Figure 1 ( A ) Representative images of WT , Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria - targeted RFP ( mt - RFP ) . Scale bar : 5 μm .

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Figure 1(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm.


Figure 2 ( A ) Representative images of WT and Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP and ER - targeted GFP . Scale bar : 5 μm . ( B ) After 48 h , the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander ' s coefficient ( n = 30 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . * p < 0 . 05 , two - tailed Student ' s t - test . I - K ) Mitochondrial Ca2 + was determined in WT and Mfn2 KO MEFs loaded with Rhod2 . The effect of ( I ) caffeine - induced ( 20 mM ) , ( J ) IP3R activation with ATP - induced ( 100 μM ) and ( K ) histamine - induced ( 100 μM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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Figure 2(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm.(B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.


Figure 3WT and Mfn2 KO cells were transfected with plasmids encoding : GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( R ) MMP was analyzed ( n = 90 cells analyzed in 3 independent experiments ) . WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( S ) ATP levels of GFP + cells was analyzed after 24 h treatment with 2DG ( 10 mM ) ( n = 3 independent experiments ) .

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Figure 3WT and Mfn2 KO cells were transfected with plasmids encoding: GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (R) MMP was analyzed (n= 90 cells analyzed in 3 independent experiments).WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S) ATP levels of GFP+ cells was analyzed after 24 h treatment with 2DG (10 mM) (n= 3 independent experiments).


Figure 4WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : mt - RFP and ER - Mfn2 or control ( CT ) plasmid . After 48 h , the cells were fixed . ( C ) Representative images . Scale bar = 5 μm . WT and Mfn2 KO MEFs were co - transfected with plasmids encoding : J - N ) GFP and ER - Mfn2 or control ( CT ) plasmid . ( J ) MMP was determined . The values were normalized to surrounding untransfected cells . ( n = 90 cells analyzed in 3 independent experiments ) . ( K ) After 24 h , the transfected cells were sorted and plated for another 24 h , after which cells were treated with 2 - DG ( 10 mM ) for 6 h , and ATP levels were measured ( n = 3 independent experiments ) . ( L ) Oxygen consumption was measured and ( M ) RCR and ( N ) SRC was calculated ( n = 4 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity .

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Figure 4WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm.WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


Figure 5WT and Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP and plasmids expressing the indicated proteins . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 cells analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . B ) GFP and ERMITO - RLuc and indicated expression vectors . After 24 h , the transfected cells were sorted and plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± SEM . C ) Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : C ) GFP and the indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 and the indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 cells from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : The red dashed line indicates the WT cells results . * p < 0 . 05 versus Mfn1 / 2 DKO cells , one - way ANOVA followed by Tukey ' s post hoc test . Note that experiments were performed at the same time , so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity . WT and Mfn1 / 2 double KO were transfected with : E ) GFP and the indicated plasmid . After 48 h , the effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 cells from 3 independent experiments ) . Data are presented as mean ± SEM .

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Figure 5WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


Figure 6B ) Representative Western blots of the indicated proteins during the neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D D ) Primary neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus the indicated plasmids . Tamoxifen - treated ( for 72 h ) and vehicle - treated neurons ( WT ; red dashed line ) were fixed and mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm . ( F ) neurite length measurement and representative tracings ( G ) of primary cortical neurons of WT and Mfn2 KO mice co - transfected at DIV4 with GFP and the indicated plasmids . After 72 h , the neurons were fixed , immunofluorescence with anti - GFP antibodies was performed and neurite length was measured ( n = 30 neurons from 3 independent experiments ) . Data are presented as mean ± SEM . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc test . Scale bar : 500 μm . E

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Figure 6B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E


Figure 1A . Immunostaining of P5 cerebellum with indicated antibodies . ( A ) low magnification . ( A ' - A ' ' ' ' ) high magnification . The solid line indicates the pial edge of the EGL . The dashed line demarcates the bottom of the KI67 - stained region . The outer EGL corresponds to the area between the solid and dashed lines , which contain GCPs . Atoh1 KD analysis . ( G , H ) Atoh1 shRNA was introduced to P5 mice EGL together with H2B - EGFP plasmid and immunostaining for NEUROD1 ( Cyan ) and KI67 ( magenta ) was performed 24 hrs later . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of Control ( G ) and Atoh1 KD ( H ) . Arrows indicate the cells of interest where GFP signals are co - localized with NEUROD1 and KI67 . ( I ) Proportions of NEUROD1 + , KI67 + cells ( ND + GCPs ) among the electroporated GCPs ( GFP + , KI67 + cells ) were increased in the Atoh1 KD mice . ( J ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 4 ) and Atoh1 shRNA ( sh # 1 : N = 3 , sh # 2 : N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 , N . S . : Not Significant . Number of cells : 98 , 71 , 81 and 60 cells from N = 4 mice ( sclamble ) , 127 , 93 and 51 cells from N = 3 mice ( sh # 1 ) , 75 , 111 , 38 cells from N = 3 mice ( sh # 2 ) .

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Figure 1A. Immunostaining of P5 cerebellum with indicated antibodies. (A) low magnification. (A'-A'''') high magnification. The solid line indicates the pial edge of the EGL. The dashed line demarcates the bottom of the KI67-stained region. The outer EGL corresponds to the area between the solid and dashed lines, which contain GCPs.Atoh1 KD analysis. (G, H) Atoh1 shRNA was introduced to P5 mice EGL together with H2B-EGFP plasmid and immunostaining for NEUROD1 (Cyan) and KI67 (magenta) was performed 24 hrs later. Nuclei of electroporated cells were labeled with GFP (green). Images of Control (G) and Atoh1 KD (H). Arrows indicate the cells of interest where GFP signals are co-localized with NEUROD1 and KI67.(I) Proportions of NEUROD1+, KI67+ cells (ND+GCPs) among the electroporated GCPs (GFP+, KI67+ cells) were increased in the Atoh1 KD mice. (J) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=4) and Atoh1 shRNA (sh#1: N=3, sh#2: N=3). Bars and error bars are representing as Mean ± SEM. *p < 0.05, N.S.: Not Significant. Number of cells: 98,71,81 and 60 cells from N=4 mice (sclamble), 127,93 and 51 cells from N=3 mice (sh#1), 75,111,38 cells from N=3 mice (sh#2).


Figure 2B . Immunostaining of P5 cerebellum with indicated antibodies . Boxplots of the intensity of CCND1 immuno - signal ( B ' ' ' ' , normalized to the mean intensity of AT + GCPs ) . We measured the intensities of CCND1 in ATOH1 + / NEUROD1 - cells and NEUROD1 + / ATOH1 - cells . ATOH1 + / NEUROD1 + cells were not included in this quantification . These boxplots are showing the median ( central band ) , 25 % / 75 % quartiles ( boxes ) and the ranges ( lower whiskers represent the regions of lower box - 1 . 5 * IQR ( inter - quartile range ) , upper whisker represent the regions of upper box - 1 . 5 * IQR ) . Wilcoxon test were performed between AT + GCPs and ND + GCPs . * * * p < 0 . 001 . C - F . Ccnd1 KD analysis . ( C , D ) Ccnd1 shRNAs together with H2B - EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( Cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( C ) and Ccnd1 KD ( D ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( E ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were decreased in the Ccnd1 KD mice . ( F ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Student T - test was performed between Control ( N = 3 ) and Ccnd1 KD ( sh # 1 : N = 3 , sh # 2 : N = 3 ) mice . Bars and error bars are representing as Mean ± SEM . * * * p < 0 . 001 , N . S . : Not Significant . Number of cells : 52 , 64 and 107 cells from N = 3 mice ( scramble ) , 72 , 79 and 75 cells from N = 3 mice ( sh # 1 ) , 84 , 70 and 40 cells from N = 3 mice ( sh # 2 ) . G - K . Over - expression analysis of Ccnd1 with or without Cdk4 . ( G , H , I ) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 ( magenta ) and KI67 ( cyan ) was performed the following day . Nuclei of electroporated cells were labeled with GFP ( yellow ) . Images of Control ( G ) , CCND1 over expression ( H ) and CCND1 and CDK4 over expression ( I ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( J ) Proportions of ATOH1 + , KI67 + cells ( AT + GCPs ) among the electroporated cells were not significantly changed between Control and CCND1 . Forced expression of CCND1 and CDK4 ( 4D ) significantly increased the proporations of AT + GCPs compared to Control . ( K ) Proportions of KI67 + cells ( total GCPs ) were not significantly changed . Tukey - Kramer test was performed on Control ( N = 6 ) , CCND1 ( N = 4 ) , CCND1 and CDK4 ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , N . S . : Not Significant . Number of cells : 52 , 64 , 107 , 244 , 127 and 146 from N = 6 mice ( Control ) , 126 , 137 , 80 and 336 from N = 4 mice ( Ccnd1 ) , 212 , 143 and 38 from N = 3 mice ( 4D ) .

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

Figure 2B. Immunostaining of P5 cerebellum with indicated antibodies. Boxplots of the intensity of CCND1 immuno-signal (B'''', normalized to the mean intensity of AT+GCPs). We measured the intensities of CCND1 in ATOH1+/NEUROD1- cells and NEUROD1+/ ATOH1- cells. ATOH1+/ NEUROD1+ cells were not included in this quantification. These boxplots are showing the median (central band), 25% / 75% quartiles (boxes) and the ranges (lower whiskers represent the regions of lower box - 1.5 * IQR (inter-quartile range), upper whisker represent the regions of upper box - 1.5 * IQR). Wilcoxon test were performed between AT+GCPs and ND+GCPs. ***p < 0.001.C-F. Ccnd1 KD analysis. (C, D) Ccnd1 shRNAs together with H2B-EGFP plasmid were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (Cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (C) and Ccnd1 KD (D). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (E) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were decreased in the Ccnd1 KD mice. (F) Proportions of KI67+ cells (total GCPs) were not significantly changed. Student T-test was performed between Control (N=3) and Ccnd1 KD (sh#1: N=3, sh#2: N=3) mice. Bars and error bars are representing as Mean ± SEM. ***p < 0.001, N.S.: Not Significant. Number of cells: 52, 64 and 107 cells from N=3 mice (scramble), 72,79 and 75 cells from N=3 mice (sh#1), 84, 70 and 40 cells from N=3 mice (sh#2).G-K. Over-expression analysis of Ccnd1 with or without Cdk4. (G, H, I) Ccnd1 and Cdk4 expression plasmids were introduced to P5 mice EGL and immunostaining for ATOH1 (magenta) and KI67 (cyan) was performed the following day. Nuclei of electroporated cells were labeled with GFP (yellow). Images of Control (G), CCND1 over expression (H) and CCND1 and CDK4 over expression (I). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (J) Proportions of ATOH1+, KI67+ cells (AT+GCPs) among the electroporated cells were not significantly changed between Control and CCND1. Forced expression of CCND1 and CDK4 (4D) significantly increased the proporations of AT+GCPs compared to Control. (K) Proportions of KI67+ cells (total GCPs) were not significantly changed. Tukey-Kramer test was performed on Control (N=6), CCND1 (N=4), CCND1 and CDK4 (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, N.S.: Not Significant. Number of cells: 52, 64, 107,244,127 and 146 from N=6 mice (Control), 126, 137, 80 and 336 from N=4 mice (Ccnd1), 212, 143 and 38 from N=3 mice (4D).


Figure 3C . N2a cells transfected with Atoh1 - GST plasmids were treated with DMSO ( Control ) , Palbociclib ( Palb ) or Palb and 10 uM MG132 ( MG132 ) for 6hrs before harvest . Lysates of N2a were subjected to immunoblotting with anti - Atoh1 or β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between Control , Palb , MG132 . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * * p < 0 . 001 , * p < 0 . 05 , N . S . : Not Significant . Three slots of each experimental condition represent the three biological replicants of each condition . G . Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids ( 4D ) . N2a cells were treated with 10 nM , 100 nM , 1000 nM Palb for 6 hrs before harvest . Lysates from N2a cells were subjected to immunoblotting with anti - Atoh1 , p - S309 - Ab and β - actin . Quantification of relative ATOH1 to the β - ACTIN ( right panel ) . Tukey - Kramer test was performed between 0 , 10 , 100 , 1000nM . Bars and error bars are representing as Mean ± SEM from three trials of each condition . * * p < 0 . 01 , * p < 0 . 05 , N . S . : 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 3C. N2a cells transfected with Atoh1-GST plasmids were treated with DMSO(Control), Palbociclib (Palb) or Palb and 10 uM MG132 (MG132) for 6hrs before harvest. Lysates of N2a were subjected to immunoblotting with anti-Atoh1 or β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between Control, Palb, MG132. Bars and error bars are representing as Mean ± SEM from three trials of each condition. ***p < 0.001, *p < 0.05, N.S.: Not Significant. Three slots of each experimental condition represent the three biological replicants of each condition.G. Atoh1 expression plasmids were transfected into N2a cells with Ccnd1 and Cdk4 expression plasmids (4D). N2a cells were treated with 10 nM, 100 nM, 1000 nM Palb for 6 hrs before harvest. Lysates from N2a cells were subjected to immunoblotting with anti-Atoh1, p-S309-Ab and β-actin. Quantification of relative ATOH1 to the β-ACTIN (right panel). Tukey-Kramer test was performed between 0, 10, 100, 1000nM. Bars and error bars are representing as Mean ± SEM from three trials of each condition. **p < 0.01, *p < 0.05, N.S.: Not Significant.


Figure 4B . Lysates of N2a cells transfected with either WT or a phospho - mimic form of ATOH1 ( S309D ) were immunoblotted with anti - Atoh1 or β - actin . Quantification of relative levels of ATOH1 to the β - ACTIN ( right panel ) . Mean ± s . e . m . Student T - test between WT and S309D was performed . * * * p < 0 . 001 . Three slots of each experimental condition represent the three biological replicants of each condition . Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1 . ( F - I ) Scramble ( Control ) , Atoh1 shRNA ( Atoh1 KD ) , shRNA and WT ( KD + WT ) or shRNA and S309A ( KD + S309A ) plasmids were introduced to P5 mice EGL with H2B - EGFP plasmid and immunostaining for NEUROD1 ( magenta ) was performed at P7 . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest which GFP signals are not co - localized with NEUROD1 ( AT + GCPs ) .

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 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

Figure 4B. Lysates of N2a cells transfected with either WT or a phospho-mimic form of ATOH1 (S309D) were immunoblotted with anti-Atoh1 or β-actin. Quantification of relative levels of ATOH1 to the β-ACTIN (right panel). Mean± s.e.m. Student T-test between WT and S309D was performed. ***p < 0.001. Three slots of each experimental condition represent the three biological replicants of each condition.Rescue experiment of Atoh1 KD with WT or S309A form of Atoh1. (F-I) Scramble (Control), Atoh1 shRNA (Atoh1 KD), shRNA and WT (KD + WT) or shRNA and S309A (KD + S309A) plasmids were introduced to P5 mice EGL with H2B-EGFP plasmid and immunostaining for NEUROD1(magenta) was performed at P7. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest which GFP signals are not co-localized with NEUROD1 (AT+GCPs).


Figure 5B - D . Overexpression analysis of PROX1 . ( B , C ) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of Control ( B ) , PROX1 overexpression ( C ) . Arrow heads indicates the CCND1 positive cells . White lines are representing as pial surfaces . ( D ) The proportion of CCND1 - positive cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and PROX1 overexpression ( N = 3 ) . * * p < 0 . 01 . Number of cells 382 , 147 and 88 cells from N = 3 mice ( control ) , 187 , 108 and 94 cells from N = 3 mice ( Prox1 OE ) . E - G . Prox1 acute KO analysis . ( E , F ) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl / fl mice and immunostaining for CCND1 ( magenta ) was performed at P8 . Nuclei of electroporated cells were labeled with GFP ( green ) . Images of H2B - GFP electroporation ( E , Control ) , Cre electroporation ( F , Cre ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( G ) The proportions of CCND1 + cells among the electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control ( N = 3 ) and Cre ( N = 3 ) . * * p < 0 . 01 . Number of cells : 238 , 129 and 529 cells from N = 3 mice ( Control ) , 169 , 210 and 216 cells from N = 3 mice ( Cre ) . H - K . Rescue experiment of Prox1 with Ccnd1 and Cdk4 - expressing plasmids . ( H - J ) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids ( Prox1 + 4D ) to P5 EGL together with H2B - GFP plasmid . Immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . ( K ) Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . The Tukey - Kramer test was performed on Control ( N = 6 ) , Prox1 ( N = 3 ) and Prox1 + 4D ( N = 4 ) mice . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Bars and error bars are representing as Mean ± SEM . Control value is the same as Fig 2G , K . Number of cells : 54 , 72 and 59 cells from N = 3 mice ( Prox1 ) , 26 , 53 , 68 and 53 cells from N = 4 mice ( Prox1 + 4D ) . L . ChIP assay in P7 mouse cerebellum with anti - Prox1 or goat IgG as a negative control . Primer set used in this experiment was designed to amplify Ccnd1 promoter region . M - R . Administration of Valproic acid ( VPA ) to electroporated mice . ( M ) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation . Immunostaining for CCND1 ( magenta ) was performed the day following the last VPA treatment . Nuclei of electroporated cells were detected with GFP ( green ) . Arrows indicate the cells of interest where GFP signals are co - localized with CCND1 . ( N - Q ) Images of H2B - GFP with Saline ( N ) , PROX1 with Saline ( O ) , H2B - GFP with VPA ( P ) , and PROX1 with VPA ( Q ) . ( R ) The relative proportions of CCND1 + cells among the electroporated cells ( normalized to the Control ) . Tukey - Kramer test among Control + saline ( N = 4 ) , PROX1 + saline ( N = 3 ) , Control + VPA ( N = 3 ) and PROX1 + VPA ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * * p < 0 . 01 , * p < 0 . 05 , N . S . : Not Significant . Number of cells : 120 , 488 , 934 and 211 from N = 4 mice ( Control + saline ) , 630 , 356 and 545 from N = 3 mice ( Control + VPA ) , 363 , 127 and 219 from N = 3 mice ( Prox1 + saline ) , 110 , 116 and 202 from N = 3 mice ( Prox1 + VPA ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B-D. Overexpression analysis of PROX1. (B, C) Prox1 expression plasmids were introduced to mouse P5 EGL and immunostaining for CCND1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of Control (B), PROX1 overexpression (C). Arrow heads indicates the CCND1 positive cells. White lines are representing as pial surfaces. (D) The proportion of CCND1-positive cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and PROX1 overexpression (N=3). **p < 0.01. Number of cells 382, 147 and 88 cells from N=3 mice (control), 187, 108 and 94 cells from N=3 mice (Prox1 OE).E-G. Prox1 acute KO analysis. (E, F) Expression plasmids of Cre recombinase were introduced to P5 EGL of Prox1fl/fl mice and immunostaining for CCND1 (magenta) was performed at P8. Nuclei of electroporated cells were labeled with GFP (green). Images of H2B-GFP electroporation (E, Control), Cre electroporation (F, Cre). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (G) The proportions of CCND1+ cells among the electroporated cells. Bars and error bars are representing as Mean ± SEM. Student T-test between Control (N=3) and Cre (N=3). **p < 0.01. Number of cells: 238, 129 and 529 cells from N=3 mice (Control), 169, 210 and 216 cells from N=3 mice (Cre).H-K. Rescue experiment of Prox1 with Ccnd1 and Cdk4-expressing plasmids. (H-J) Prox1 expressing plasmids were introduced with Ccnd1 and Cdk4 expression plasmids (Prox1+4D) to P5 EGL together with H2B-GFP plasmid. Immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1. (K) Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. The Tukey-Kramer test was performed on Control (N=6), Prox1 (N=3) and Prox1+4D (N=4) mice. **p < 0.01, *p <0.05, N.S.: Not Significant. Bars and error bars are representing as Mean ± SEM. Control value is the same as Fig 2G,K. Number of cells: 54, 72 and 59 cells from N=3 mice (Prox1), 26, 53, 68 and 53 cells from N=4 mice (Prox1+4D).L. ChIP assay in P7 mouse cerebellum with anti-Prox1 or goat IgG as a negative control. Primer set used in this experiment was designed to amplify Ccnd1 promoter region. M-R. Administration of Valproic acid (VPA) to electroporated mice. (M) Expression plasmids of Prox1 were introduced to P5 EGL and VPA or Saline was administered intraperitoneally 2 days after electroporation. Immunostaining for CCND1 (magenta) was performed the day following the last VPA treatment. Nuclei of electroporated cells were detected with GFP (green). Arrows indicate the cells of interest where GFP signals are co-localized with CCND1. (N-Q) Images of H2B-GFP with Saline (N), PROX1 with Saline (O), H2B-GFP with VPA (P), and PROX1 with VPA (Q). (R) The relative proportions of CCND1+ cells among the electroporated cells (normalized to the Control).Tukey-Kramer test among Control+saline (N=4), PROX1+saline (N=3), Control +VPA (N=3) and PROX1 +VPA (N=3). Bars and error bars are representing as Mean ± SEM. **p < 0.01, *p <0.05, N.S.: Not Significant. Number of cells: 120, 488, 934 and 211 from N=4 mice (Control+saline), 630, 356 and 545 from N=3 mice (Control+VPA), 363, 127 and 219 from N=3 mice (Prox1+saline), 110, 116 and 202 from N=3 mice (Prox1+VPA).


Figure 6H - J . Whole cerebellum lysates of P6 , P9 and P12 mice were subjected to immunoblotting with anti - pAtoh1 S309 , Atoh1 , Ccnd1 , Prox1 , β - actin . ( I ) The protein levels of CCND1 and PROX1 normalized by β - ACTIN . ( J ) Quantification of p - L - P . Over - expression analysis of WT or S309D form of Atoh1 . ( L ) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 ( magenta ) was performed in P16 mouse cerebellum . Nuclei of electroporated cells were detected by GFP ( green ) . Images of H2B - GFP ( M , Control ) , WT ( N ) and S309D ( O ) . Arrows indicate the cells of interest where GFP signals are co - localized with KI67 . ( GCPs ) . ( P ) Proportions of KI67 + cells among the electroporated cells . Tukey - Kramer test among Control ( N = 3 ) , Atoh1 WT ( N = 3 ) and Atoh1 S309D ( N = 3 ) . Bars and error bars are representing as Mean ± SEM . * p < 0 . 05 . Number of cells : 43 , 68 and 46 from N = 3 mice ( Control ) , 104 , 215 and 157 cells from N = 3 mice ( WT ) , 242 , 119 and 111 cells from N = 3 mice ( S309D ) .

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 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

Figure 6H-J. Whole cerebellum lysates of P6, P9 and P12 mice were subjected to immunoblotting with anti-pAtoh1 S309, Atoh1, Ccnd1, Prox1, β-actin. (I) The protein levels of CCND1 and PROX1 normalized by β-ACTIN. (J) Quantification of p-L-P. Over-expression analysis of WT or S309D form of Atoh1. (L) Expression plasmids of either WT or S309D form of Atoh1 were introduced to P10 mice EGL and immunostaining for KI67 (magenta) was performed in P16 mouse cerebellum. Nuclei of electroporated cells were detected by GFP (green). Images of H2B-GFP (M, Control), WT(N) and S309D (O). Arrows indicate the cells of interest where GFP signals are co-localized with KI67. (GCPs). (P) Proportions of KI67+ cells among the electroporated cells. Tukey-Kramer test among Control (N=3), Atoh1 WT (N=3) and Atoh1 S309D (N=3). Bars and error bars are representing as Mean ± SEM. *p <0.05. Number of cells: 43, 68 and 46 from N=3 mice (Control), 104, 215 and 157 cells from N=3 mice (WT), 242, 119 and 111 cells from N=3 mice (S309D).


Figure 7D - F . Over - expression analysis of the activated form of β - catenin ( βcat S33Y ) . ( D , E ) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected with GFP ( green ) . Images of H2B - GFP ( D , Control ) and βcat S33Y ( E , S33Y ) . Arrows indicate the cells of interest where GFP signals are co - localized with PROX1 . ( F ) Proportions of PROX1 - positive cells in electroporated cells . Bars and error bars are representing as Mean ± SEM . Student T - test between Control and S33Y was performed . * * * p < 0 . 001 . Number of cells : 89 , 90 and 55 cells from N = 3 mice ( Control ) , 32 , 111 and 56 cells from N = 3 mice ( S33Y ) . J - L . Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids ( 4D ) to P5 mice EGL and immunostaining for ATOH1 ( magenta ) was performed the following day . Nuclei of electroporated cells were detected by GFP ( green ) . Images of βcat S33Y ( J , S33Y ) and βcat S33Y with Ccnd1 and Cdk4 ( K ) . Arrows indicate the cells of interest where GFP signals are co - localized with ATOH1 . Proportions of ATOH1 + cells ( AT + GCPs ) among the electroporated cells . Control value is the same as Fig 2G - K . Bars and error bars are representing as Mean ± SEM . Tukey - Kramer test was performed between Control , S33Y and S33Y + 4D ( L ) . * * * p < 0 . 001 , N . S : Not Significant . Number of cells : 123 , 138 and 113 cells from N = 3 mice ( S33Y ) , 144 , 223 and 180 cells from N = 3 mice ( S33Y + 4D ) .

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 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

Figure 7D-F. Over-expression analysis of the activated form of β-catenin (βcat S33Y). (D, E) Expression plasmids of βcat S33Y were introduced to P5 mice EGL and immunostaining for PROX1 (magenta) was performed the following day. Nuclei of electroporated cells were detected with GFP (green). Images of H2B-GFP (D, Control) and βcat S33Y (E, S33Y). Arrows indicate the cells of interest where GFP signals are co-localized with PROX1. (F) Proportions of PROX1-positive cells in electroporated cells. Bars and error bars are representing as Mean± SEM. Student T-test between Control and S33Y was performed. ***p < 0.001. Number of cells: 89, 90 and 55 cells from N=3 mice (Control), 32, 111 and 56 cells from N=3 mice (S33Y).J-L. Expression plasmids of βcat S33Y were introduced with or without Ccnd1 and Cdk4 expression plasmids (4D) to P5 mice EGL and immunostaining for ATOH1 (magenta) was performed the following day. Nuclei of electroporated cells were detected by GFP (green). Images of βcat S33Y (J, S33Y) and βcat S33Y with Ccnd1 and Cdk4 (K). Arrows indicate the cells of interest where GFP signals are co-localized with ATOH1.Proportions of ATOH1+ cells (AT+GCPs) among the electroporated cells. Control value is the same as Fig 2G-K. Bars and error bars are representing as Mean± SEM. Tukey-Kramer test was performed between Control, S33Y and S33Y+4D (L). ***p < 0.001, N.S: Not Significant. Number of cells: 123, 138 and 113 cells from N=3 mice (S33Y), 144, 223 and 180 cells from N=3 mice (S33Y+4D).


Figure 1A . Assessment of protein levels in different AIFM1 knockout clones . Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells . D . Assessment of protein levels in HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA ( expression of AIFM1 - HA induced for one day ) . Different amounts of lysates from the indicated cells were analyzed by reducing SDS - PAGE and immunoblotting . While levels of MIA40 were largely unchanged , levels of many MIA40 / CHCHD4 substrates were diminished in AIFM1 knockout cells . N = 3 biological replicates . F . BN - PAGE analyses of HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1 - HA or AIFM1 . Complex I was resolved from isolated mitochondria with blue native electrophoresis ( BN - PAGE ) followed by immunoblotting or in gel determination of the complex I specific activity . Levels of complex I were decreased in AIFM1 knockout cells . N = 3 biological

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 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

Figure 1A. Assessment of protein levels in different AIFM1 knockout clones. Lysates from different AIFM1 knockout clones and wild type cells were analyzed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 levels are not changed in AIFM1 knockout HEK293 cells.D. Assessment of protein levels in HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA (expression of AIFM1-HA induced for one day). Different amounts of lysates from the indicated cells were analyzed by reducing SDS-PAGE and immunoblotting. While levels of MIA40 were largely unchanged, levels of many MIA40/CHCHD4 substrates were diminished in AIFM1 knockout cells. N = 3 biological replicates.F. BN-PAGE analyses of HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells with reintroduced AIFM1-HA or AIFM1. Complex I was resolved from isolated mitochondria with blue native electrophoresis (BN-PAGE) followed by immunoblotting or in gel determination of the complex I specific activity. Levels of complex I were decreased in AIFM1 knockout cells. N = 3 biological replicates


Figure 3A . In organello import assay with NDUFS5 , NDUFB7 and as control the matrix protein SOD2 . In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells . Non - imported proteins were removed by treatment with Proteinase K . Imported proteins were analyzed by reducing SDS - PAGE and autoradiography . Signals were quantified using ImageQuantTL and the amount of imported protein was plotted . NDUFS5 import is strongly affected by loss of AIFM1 . Import of NDUFB7 is less affected and SOD2 import remained unaffected . This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar . B . Oxidation kinetics of endogenous NDUFB7 and NDUFS5 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analyzed by pulse - chase experiments coupled to redox state determination . To this end , cells were incubated with 35S - Met for 5 min ( pulse ) . After removal of 35S - Met , cells were left in " cold " Met for different chase times . Further oxidation was stopped by addition of trichloroacetic acid ( TCA ) . Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells . NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed . N = 4 biological replicates for NDUFB7 and NDUFS5 . C . Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells . Cells were incubated with 35S - Met for 5 min ( pulse ) , and then MIA40 / CHCHD4 substrates were enriched by immunoprecipitation ( IP ) , and analyzed by non - reducing SDS - PAGE and autoradiography . NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells . D . Interaction between endogenous MIA40 / CHCHD4 and its substrates in wild type and AIFM1 knockout cells . Cells were subjected to native immunoprecipitation ( IP ) against endogenous MIA40 / CHCHD4 . Precipitates were analyzed by SDS - PAGE and immunoblotting against the indicated proteins . In AIFM1 knockout cell interaction between MIA40 / CHCHD4 and its substrates is strongly affected with the interaction between MIA40 / CHCHD4 and NDUFS5 almost completely abolished . This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells , indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40 / CHCHD4 rather than the cause . Quantification using Image lab . Data from 2 experiments were combined and standard deviations are presented . Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells , respectively .

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

Figure 3A. In organello import assay with NDUFS5, NDUFB7 and as control the matrix protein SOD2. In vitro translated radioactive proteins were incubated with mitochondria isolated from HEK293 and AIFM1 knockout cells. Non-imported proteins were removed by treatment with Proteinase K. Imported proteins were analyzed by reducing SDS-PAGE and autoradiography. Signals were quantified using ImageQuantTL and the amount of imported protein was plotted. NDUFS5 import is strongly affected by loss of AIFM1. Import of NDUFB7 is less affected and SOD2 import remained unaffected. This is despite the fact that the core domains important for oxidative folding of NDUFS5 an NDUFB7 are very similar.B. Oxidation kinetics of endogenous NDUFB7 and NDUFS5. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analyzed by pulse-chase experiments coupled to redox state determination. To this end, cells were incubated with 35S-Met for 5 min (pulse). After removal of 35S-Met, cells were left in "cold" Met for different chase times. Further oxidation was stopped by addition of trichloroacetic acid (TCA). Free thiols but not thiols in disulfide bonds were modified using mmPEG24 and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. Occurrence of oxidized NDUFB7 was slightly delayed in AIFM1 knockout cells. NDUFS5 levels dropped rapidly and only little oxidized NDUFS5 could be observed. N = 4 biological replicates for NDUFB7 and NDUFS5.C. Synthesis of NDUFS5 and NDUFB7 in wild type and AIFM1 knockout cells. Cells were incubated with 35S-Met for 5 min (pulse), and then MIA40/CHCHD4 substrates were enriched by immunoprecipitation (IP), and analyzed by non-reducing SDS-PAGE and autoradiography. NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells.D. Interaction between endogenous MIA40/CHCHD4 and its substrates in wild type and AIFM1 knockout cells. Cells were subjected to native immunoprecipitation (IP) against endogenous MIA40/CHCHD4. Precipitates were analyzed by SDS-PAGE and immunoblotting against the indicated proteins. In AIFM1 knockout cell interaction between MIA40/CHCHD4 and its substrates is strongly affected with the interaction between MIA40/CHCHD4 and NDUFS5 almost completely abolished. This is despite the fact that NDUFS5 and NDUFB7 are synthesized in equal amounts in wildtype and AIFM1 knockout cells, indicating that lowered steady state levels of these proteins are consequence of a lacking interaction with MIA40/CHCHD4 rather than the cause. Quantification using Image lab. Data from 2 experiments were combined and standard deviations are presented. Asterisk indicates background band orange arrow indicates signal of NDUFS5 and NDUFB7 in AIFM1 KO cells, respectively.


Figure 4A . Assessment of MIA40 / CHCHD4 variant - AIFM1 interaction . The indicated MIA40 / CHCHD4 - Strep variants were affinity precipitated ( AP ) under native conditions after stopping thiol - disulfide exchange reactions by NEM incubation . Precipitates were tested for AIFM1 , ALR and MIA40 / CHCHD4 by reducing SDS - PAGE and immunoblotting . 1 % of the total lysate was loaded as input control . M , mock control not expressing MIA40 - Strep . Both , wildtype MIA40 / CHCHD4 and MTS - MIA40 / CHCHD4 but not MIA40 / CHCHD4 ∆ 1 - 40 variants coprecipitated AIFM1 . All mitochondria - localized variants precipitate ALR indicating redox functionality . Arrowhead indicates endogenous MIA40 / CHCHD4 . Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . HEK293 cells stably expressing MIA40 / CHCHD4 - Strep were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and MIA40 / CHCHD4 - Strep ( B ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 interact over a period of at least 6 - 8 hours while the two MIA40 / CHCHD4 substrates , NDUFB7 and AK2 , are quickly released from MIA40 / CHCHD4 ( B , gray box ) . AP , affinity precipitation ; asterisk , background band . C . Emetine - chase experiments to assess the stability of the AIFM1 / CHCHD4 complex . AIFM1 knockout cells stably expressing AIFM1 - HA ( C ) were treated for the indicated times with the ribosome inhibitor emetine . Then , cells were lysed under native conditions ( triton X - 100 ) , and AIFM1 - HA ( C ) were precipitated . Precipitates were analysed by reducing SDS - PAGE and immunoblotting . MIA40 / CHCHD4 and AIFM1 interact over a period of at least 6 - 8 hours AP , affinity precipitation ; asterisk , background band . Eluted fractions were subjected to SDS - PAGE and TCE staining ( E ) . AIFM1 and full - length MIA40 / CHCHD4 migrate together indicating formation of a complex . MIA40 / CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40 / CHCHD4 . AIFM1 and ∆ 1 - 40 MIA40 / CHCHD4 do not migrate together emphasizing the importance of the N - terminal amino acids in MIA40 for interaction wth AIFM1 .

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 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 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 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

Figure 4A. Assessment of MIA40/CHCHD4 variant-AIFM1 interaction. The indicated MIA40/CHCHD4-Strep variants were affinity precipitated (AP) under native conditions after stopping thiol-disulfide exchange reactions by NEM incubation. Precipitates were tested for AIFM1, ALR and MIA40/CHCHD4 by reducing SDS-PAGE and immunoblotting. 1% of the total lysate was loaded as input control. M, mock control not expressing MIA40-Strep. Both, wildtype MIA40/CHCHD4 and MTS-MIA40/CHCHD4 but not MIA40/CHCHD4∆1-40 variants coprecipitated AIFM1. All mitochondria-localized variants precipitate ALR indicating redox functionality. Arrowhead indicates endogenous MIA40/CHCHD4.Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. HEK293 cells stably expressing MIA40/CHCHD4-Strep were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and MIA40/CHCHD4-Strep (B) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 interact over a period of at least 6-8 hours while the two MIA40/CHCHD4 substrates, NDUFB7 and AK2, are quickly released from MIA40/CHCHD4 (B, gray box). AP, affinity precipitation; asterisk, background band.C. Emetine-chase experiments to assess the stability of the AIFM1/CHCHD4 complex. AIFM1 knockout cells stably expressing AIFM1-HA (C) were treated for the indicated times with the ribosome inhibitor emetine. Then, cells were lysed under native conditions (triton X-100), and AIFM1-HA (C) were precipitated. Precipitates were analysed by reducing SDS-PAGE and immunoblotting. MIA40/CHCHD4 and AIFM1 interact over a period of at least 6-8 hours AP, affinity precipitation; asterisk, background band.Eluted fractions were subjected to SDS-PAGE and TCE staining (E). AIFM1 and full-length MIA40/CHCHD4 migrate together indicating formation of a complex. MIA40/CHCHD4 was present in excess over AIFM1 resulting in the presence of free MIA40/CHCHD4. AIFM1 and ∆1-40 MIA40/CHCHD4 do not migrate together emphasizing the importance of the N-terminal amino acids in MIA40 for interaction wth AIFM1.


Figure 5A . Gel filtration analysis to assess the AIFM1 - MIA40 complex in HEK293 cells . HEK293 cells , cells lacking AIFM1 ( AIFM1 knockout , AIFM1 KO ) or AIFM1 knockout cells complemented with AIFM1 - HA were lysed under native conditions ( triton X - 100 ) , and the cleared lysates subjected to gel filtration analysis . Eluted fractions were subjected to TCA precipitation , resuspension in loading buffer containing SDS and DTT , and subsequent immunoblotting against AIFM1 and MIA40 . Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa ( as judged by comparison to protein markers : apoferritin 443 kDa ; β - amylase 200 kDa ; alcohol dehydrogenase 150 kDa ; bovine serum albumin 66 kDa ; carbonic anhydrase 29 kDa ) . Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40 . This behavior is partially rescued by complementation with AIFM1 - HA . B . Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids ( ∆ 1 - 40 MIA40 / CHCHD4 - Strep ) , which constitute the AIFM1 interaction motif . Experiment was performed as described in ( A ) . AIFM1 and endogenous MIA40 / CHCHD4 migrate in a complex with a size larger than 150 kDa , while N - terminally truncated MIA40 / CHCHD4 migrates as monomer . For number of biological replicates and quantifications , see 5C . C . Quantification of 5A , B and Appendix Figure S9 . Quantification of the areas representing the region of monomeric MIA40 / CHCHD4 and the AIFM1 - MIA40 / CHCHD4 complex , respectively was performed . Quantifications consistently show that AIFM1 knockout , siRNA - mediated depletion of AIFM1 and overexpression of MIA40 / CHCHD4 all resulted in an increase of monomeric MIA40 / CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40 / CHCHD4 are tightly regulated . The numbers below the bars indicate the numbers of biological replicates performed . Averages and standard deviations are presented . D . Levels of AIFM1 and MIA40 / CHCHD4 in mitochondria isolated from the indicated tissues . Ratio of AIFM and MIA40 / CHCHD4 was formed and normalized to ratio in HEK293 cells ( = 1 ) . AIFM1 is in liver and kidney more abundant than MIA40 / CHCHD4 . E . Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells . Experiment was performed as described in ( A ) . AIFM1 and MIA40 / CHCHD4 co - migrate in all samples . Excess AIFM1 migrates at lower molecular mass . Number of biological replicates in quantification plot . Where appropriate , standard deviations are presented . F . Assessing the solubility of MIA40 / CHCHD4 in the IMS . Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling . MIA40 / CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1 - HA . It was released from cells lacking AIFM1 . This indicates that MIA40 / CHCHD4 in wildtype cells is membrane - associated via AIFM1 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 5A. Gel filtration analysis to assess the AIFM1-MIA40 complex in HEK293 cells. HEK293 cells, cells lacking AIFM1 (AIFM1 knockout, AIFM1 KO) or AIFM1 knockout cells complemented with AIFM1-HA were lysed under native conditions (triton X-100), and the cleared lysates subjected to gel filtration analysis. Eluted fractions were subjected to TCA precipitation, resuspension in loading buffer containing SDS and DTT, and subsequent immunoblotting against AIFM1 and MIA40. Endogenous AIFM1 and all endogenous MIA40 migrate in a complex with a size larger than 150 kDa (as judged by comparison to protein markers: apoferritin 443 kDa; β-amylase 200 kDa; alcohol dehydrogenase 150 kDa; bovine serum albumin 66 kDa; carbonic anhydrase 29 kDa). Absence of AIFM1 results in migration of MIA40 at the height of monomeric MIA40. This behavior is partially rescued by complementation with AIFM1-HA. B. Gel filtration analysis of a MIA40 variant lacking the first 40 amino acids (∆1-40 MIA40/CHCHD4-Strep), which constitute the AIFM1 interaction motif. Experiment was performed as described in (A). AIFM1 and endogenous MIA40/CHCHD4 migrate in a complex with a size larger than 150 kDa, while N-terminally truncated MIA40/CHCHD4 migrates as monomer. For number of biological replicates and quantifications, see 5C. C. Quantification of 5A,B and Appendix Figure S9. Quantification of the areas representing the region of monomeric MIA40/CHCHD4 and the AIFM1-MIA40/CHCHD4 complex, respectively was performed. Quantifications consistently show that AIFM1 knockout, siRNA-mediated depletion of AIFM1 and overexpression of MIA40/CHCHD4 all resulted in an increase of monomeric MIA40/CHCHD4 indicating that the amounts of available AIFM1 with respect to the amounts of MIA40/CHCHD4 are tightly regulated. The numbers below the bars indicate the numbers of biological replicates performed. Averages and standard deviations are presented.D. Levels of AIFM1 and MIA40/CHCHD4 in mitochondria isolated from the indicated tissues. Ratio of AIFM and MIA40/CHCHD4 was formed and normalized to ratio in HEK293 cells (= 1). AIFM1 is in liver and kidney more abundant than MIA40/CHCHD4.E. Gel filtration analysis of lysates prepared from mitochondria isolated from the indicated tissues and from HepG2 and HEK293 cells. Experiment was performed as described in (A). AIFM1 and MIA40/CHCHD4 co-migrate in all samples. Excess AIFM1 migrates at lower molecular mass. Number of biological replicates in quantification plot. Where appropriate, standard deviations are presented.F. Assessing the solubility of MIA40/CHCHD4 in the IMS. Mitochondria were isolated form the indicated cell lines and the outer membrane destroyed by hypoxic swelling. MIA40/CHCHD4 remains bound to these mitoplasts when they were isolated from wildtype cells or AIFM1 knockout cells that had been complemented with AIFM1-HA. It was released from cells lacking AIFM1. This indicates that MIA40/CHCHD4 in wildtype cells is membrane-associated via AIFM1.


Figure 6A . Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132 . HEK293 cells , AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1 - HA were analysed by pulse - chase experiments coupled to redox state determination . Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132 . Conversely , NDUFS5 levels were strongly increased . In AIFM1 knockout cells , large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation - dependent protein import . The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132 . N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5 .

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Figure 6A. Oxidation kinetics of NDUFB7 and NDUFS5 in the presence of the proteasome inhibitor MG132. HEK293 cells, AIFM1 knockout cells and AIFM1 knockout cells complemented with AIFM1-HA were analysed by pulse-chase experiments coupled to redox state determination. Levels and occurrence of oxidized NDUFB7 was not changed in the presence of MG132. Conversely, NDUFS5 levels were strongly increased. In AIFM1 knockout cells, large amounts of NDUFS5 accumulated in their reduced state indicating impaired oxidation-dependent protein import. The fraction of oxidized protein accumulating is slightly larger than in the absence of MG132. N = 1 biological replicate for NDUFB7 and 2 biological replicates for NDUFS5.


Figure 2 The heat map shows the position ( left to right ) , identity ( top row ) and frequency ( color code ) of variant nucleotide positions detected by SARS - CoV - 2 full genome amplicon sequencing in ( A ) 20 samples of MPP - R workers tested positive on May 20 or 25 , ( B ) 15 samples of MPP - R workers tested positive between June 5 and 17 , and ( C ) two workers from MPP - D who may have transmitted the virus to cases B1 and / or B2 . Individual collection dates are shown to the right of each sample . Variant sequences are given relative to the Wuhan reference strain NC _ 045512 . The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk . Frequencies below 100 % mean that only a fraction of viral genomes shows nucleotide variations , indicating the presence of viral intra - host sub - populations . White rectangles denote nucleotide positions which were not covered by amplicon - seq reads within the respective sample . For nucleotide positions in coding regions , the corresponding viral ORF ( s ) are shown above the variant position . Variants without such information are located in non - coding regions .

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Figure 2  The heat map shows the position (left to right), identity (top row) and frequency (color code) of variant nucleotide positions detected by SARS-CoV-2 full genome amplicon sequencing in (A) 20 samples of MPP-R workers tested positive on May 20 or 25, (B) 15 samples of MPP-R workers tested positive between June 5 and 17, and (C) two workers from MPP-D who may have transmitted the virus to cases B1 and/or B2. Individual collection dates are shown to the right of each sample. Variant sequences are given relative to the Wuhan reference strain NC_045512. The two silent mutations which define the prototype of the investigated outbreak are marked with an asterisk. Frequencies below 100% mean that only a fraction of viral genomes shows nucleotide variations, indicating the presence of viral intra-host sub-populations. White rectangles denote nucleotide positions which were not covered by amplicon-seq reads within the respective sample. For nucleotide positions in coding regions, the corresponding viral ORF(s) are shown above the variant position. Variants without such information are located in non-coding regions.


Figure 3 ( A ) Distance ( in meters ) of PCR - tested workers from the suspected index case B1 at the workplace . For workers without fixed position in the beef processing plant ( marked with an asterisk ) coordinates indicate estimated average location during the early shift . Squares and diamonds denote prototype or variant SARS - CoV - 2 genotypes , respectively . Filled blue circles denote cases for which viral genomes were not sequenced ( i . e . , workers tested positive after 5 / 25 / 2020 ) . ( B ) Top panel : Observed accumulated percentage of positive cases ( red line ) within the indicated distance from the suspected index case . The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases . Bottom panel : - log10 p - value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation .

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Figure 3 (A) Distance (in meters) of PCR-tested workers from the suspected index case B1 at the workplace. For workers without fixed position in the beef processing plant (marked with an asterisk) coordinates indicate estimated average location during the early shift. Squares and diamonds denote prototype or variant SARS-CoV-2 genotypes, respectively. Filled blue circles denote cases for which viral genomes were not sequenced (i.e., workers tested positive after 5/25/2020). (B) Top panel: Observed accumulated percentage of positive cases (red line) within the indicated distance from the suspected index case. The gray dashed line shows the average infection rate that would be expected for a random spatial distribution of positive cases. Bottom panel: -log10 p-value for the frequency of accumulated positive cases within the given distance being significantly higher than expected based on a random spatial distribution of positive cases Only employees with fixed work positions were included in the calculation.


Figure 1Immunoblot analysis of proteins biotinylated and isolated as in ( A ) . Parental HeLa - T - REx cells or cells expressing H3 . 1 - BirA * - HA and treated with biotin for indicated times . Immunoblot analysis of TAP - H3 . 1 or - CENP - A pulldown of UBR7 - LAP ( schematic , upper ) . 293T cells were co - transfected with UBR7 - LAP and TAP - tagged constructs , which were purified on IgG - conjugated sepharose beads .

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Figure 1Immunoblot analysis of proteins biotinylated and isolated as in (A). Parental HeLa-T-REx cells or cells expressing H3.1-BirA*-HA and treated with biotin for indicated times.Immunoblot analysis of TAP-H3.1 or -CENP-A pulldown of UBR7-LAP (schematic, upper). 293T cells were co-transfected with UBR7-LAP and TAP-tagged constructs, which were purified on IgG-conjugated sepharose beads.


Figure 2Immunoblot of anti - FLAG IP of 3x - FLAG tagged GFP , or FLAG - HA tagged H3 . 1 or H2B , from the non - nucleosomal fraction of 293T cells . Representative images of HeLa cells transfected with UBR7 - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . The area within yellow dotted box in ( E ) is magnified 4x in ( E ' ) . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( E ) . Error bars represent mean + / - standard deviation . One way ANOVA was used to calculate p - values ( italics ) associated with differences between treatments . Experiment in ( E - F ) was performed twice .

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Figure 2Immunoblot of anti-FLAG IP of 3x-FLAG tagged GFP, or FLAG-HA tagged H3.1 or H2B, from the non-nucleosomal fraction of 293T cells.Representative images of HeLa cells transfected with UBR7-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. The area within yellow dotted box in (E) is magnified 4x in (E'). Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (E). Error bars represent mean +/- standard deviation. One way ANOVA was used to calculate p-values (italics) associated with differences between treatments. Experiment in (E-F) was performed twice.


Figure 3Immunoblot analysis of anti - FLAG pulldown of full length UBR7 - FLAG ( residues 1 - 425 ) mutant UBR7 bearing deletion of UBR7 C - terminus ( ∆ C - term , residues 1 - 215 ) , UBR box ( ∆ UBR , residues 117 - 425 ) , UBR box and PHD ( ∆ UBR ∆ PHD , residues 216 - 425 ) , or PHD and C - terminus ( ∆ PHD ∆ C - term , residues 1 - 116 ) . 293T cells were co - transfected with H3 . 1 - HA and UBR7 - FLAG constructs ( schematic , upper ) . Asterisk represents non - specific band . Numbers below blot indicate FLAG - normalized anti - HA immunoblot signal relative to full length UBR7 - FLAG . Immunoblot analysis of anti - H3 . 1 - HA pulldown of full - length UBR7 - LAP and UBR7PHD fragment ( residues 117 - 215 ) ( schematic , upper ) . 293T cells were transfected as in ( A ) . Values below pulldown represent H3 . 1 - HA - normalized GFP - UBR7 immunoblot signal relative to UBR7 - FL . Data were acquired from a continuous membrane , and the image is cropped to omit irrelevant samples . Numbers below blot indicate HA - normalized anti - GFP immunoblot signal relative to full length UBR7 - LAP .

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Figure 3Immunoblot analysis of anti-FLAG pulldown of full length UBR7-FLAG (residues 1-425) mutant UBR7 bearing deletion of UBR7 C-terminus (∆C-term, residues 1-215), UBR box (∆UBR, residues 117-425), UBR box and PHD (∆UBR∆PHD, residues 216-425), or PHD and C-terminus (∆PHD∆C-term, residues 1-116). 293T cells were co-transfected with H3.1-HA and UBR7-FLAG constructs (schematic, upper). Asterisk represents non-specific band. Numbers below blot indicate FLAG-normalized anti-HA immunoblot signal relative to full length UBR7-FLAG.Immunoblot analysis of anti-H3.1-HA pulldown of full-length UBR7-LAP and UBR7PHD fragment (residues 117-215) (schematic, upper). 293T cells were transfected as in (A). Values below pulldown represent H3.1-HA-normalized GFP-UBR7 immunoblot signal relative to UBR7-FL. Data were acquired from a continuous membrane, and the image is cropped to omit irrelevant samples. Numbers below blot indicate HA-normalized anti-GFP immunoblot signal relative to full length UBR7-LAP.


Figure 4Anti - GFP immunoblot analysis of streptavidin pulldown of the indicated biotin - conjugated H3 peptides following incubation with 293T - GFP - UBR7 cell lysate after mutation of residues highlighted in ( C ) . Immunoblot analysis for total and modified histones in anti - FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post - nucleosomal H3 / H4 . Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in ( G ) . 4 . 5 h represent newly synthesized histones and 48 h represent pre - existing histones . Wedge represents loading titration : 1x > 0 . 1x > 0 . 01x > 0 . 001x . All other lanes are loaded at 1x concentration . Immunoblot signals were normalized to anti - FLAG signal . Quantification of anti - FLAG ( H3 . 1 - APEX2 - 3xFLAG ) and anti - UBR7 immunoblot represented in ( G - H ) . Lanes 8 and 11 were quantified . Error bar represents mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) .

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Figure 4Anti-GFP immunoblot analysis of streptavidin pulldown of the indicated biotin-conjugated H3 peptides following incubation with 293T-GFP-UBR7 cell lysate after mutation of residues highlighted in (C).Immunoblot analysis for total and modified histones in anti-FLAG pulldown of constructs from soluble cellular fractions demonstrates that UBR7 UBR box and PHD are necessary for binding to post-nucleosomal H3/H4.Immunoblot analysis of streptavidin pulldown from proximity biotinylation described in (G). 4.5 h represent newly synthesized histones and 48 h represent pre-existing histones. Wedge represents loading titration: 1x > 0.1x > 0.01x > 0.001x. All other lanes are loaded at 1x concentration. Immunoblot signals were normalized to anti-FLAG signal.   Quantification of anti-FLAG (H3.1-APEX2-3xFLAG) and anti-UBR7 immunoblot represented in (G-H). Lanes 8 and 11 were quantified. Error bar represents mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line).


Figure 5Immunoblot analysis for total and modified H3 and H4 in anti - FLAG IP . Experiment was performed as in ( D ) . Dot plot error bars represent mean + / - standard deviation . Immunoblot signals were normalized to anti - H4 signal . A two - tailed one sample t - test was used to calculate p - value associated with group mean difference from a hypothetical value of 1 . 0 . Experiment was performed twice . Representative images of HeLa cells transfected with sNASP - FL . DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green . Scale bar = 5 μm . Quantification of the nuclear anti - FLAG immunofluorescence intensity in ( G ) demonstrating that sNASP - FL is highly susceptible to pre - extraction with Triton X - 100 . One way ANOVA was used to determine p - values ( italics ) associated with differences between treatments . Error bars represent mean + / - standard deviation . Experiment in ( G - H ) was performed twice .

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Figure 5Immunoblot analysis for total and modified H3 and H4 in anti-FLAG IP. Experiment was performed as in (D). Dot plot error bars represent mean +/- standard deviation. Immunoblot signals were normalized to anti-H4 signal. A two-tailed one sample t-test was used to calculate p-value associated with group mean difference from a hypothetical value of 1.0. Experiment was performed twice.Representative images of HeLa cells transfected with sNASP-FL. DNA is visualized via DAPI staining shown in magenta and immunofluorescence for 3xFLAG is shown in green. Scale bar = 5 μm.   Quantification of the nuclear anti-FLAG immunofluorescence intensity in (G) demonstrating that sNASP-FL is highly susceptible to pre-extraction with Triton X-100. One way ANOVA was used to determine p-values (italics) associated with differences between treatments. Error bars represent mean +/- standard deviation. Experiment in (G-H) was performed twice.


Figure 6A , C . Immunoblot analysis for modified histone in anti - FLAG pulldown of UBR7 - FL ( A ) or sNASP - FL ( C ) from non - nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors ( hydroxyurea , aphidicolin ) or transcription inhibitors ( α - amanitin , triptolide ) . B , D . Plots represent either normalized immunoblot signals or cell counts prior to fractionation . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation , with grey dotted line marking mean of No Treat condition . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 . Experiments were performed three times . Cells numbers were counted in two of the three experiments .

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Figure 6A,C. Immunoblot analysis for modified histone in anti-FLAG pulldown of UBR7-FL (A) or sNASP-FL (C) from non-nucleosomal fractions of untreated cells or cells treated with DNA replication inhibitors (hydroxyurea, aphidicolin) or transcription inhibitors (α-amanitin, triptolide). B,D. Plots represent either normalized immunoblot signals or cell counts prior to fractionation. Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation, with grey dotted line marking mean of No Treat condition. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0. Experiments were performed three times. Cells numbers were counted in two of the three experiments.


Figure 7Immunoblot analysis of H3 , UBR7 , and GAPDH in the non - nucleosomal fraction of parental and 293T - UBR7KO cells . Quantification of anti - H3 immunoblot represented in ( A ) . Immunoblot signals were normalized to anti - GAPDH signal . Error bars represent mean + / - standard deviation . A two - tailed one - sample t - test was used to calculate p - value ( italics ) associated with group mean difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiment in ( A - B ) was performed in three times . Immunoblot analysis of anti - FLAG pulldown of sNASP - FL in parental or 293T - UBR7KO cells . 3xFLAG tagged sNASP - FL was immunoprecipitated from non - nucleosomal fraction and eluted with FLAG peptide . Experiment was performed three times . Quantification of anti - FLAG ( sNASP - FL ) , and anti - histone immunoblots represented in ( C ) . Immunoblot signals were normalized to anti - FLAG signal . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments were performed two ( sNASP , H4 ) to three ( all other targets ) times .

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Figure 7Immunoblot analysis of H3, UBR7, and GAPDH in the non-nucleosomal fraction of parental and 293T-UBR7KO cells.   Quantification of anti-H3 immunoblot represented in (A). Immunoblot signals were normalized to anti-GAPDH signal. Error bars represent mean +/- standard deviation. A two-tailed one-sample t-test was used to calculate p-value (italics) associated with group mean difference from a hypothetical value of 1.0 (represented by dashed line). Experiment in (A-B) was performed in three times.  Immunoblot analysis of anti-FLAG pulldown of sNASP-FL in parental or 293T-UBR7KO cells. 3xFLAG tagged sNASP-FL was immunoprecipitated from non-nucleosomal fraction and eluted with FLAG peptide. Experiment was performed three times.   Quantification of anti-FLAG (sNASP-FL), and anti-histone immunoblots represented in (C). Immunoblot signals were normalized to anti-FLAG signal. Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments were performed two (sNASP, H4) to three (all other targets) times.


Figure 8Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7 - FL ( left ) or sNASP - FL ( right ) following treatment with siCTRL , siLAMP2A , or MG - 132 . Quantification of data represented in ( C , D ) . Error bars represent mean + / - standard deviation . A two - tailed one sample t - test was used to calculate p - values ( italics ) associated with group means difference from a hypothetical value of 1 . 0 ( represented by dashed line ) . Experiments in ( C - E ) were performed three times .

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Figure 8Immunoblot for LAMP2A in parental 293T cells or cells expressing UBR7-FL (left) or sNASP-FL (right) following treatment with siCTRL, siLAMP2A, or MG-132.Quantification of data represented in (C, D). Error bars represent mean +/- standard deviation. A two-tailed one sample t-test was used to calculate p-values (italics) associated with group means difference from a hypothetical value of 1.0 (represented by dashed line). Experiments in (C-E) were performed three times.


Figure 9Heatmaps of ATAC - seq , anti - GFP ( GFP - UBR7 ) ChIP - seq , and anti - H3K4me3 CUT & RUN in parental and UBR7KO cells . Plots are centered on TSS that show changes in ATAC - seq signal in UBR7KO versus parental cells . Sites are clustered by unsupervised k - means analysis . Cluster color key is defined by cluster labels .

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Figure 9Heatmaps of ATAC-seq, anti-GFP (GFP-UBR7) ChIP-seq, and anti-H3K4me3 CUT&RUN in parental and UBR7KO cells. Plots are centered on TSS that show changes in ATAC-seq signal in UBR7KO versus parental cells. Sites are clustered by unsupervised k-means analysis. Cluster color key is defined by cluster labels.


Figure 1F , Chow fed hIGFREO had similar body mass to WT , however , hIGFREO did not gain as much weight as WT after 8 weeks of HFD ( n = 6 - 10 mice per group ) . G , Representative images of difference in fat and water distribution shown by magnetic resonance ( MR ) imaging in hIGFREO and WT . Scale bar = 1cm .

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Figure 1F, Chow fed hIGFREO had similar body mass to WT, however, hIGFREO did not gain as much weight as WT after 8 weeks of HFD (n=6-10 mice per group).G, Representative images of difference in fat and water distribution shown by magnetic resonance (MR) imaging in hIGFREO and WT. Scale bar = 1cm.


Figure 2hIGFREO glucose intolerance measured by glucose tolerance test ( n = 5 - 7 mice per group ) . G , H , Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve ( n = 10 - 12 mice per group ) .

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Figure 2hIGFREO glucose intolerance measured by glucose tolerance test (n=5-7 mice per group).G, H, Percentage change in plasma levels of triglycerides after an olive oil oral gavage was reduced over the 3hr period post gavage in hIGFREO compared to WT and shown as area under the curve (n=10-12 mice per group).


Figure 3hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates ( WT ) after HFD . ( n = 4 per genotype ) . D , Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT ( n = 6 per genotype ) .

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Figure 3hIGFREO , food consumption using indirect calorimeter assessment after HFD compared to wildtype littermates (WT) after HFD. (n=4 per genotype).D, Adipose expression of browning markers are also no different in white epididymal adipose tissue and brown adipose tissue compared to WT (n= 6 per genotype).


Figure 4Faith ' s phylogenetic diversity ( PD ) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF - 1R endothelial overexpressing mice ( hIGFREO ) mice and wildtype littermates ( WT ) mice after high fat diet feeding ( n = 4 - 5 mice per group ) . Chao - 1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT ( n = 4 - 5 mice per group ) .

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Figure 4Faith's phylogenetic diversity (PD) was used to measure the fecal microbial diversity and demonstrates a significant difference between human IGF-1R endothelial overexpressing mice (hIGFREO) mice and wildtype littermates (WT) mice after high fat diet feeding (n=4-5 mice per group). Chao-1 analysis was used to measure the fecal microbial diversity and abundance and demonstrates a significant difference between hIGFREO and WT (n=4-5 mice per group).


Figure 5B , Bi , human IGF - 1R endothelial overexpressing mice ( hIGFREO ) had comparable weight gain as wildtype littermates ( WT ) after 8 weeks of HFD + antibiotics ( ABs ) when compared to WT . Bii , both hIGFREO and WT gained significant weight compared to chow fed mice ( n = 7 = 9 mice per group ) . D , E , There was no difference in hIGFREO and WT glucose tolerance ( as measured by glucose tolerance test and area under the curve ( AUC ) ) ( n = 7 - 9 mice per group ) .

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Figure 5B, Bi, human IGF-1R endothelial overexpressing mice (hIGFREO) had comparable weight gain as wildtype littermates (WT) after 8 weeks of HFD + antibiotics (ABs) when compared to WT. Bii, both hIGFREO and WT gained significant weight compared to chow fed mice (n=7=9 mice per group).D, E, There was no difference in hIGFREO and WT glucose tolerance (as measured by glucose tolerance test and area under the curve (AUC)) (n=7-9 mice per group).