers of antigen specific IgA1 and IgA2 are expressed in IU/ml and AU, respectively (see Methods). LoD is indicated by a dotted line: LoD (IgA1)=74.64; LoD (IgA2)=0.009. For saliva samples the titers of antigen specific Ig were normalized by dividing the values of SARS-CoV-2 specific Ig by total IgA concentration of each sample. The normalization was applied only to values higher than LoD. The adjusted values are expressed in AU.B, IgA1 and IgA2 subtypes specific for spike protein were measured in plasma and saliva of vaccinated naïve and SARS-CoV-2-Exp subjects 7-10 days after second dose (T2) and the antibody response was compared to that obtained in COVID-19 patients (plasma: n=28; saliva: n=26) and in non-vaccinated subjects (n=19, Control).


Figure 1 ( B ) P2 MRI images ( axial , sagittal and coronal plane ) showing ventriculomegaly ( red asterisk ) , slightly thickened cortex and bilateral enlarged gyri ( white asterisk ) . ( D ) FIBCD1 expression in various human visceral tissues and brain regions ( inset ) . Expression is plotted relative to the tissue with lowest detectable expression ( trachea ; inset , choroid plexus ) . n represents technical replicates ( n = 2 ) .

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 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

Figure 1(B) P2 MRI images (axial, sagittal and coronal plane) showing ventriculomegaly (red asterisk), slightly thickened cortex and bilateral enlarged gyri (white asterisk).(D) FIBCD1 expression in various human visceral tissues and brain regions (inset). Expression is plotted relative to the tissue with lowest detectable expression (trachea; inset, choroid plexus). n represents technical replicates (n = 2).


Figure 2 ( A ) Immunofluorescent images of control and neuronal ( Nsyb ) CG10359 ( dFibcd1 ) RNAi - mediated knockdown D . melanogaster , 3rd instar larvae NMJ ( NMJ6 / 7 ) stained with anti - horseradish peroxidase antibodies . Empty control and RNAi - mediated knockdown of CG10359 ( dFibcd1 - i ) lines # 1 , 2 and 3 shown . Scale bar = 20µm . Representative images of 3 independent experiments . Quantification of ( A ) , control and CG10359 knockdown lines NMJ neuron bouton number ( B ) n ( empty ) = 12 ; n ( line # 1 ) = 20 ; n ( line # 2 ) = 11 ; n ( line # 3 ) = 14 . Data information : each data point represents an individual NMJ Data is represented as mean and error bars represent SEM . p values were calculated using 2 - way ANOVA ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . Quantification of NMJ neuron axon branch points ( C ) . n ( empty ) = 12 ; n ( line # 1 ) = 20 ; n ( line # 2 ) = 11 ; n ( line # 3 ) = 14 . Data information : each data point represents an individual NMJ Data is represented as mean and error bars represent SEM . p values were calculated using 2 - way ANOVA ( panels C ) , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ( E ) Representative coronal section images of Golgi - Cox staining of Fibcd1 WT and KO hippocampi ( left ) , Neurolucida tracing of hippocampal CA1 pyramidal neurons ( middle ) and apical dendrites with spines ( right ) . Scale bars as indicated . ( F - I ) Quantifications of ( F ) dendritic spine density , ( G ) total length of apical as well as basal dendrites , ( H ) dendritic nodes in apical as well as basal dendrites and ( I ) number of dendrites ( n = 5 ) . Data information : for panels F - I , each data point represents an individual mouse . Data is represented as mean and error bars represent SEM . p values were calculated using unpaired Student ' s t - test ( panels F - I , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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

Figure 2(A) Immunofluorescent images of control and neuronal (Nsyb) CG10359 (dFibcd1) RNAi-mediated knockdown D. melanogaster, 3rd instar larvae NMJ (NMJ6/7) stained with anti-horseradish peroxidase antibodies. Empty control and RNAi-mediated knockdown of CG10359 (dFibcd1-i) lines #1, 2 and 3 shown. Scale bar = 20µm. Representative images of 3 independent experiments. Quantification of (A), control and CG10359 knockdown lines NMJ neuron bouton number (B) n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.Quantification of NMJ neuron axon branch points (C). n(empty) = 12; n(line #1) = 20; n(line #2) = 11; n(line #3) = 14. Data information: each data point represents an individual NMJ Data is represented as mean and error bars represent SEM. p values were calculated using 2-way ANOVA (panels C), * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.(E) Representative coronal section images of Golgi-Cox staining of Fibcd1 WT and KO hippocampi (left), Neurolucida tracing of hippocampal CA1 pyramidal neurons (middle) and apical dendrites with spines (right). Scale bars as indicated.(F-I) Quantifications of (F) dendritic spine density, (G) total length of apical as well as basal dendrites, (H) dendritic nodes in apical as well as basal dendrites and (I) number of dendrites (n = 5). Data information: for panels F-I, each data point represents an individual mouse. Data is represented as mean and error bars represent SEM. p values were calculated using unpaired Student's t-test (panels F-I, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


Figure 4 ( B ) Immunoblot analysis ( left ) and quantification of signal intensity ( right ) of Fibcd1 WT ( blue ) versus Fibcd1 KO littermates ( red ) adult hippocampi with antibodies against CS - 0S , CS - 4S , CS - 6S and actin as a loading control . Each lane represents an independent animal ( n = 3 ) . Protein marker sizes are indicated . Data information : For panel B , each data point represent hippocampal protein isolates from an individual mouse , Data are shown as mean values + / - SEM . p values were calculated using paired Student ' s t - test ( panels B * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ( F ) Confocal images depicting internalisation of FITC - tagged CS - 4S by FIBCD1 - overexpressing HEK293T lines compared to untransduced cells and unstained cells . Left , representative images ; right , quantification . Data is plotted as total puncta per condition ( n = 5 ) . Cells are further stained with CellMask Orange ( cellular membrane ) and Hoechst ( nuclei ) . Scale bar = 50µm . Data information : for panels F , each data point represents an individual cell preparation . Data are shown as mean values + / - SEM . p values were calculated using 1 - way ANOVA ( panels F , * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 .

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 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

Figure 4(B) Immunoblot analysis (left) and quantification of signal intensity (right) of Fibcd1 WT (blue) versus Fibcd1 KO littermates (red) adult hippocampi with antibodies against CS-0S, CS-4S, CS-6S and actin as a loading control. Each lane represents an independent animal (n = 3). Protein marker sizes are indicated. Data information: For panel B, each data point represent hippocampal protein isolates from an individual mouse, Data are shown as mean values +/- SEM. p values were calculated using paired Student's t-test (panels B * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.(F) Confocal images depicting internalisation of FITC-tagged CS-4S by FIBCD1-overexpressing HEK293T lines compared to untransduced cells and unstained cells. Left, representative images; right, quantification. Data is plotted as total puncta per condition (n = 5). Cells are further stained with CellMask Orange (cellular membrane) and Hoechst (nuclei). Scale bar = 50µm. Data information: for panels F, each data point represents an individual cell preparation. Data are shown as mean values +/- SEM. p values were calculated using 1-way ANOVA (panels F, * = p≤0.05; ** =p≤0.01; *** =p≤0.001; **** p≤0.0001.


Figure 5 ( A ) Left , representative image of immunofluorescent staining ( MAP2 , red ; DAPI , blue ) of primary hippocampal cultures at 2 days in vitro ( DIV ) , plated on + / - CSPG coating with and without prior digestion with ChABC , as indicated . Right , quantification of DIV2 images , showing the number of protruding cells per field normalised to untreated condition . n ( Fibcd1 WT ) = 4 ; n ( Fibcd1 KO ) = 3 . Scale bar = 250µm . ( B ) Left , representative images of DIV14 neurons , same conditions as in ( A ) . Right , quantification of DIV14 images , representing the percentage of cells per field that are clumped . n ( Fibcd1 WT ) = 3 ; n ( Fibcd1 KO ) = 2 . Scale bar = 250µm . Data information : For panels A and B each data point represents an individual preparations of primary cell culture . Data are represented as mean and error bars represent SEM . p values were calculated by 1 - way ANOVA . * = p ≤ 0 . 05 ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 .

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

Figure 5(A) Left, representative image of immunofluorescent staining (MAP2, red; DAPI, blue) of primary hippocampal cultures at 2 days in vitro (DIV), plated on +/- CSPG coating with and without prior digestion with ChABC, as indicated. Right, quantification of DIV2 images, showing the number of protruding cells per field normalised to untreated condition. n(Fibcd1 WT) = 4; n(Fibcd1 KO) = 3. Scale bar = 250µm. (B) Left, representative images of DIV14 neurons, same conditions as in (A). Right, quantification of DIV14 images, representing the percentage of cells per field that are clumped. n(Fibcd1 WT) = 3; n(Fibcd1 KO) = 2. Scale bar = 250µm. Data information: For panels A and B each data point represents an individual preparations of primary cell culture. Data are represented as mean and error bars represent SEM. p values were calculated by 1-way ANOVA. * = p≤0.05; ** =p≤0.01; *** =p≤0.001.


Figure 1 ( C ) MRM mass spectrometry verification of the unique peptide ( heavy ) of SMIM26 in HEK293T cells . ( D ) PRM mass spectrometry verification of the unique peptide ( heavy ) of SMIM26 in HEK293T cells . ( G ) Immunofluorescence detected subcellular localization of SMIM26 ( green ) with mitochondria , F - actin , ER and Golgi . Nuclei were stained with DAPI ( blue ) , and the colocalization between red ( mitochondria , F - actin , ER , Golgi ) and green ( SMIM26 ) was measured . Scale bar , 10 μm . The colocalization of SMIM26 with mitochondria , F - actin , ER and Golgi in cells was measured by fluorescence intensity analysis with ImageJ software . The red line of statistical analysis centers on the co - location of SMIM26 and indicate cell organelle . ( H , I ) Mitochondria were purified and SMIM26 was enriched in mitochondria fractions ( H ) , SMIM26 is a mitochondrial outer membrane protein as detected by proteinase K and Triton X - 100 assayin HEK293T cells ( I ) . ( J ) The degradation half - life of SMIM26 was detected in HEK293T . Three biological replicates . Data are shown as 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 1(C) MRM mass spectrometry verification of the unique peptide (heavy) of SMIM26 in HEK293T cells. (D) PRM mass spectrometry verification of the unique peptide (heavy) of SMIM26 in HEK293T cells.(G) Immunofluorescence detected subcellular localization of SMIM26 (green) with mitochondria, F-actin, ER and Golgi. Nuclei were stained with DAPI (blue), and the colocalization between red (mitochondria, F-actin, ER, Golgi) and green (SMIM26) was measured. Scale bar, 10 μm. The colocalization of SMIM26 with mitochondria, F-actin, ER and Golgi in cells was measured by fluorescence intensity analysis with ImageJ software. The red line of statistical analysis centers on the co-location of SMIM26 and indicate cell organelle.(H, I) Mitochondria were purified and SMIM26 was enriched in mitochondria fractions (H), SMIM26 is a mitochondrial outer membrane protein as detected by proteinase K and Triton X-100 assayin HEK293T cells (I).(J) The degradation half-life of SMIM26 was detected in HEK293T. Three biological replicates. Data are shown as mean ± SEM.


Figure 2 ( A ) Representative immunohistochemistry ( IHC ) images of SMIM26 protein level in multiple types of tumoral tissues ( T ) and adjacent non - tumorous tissues ( N ) , showing that SMIM26 has significantly decreased expression in kidney cancer only . Mann - Whitney test , * * P < 0 . 01 ; n = 5 ; Bar , SEM ; Scale bar , 100 μm . ( B ) Representative IHC images of SMIM26 protein levels in 368 ccRCC tissues ( T ) and 281 non - tumorous renal tissues ( N ) . Mann - Whitney test , * * * P < 0 . 001 ; Scale bar , 200 μm . ( C ) A Kaplan - Meier survival analysis of ccRCC patients with high or low expression levels of SMIM26 ( P < 0 . 05 , log - rank test ) . ( E , The protein level ( E ) of SMIM26 were detected in five ccRCC cell lines by western blotting ( G ) The protein level of SMIM26 ( up ) and RNA level of LINC00493 ( down ) in ccRCC tissues ( T ) and their adjacent non - tumoral tissues ( N ) were detected by IHC staining and RNA in situ hybridization ( ISH ) staining . The right scatter plot shows no significant correlation between SMIM26 protein expression and LINC00493 RNA expression in ccRCC tissues ( green dots , P = 0 . 94 ) and adjacent non - tumorous renal tissues ( purple dots , P = 0 . 53 ) . n = 72 . Scale bar , 200 μm .

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

Figure 2(A) Representative immunohistochemistry (IHC) images of SMIM26 protein level in multiple types of tumoral tissues (T) and adjacent non-tumorous tissues (N), showing that SMIM26 has significantly decreased expression in kidney cancer only. Mann-Whitney test, **P < 0.01; n = 5; Bar, SEM; Scale bar,100 μm.(B) Representative IHC images of SMIM26 protein levels in 368 ccRCC tissues (T) and 281 non-tumorous renal tissues (N). Mann-Whitney test, *** P < 0.001; Scale bar, 200 μm.(C) A Kaplan-Meier survival analysis of ccRCC patients with high or low expression levels of SMIM26 (P < 0.05, log-rank test).(E, The protein level (E) of SMIM26 were detected in five ccRCC cell lines by western blotting(G) The protein level of SMIM26 (up) and RNA level of LINC00493 (down) in ccRCC tissues (T) and their adjacent non-tumoral tissues (N) were detected by IHC staining and RNA in situ hybridization (ISH) staining. The right scatter plot shows no significant correlation between SMIM26 protein expression and LINC00493 RNA expression in ccRCC tissues (green dots, P=0.94) and adjacent non-tumorous renal tissues (purple dots, P=0.53). n = 72. Scale bar, 200 μm.


Figure 3 ( D ) The expression of PABPC4 and SMIM26 in ccRCC tissues compared with adjacent non - tumor tissues was detected by western blotting . ( F ) RIP assay showing a strong binding of LINC00493 with PABPC4 in HEK293T and ACHN cells . ( G ) RNA levels of SMIM26 were determined by qRT - PCR in PABPC4 knockdown cells . Data are shown as mean ± SEM of three biological replicates . Unpaired two - tailed Student ' s t - test . n . s , nonsignificant . Bars , SEM . ( I ) Knocking down PABPC4 could not significantly alter the degradation half - life of SMIM26 protein . Data are shown as mean ± SEM of three biological replicates . ( J ) RNC of SMIM26 was detected in HEK293T and ACHN cells transfected with anti - PABPC4 siRNAs . Unpaired two - tailed Student ' s t - test , * * * P < 0 . 001 . Data are shown as mean ± SEM of three biological replicates .

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 B-PANEL_START 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 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 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

Figure 3(D) The expression of PABPC4 and SMIM26 in ccRCC tissues compared with adjacent non-tumor tissues was detected by western blotting.(F) RIP assay showing a strong binding of LINC00493 with PABPC4 in HEK293T and ACHN cells.(G) RNA levels of SMIM26 were determined by qRT-PCR in PABPC4 knockdown cells. Data are shown as mean ± SEM of three biological replicates. Unpaired two-tailed Student's t-test. n.s, nonsignificant. Bars, SEM.(I) Knocking down PABPC4 could not significantly alter the degradation half-life of SMIM26 protein. Data are shown as mean ± SEM of three biological replicates.(J) RNC of SMIM26 was detected in HEK293T and ACHN cells transfected with anti-PABPC4 siRNAs. Unpaired two-tailed Student's t-test, ***P < 0.001. Data are shown as mean ± SEM of three biological replicates.


Figure 4 ( D ) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells transfected with indicated constructs . Unpaired two - tailed Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . n . s , nonsignificant . Data are shown as mean ± SEM of three biological replicates . Scale bar , 600 μm . ( E ) Immunofluorescence staining of F - actin ( red ) in ACHN and 769 - P cells transfected with indicated constructs . Nuclei were stained with DAPI ( blue ) . The intensity of F - actin fluorescence was statistically plotted . Unpaired two - tailed Student ' s t - test , * * P < 0 . 01 , n . s , nonsignificant . Data are shown as mean ± SEM of three biological replicates . Scale bar , 20 μm . ( F ) Western blotting showed that mesenchymal marker of Vimentin and Fibronectin was decreased , and E - cadherin was increased in SMIM26 - overexpressing cells . ( J ) NOD - SCID mice were transplanted with ACHN - luci cells with or without overexpression of SMIM26 via tail vein injection , respectively . Bioluminescent was visualized 5 weeks later by using an IVIS 200 Imaging System . Data are shown as mean ± SD of 5 mice per group . Unpaired two - tailed Student ' s t - test , * * * * P < 0 . 0001 . n . s , nonsignificant .

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 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 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 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

Figure 4(D) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells transfected with indicated constructs. Unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. n.s, nonsignificant. Data are shown as mean ± SEM of three biological replicates. Scale bar, 600 μm.(E) Immunofluorescence staining of F-actin (red) in ACHN and 769-P cells transfected with indicated constructs. Nuclei were stained with DAPI (blue). The intensity of F-actin fluorescence was statistically plotted. Unpaired two-tailed Student's t-test, **P < 0.01, n.s, nonsignificant. Data are shown as mean ± SEM of three biological replicates. Scale bar, 20 μm.(F) Western blotting showed that mesenchymal marker of Vimentin and Fibronectin was decreased, and E-cadherin was increased in SMIM26-overexpressing cells.(J) NOD-SCID mice were transplanted with ACHN-luci cells with or without overexpression of SMIM26 via tail vein injection, respectively. Bioluminescent was visualized 5 weeks later by using an IVIS 200 Imaging System. Data are shown as mean ± SD of 5 mice per group. Unpaired two-tailed Student's t-test, ****P < 0.0001. n.s, nonsignificant.


Figure 5Co - IP assays were performed to detect the interaction between SMIM26 and SLC25A11 . Lysates from ACHN cells expressing SMIM26 - flag were co - immunoprecipitated by anti - flag antibody ( E ) SMIM26 and SLC25A11 were detected by western blotting . ( G ) Co - localization of SMIM26 ( green ) with SLC25A11 ( red ) in ACHN was determined by immunofluorescence . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . The red line of statistical analysis centers on the co - location of SMIM26 and SLC25A11 . ( K - L ) N - ter domain of SMIM26 fused with GFP - flag interacted with SLC25A11 . The N - ter SMIM26 - GFP - flag construct was transfected into HEK293T cells , co - immunoprecipitation was performed by using anti - flag antibody ( K ) and anti - SLC25A11 antibody ( L ) . ( O ) Oxygen consumption rate profile was monitored in cells with indicated treatment by using a Seahorse XF24 analyzer . The metabolic inhibitors were injected at different time points , as indicated . Data are shown as mean ± SD of three biological replicates . ( P ) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells with indicated treatment . Unpaired two - tailed Student ' s t - test , * P < 0 . 05 , * * P < 0 . 01 . Data are shown as mean ± SEM of three biological replicates .

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 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 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 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

Figure 5Co-IP assays were performed to detect the interaction between SMIM26 and SLC25A11. Lysates from ACHN cells expressing SMIM26-flag were co-immunoprecipitated by anti-flag antibody (E) SMIM26 and SLC25A11 were detected by western blotting.(G) Co-localization of SMIM26 (green) with SLC25A11 (red) in ACHN was determined by immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. The red line of statistical analysis centers on the co-location of SMIM26 and SLC25A11.(K-L) N-ter domain of SMIM26 fused with GFP-flag interacted with SLC25A11. The N-ter SMIM26-GFP-flag construct was transfected into HEK293T cells, co-immunoprecipitation was performed by using anti-flag antibody (K) and anti-SLC25A11 antibody (L).(O) Oxygen consumption rate profile was monitored in cells with indicated treatment by using a Seahorse XF24 analyzer. The metabolic inhibitors were injected at different time points, as indicated. Data are shown as mean ± SD of three biological replicates.(P) Transwell assays were used to test the migration and invasion abilities of both ccRCC cells with indicated treatment. Unpaired two-tailed Student's t-test, *P < 0.05, **P < 0.01. Data are shown as mean ± SEM of three biological replicates.


Figure 6 ( A - B ) Immunoblotting validation of the p - AKT at Ser473 expression in ACHN and 769 - P cells with overexpression ( A ) or knockdown ( B ) of SMIM26 compared with their corresponding control . ( D ) Co - IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769 - P cells expressing SMIM26 - flag . Lysates were co - immunoprecipitated by anti - flag antibody . ( E ) Co - IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769 - P cells expressing SMIM26 - flag . Lysates were co - immunoprecipitated by anti - AGK antibody . ( G ) Immunofluorescence of SMIM26 ( purple ) , AGK ( green ) and mitochondrial ( red ) in ACHN cells with or without overexpression of SMIM26 . Scale bar , 10 μm . Fluorescence intensity analysis of colocalization of AGK and mitochondria in cells treated with control or SMIM26 . ImageJ software was used to analyze the fluorescence colocalization . The red line of statistical analysis centers on the co - location of AGK and mitochondria . The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769 - P cells with control or SMIM26 overexpressing ( K ) The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769 - P cells with control or SMIM26 knockdown ( L ) .

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

Figure 6(A-B) Immunoblotting validation of the p-AKT at Ser473 expression in ACHN and 769-P cells with overexpression (A) or knockdown (B) of SMIM26 compared with their corresponding control.(D) Co-IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769-P cells expressing SMIM26-flag. Lysates were co-immunoprecipitated by anti-flag antibody. (E) Co-IP assays were performed to detect the interaction between SMIM26 and AGK in ACHN and 769-P cells expressing SMIM26-flag. Lysates were co-immunoprecipitated by anti-AGK antibody.(G) Immunofluorescence of SMIM26 (purple), AGK (green) and mitochondrial (red) in ACHN cells with or without overexpression of SMIM26. Scale bar, 10 μm. Fluorescence intensity analysis of colocalization of AGK and mitochondria in cells treated with control or SMIM26. ImageJ software was used to analyze the fluorescence colocalization. The red line of statistical analysis centers on the co-location of AGK and mitochondria.The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769-P cells with control or SMIM26 overexpressing (K)The interaction of AGK and SLC25A11 was determined by immunoblotting in ACHN and 769-P cells with control or SMIM26 knockdown (L).


Figure 1Macrophages were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Immunoblot of co - immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β - actin as loading control . ( B ) Cells were incubated with 10 µg / mL pepstatin A for 4 h prior to lysis . Immunoblots of LC3B isoforms using antibody to LC3B or β - actin . ( C ) Cells were fixed and permeabilized then stained with antibody to LC3B ( green ) and Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative fluorescence microscopy images . When puncta are not present , LC3B staining gives rise to a diffuse cytoplasmic pattern . Right , percentage of cells with LC3B puncta . Bar charts are presented as the means ± s . e . m . , n = 6 . ( D ) Immunoblots of SQSTM1 using antibody to SQSTM1 or β - actin . * p < 0 . 05 .

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Figure 1Macrophages were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Immunoblot of co-immunoprecipitation of cell lysates with BECN1 specific antibody using antibody to PIK3C3 and BECN1 with antibody to β-actin as loading control.(B) Cells were incubated with 10 µg/mL pepstatin A for 4 h prior to lysis. Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 6.(D) Immunoblots of SQSTM1 using antibody to SQSTM1 or β-actin. * p<0.05.


Figure 2Macrophages transduced with non - specific scrambled shRNA ( shNS ) or TLR8 shRNA ( shTLR8 ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( A ) Cells were lysed and immunoblot performed with antibodies to TLR8 and β - actin . ( B ) Immunoblots of LC3B isoforms using antibody to LC3B or β - actin . ( C ) Cells were fixed and permeabilized then stained with antibody to LC3B ( green ) and Hoechst 33342 ( blue ) . Scale bars indicate 10 µm . Left , representative fluorescence microscopy images from three donors are shown . When puncta are not present , LC3B staining gives rise to a diffuse cytoplasmic pattern . Right , percentage of cells with LC3B puncta . Bar charts are presented as the means ± s . e . m . , n = 4 . * p < 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 B-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Macrophages transduced with non-specific scrambled shRNA (shNS) or TLR8 shRNA (shTLR8) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (A) Cells were lysed and immunoblot performed with antibodies to TLR8 and β-actin.(B) Immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(C) Cells were fixed and permeabilized then stained with antibody to LC3B (green) and Hoechst 33342 (blue). Scale bars indicate 10 µm. Left, representative fluorescence microscopy images from three donors are shown. When puncta are not present, LC3B staining gives rise to a diffuse cytoplasmic pattern. Right, percentage of cells with LC3B puncta. Bar charts are presented as the means ± s.e.m., n = 4. * p<0.05.


Figure 3A - B ) Macrophages were treated for 24 h with CL097 , ssRNA40 or ssRNA41 . ( A ) Top , qRT - PCR for CYP27B1 after 6 h . Bottom , immunoblots of CYP27B1 using antibody to CYP27B1 or β - actin . ( B ) Top , qRT - PCR for VDR after 6 h . Bottom , immunoblots of VDR using antibody to VDR or β - actin . ( C - D ) Macrophages transduced with non - specific scrambled shRNA ( shNS ) , CYP27B1 shRNA ( shCYP27B1 ) or VDR shRNA ( shVDR ) were treated for 24 h with CL097 , ssRNA40 , ssRNA41 or rapamycin ( Rapa ) . ( C ) Left , immunoblots performed with antibodies to CYP27B1 , VDR and β - actin . Right , immunoblots of LC3B isoforms using antibody to LC3B or β - actin . ( D ) Cells were fixed and permeabilized then stained with antibody to LC3B . Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s . e . m . , n = 4 . ( E ) Macrophages were treated for 24 h with CL097 , ssRNA40 , and rapamycin in the presence of 45 nmol / L or 100 nmol / L 25D3 and lysed . Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B , SQSTM1 or β - actin . Bar charts are reported as mean ± s . e . m . , n = 4 . * p < 0 . 05 .

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Figure 3A-B) Macrophages were treated for 24 h with CL097, ssRNA40 or ssRNA41. (A) Top, qRT-PCR for CYP27B1 after 6 h. Bottom, immunoblots of CYP27B1 using antibody to CYP27B1 or β-actin.(B) Top, qRT-PCR for VDR after 6 h. Bottom, immunoblots of VDR using antibody to VDR or β-actin.(C-D) Macrophages transduced with non-specific scrambled shRNA (shNS), CYP27B1 shRNA (shCYP27B1) or VDR shRNA (shVDR) were treated for 24 h with CL097, ssRNA40, ssRNA41 or rapamycin (Rapa). (C) Left, immunoblots performed with antibodies to CYP27B1, VDR and β-actin. Right, immunoblots of LC3B isoforms using antibody to LC3B or β-actin.(D) Cells were fixed and permeabilized then stained with antibody to LC3B. Bar charts represent the percentage of cells with LC3B puncta and are presented as the means ± s.e.m., n = 4.(E) Macrophages were treated for 24 h with CL097, ssRNA40, and rapamycin in the presence of 45 nmol/L or 100 nmol/L 25D3 and lysed. Immunoblots of LC3B isoforms and SQSTM1 using antibody to LC3B, SQSTM1 or β-actin. Bar charts are reported as mean ± s.e.m., n = 4. * p<0.05.