Figure 1B The expression of mature miR - 200b and miR - 200a in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 ; Student ' s t - test ) . C The expression of Ttll10 ( the host gene of miR - 200b / a ) mRNA in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 3540 ; Student ' s t - test ) . D The locomotor activity of the miR - 200b / a KD mice was similar to that of the NC mice ( NC : n = 13 mice , miR - 200b / a knockdown : n = 15 mice ; data represent the mean ± SEM ; p = 0 . 5030 ; Student ' s t - test ) . E The odorant habituation results shown as the ratio of odor sniffs / water of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice . These odorants included male urine ( 1 : 50 ) , female urine ( 1 : 50 ) , isopentyl acetate ( 50 μM ) , citral ( 50 μM ) , 2 - heptanone ( 50 μM ) and propyl propionate ( 50 μM ) ( n = 6 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) . The resident / intruder experiment showed that the attack frequency ( F ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . G The resident / intruder experiment showed that the attack duration ( G ) of the miR - 200b / a KD mice were impaired compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . H , I The analysis of male - female mating behaviors showed that the mating frequency ( H ) and duration ( I ) of the miR - 200b / a KD mice were significantly reduced compared to those of the NC mice ( NC : n = 11 mice , miR - 200b / a KD : n = 10 mice ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 1B The expression of mature miR-200b and miR-200a in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01; Student's t-test).C The expression of Ttll10 (the host gene of miR-200b/a) mRNA in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; p=0.3540; Student's t-test).D The locomotor activity of the miR-200b/a KD mice was similar to that of the NC mice (NC: n=13 mice, miR-200b/a knockdown: n=15 mice; data represent the mean±SEM; p=0.5030; Student's t-test).E The odorant habituation results shown as the ratio of odor sniffs/water of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice. These odorants included male urine (1:50), female urine (1:50), isopentyl acetate (50 μM), citral (50 μM), 2-heptanone (50 μM) and propyl propionate (50 μM) (n=6 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test).The resident/intruder experiment showed that the attack frequency (F) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).G The resident/intruder experiment showed that the attack duration (G) of the miR-200b/a KD mice were impaired compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; ****p<0.0001; Student's t-test).H, I The analysis of male-female mating behaviors showed that the mating frequency (H) and duration (I) of the miR-200b/a KD mice were significantly reduced compared to those of the NC mice (NC: n=11 mice, miR-200b/a KD: n=10 mice; data represent the mean±SEM; **p<0.01, ***p<0.001; Student's t-test).


Figure 2B Representative IF staining with Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . C Quantification of the numbers of Ki67 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 , * * * * p < 0 . 0001 ; Student ' s t - test ) . D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the EdU + cells and the yellow arrows indicate the cells positive for both EdU and Ki67 . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the numbers of costained Ki67 + and EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR - 200b / a KD mice compared with their NC controls [ the cell cycle exit index was calculated using the following formula : ( EdU + Ki67 − cells / total EdU + cells ) × 100 ; n = 3 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ] . G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the BrdU + cells , and the yellow arrows indicate the cells positive for both BrdU and EdU . G ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . G : Scale bars , 20 μm ; G ' : Scale bars , 2 μm . H The S phase length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( the S phase length was calculated using the following formula : 3 h × BrdU + / BrdU + EdU - ; n = 3 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . I Total cell cycle length of the GBCs in the MOE of the NC and miR - 200b / a KD mice ( total cell cycle length was calculated using the following formula : 20 h + ( Ts × EdU + BrdU - / Edu + ) ; n = 3 mice each group ; p = 0 . 9785 ; data represent the mean ± SEM ; Student ' 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 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 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 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 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

Figure 2B Representative IF staining with Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. C Quantification of the numbers of Ki67+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01, ****p<0.0001; Student's t-test). D Representative IF costaining with both EdU and Ki67 antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the EdU+ cells and the yellow arrows indicate the cells positive for both EdU and Ki67. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the numbers of costained Ki67+ and EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). F The proportion of GBCs that exited the cell cycle was significantly reduced in the miR-200b/a KD mice compared with their NC controls [the cell cycle exit index was calculated using the following formula: (EdU+ Ki67− cells/total EdU+ cells)×100; n=3 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test]. G Representative IF costaining with both EdU and BrdU antibody in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the BrdU+ cells, and the yellow arrows indicate the cells positive for both BrdU and EdU. G' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). G: Scale bars, 20 μm; G': Scale bars, 2 μm.H The S phase length of the GBCs in the MOE of the NC and miR-200b/a KD mice (the S phase length was calculated using the following formula: 3 h×BrdU+/BrdU+EdU-; n=3 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).I Total cell cycle length of the GBCs in the MOE of the NC and miR-200b/a KD mice (total cell cycle length was calculated using the following formula: 20 h+(Ts×EdU+BrdU-/Edu+); n=3 mice each group; p=0.9785; data represent the mean±SEM; Student's t-test).


Figure 3B The mRNA levels of CK14 , Trp63 , Sox2 and Mash1 in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 2135 , Trp63 : p = 0 . 1930 , Sox2 : p = 0 . 5190 , * p < 0 . 05 ; Student ' s t - test ) . C Representative IF staining for CK14 , Sox2 , and Mash1 in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . D Quantification of the number of CK14 + , basal Sox2 + , apical Sox2 + , and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; CK14 : p = 0 . 8998 , basal Sox2 : p = 0 . 7056 , apical Sox2 : p = 0 . 4455 , * * p < 0 . 01 ; Student ' s t - test ) . E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR - 200b / a KD mice ( n = 2 mice each group ) . GAPDH and Tubulin served as loading controls . The molecular weight of each band is indicated at the right . F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Sox2 and EdU . F ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . F : Scale bars , 20 μm ; F ' : Scale bars , 2 μm . G Quantification of the number of Sox2 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; p = 0 . 7988 ; Student ' s t - test ) . H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both Mash1 and EdU . H ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( right images ) . H : Scale bars , 20 μm ; H ' : Scale bars , 2 μm . I Quantification of the number of Mash1 + EdU + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B The mRNA levels of CK14, Trp63, Sox2 and Mash1 in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; CK14: p=0.2135, Trp63: p=0.1930, Sox2: p=0.5190, *p<0.05; Student's t-test).C Representative IF staining for CK14, Sox2, and Mash1 in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. D Quantification of the number of CK14+, basal Sox2+, apical Sox2+, and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; CK14: p=0.8998, basal Sox2: p=0.7056, apical Sox2: p=0.4455, **p<0.01; Student's t-test). E Western Blot analysis of the Sox2 and Mash1 protein levels in the MOE of the NC and miR-200b/a KD mice (n=2 mice each group). GAPDH and Tubulin served as loading controls. The molecular weight of each band is indicated at the right. F Representative IF costaining with Sox2 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Sox2 and EdU. F' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). F: Scale bars, 20 μm; F': Scale bars, 2 μm. G Quantification of the number of Sox2+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; p=0.7988; Student's t-test). H Representative IF costaining with Mash1 antibody and EdU in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both Mash1 and EdU. H' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (right images). H: Scale bars, 20 μm; H': Scale bars, 2 μm. I Quantification of the number of Mash1+EdU+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test).


Figure 4A Representative IF staining for Tuj1 , GAP43 , and OMP in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . B Quantification of the number of Tuj1 + , GAP43 + , and OMP + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ; Student ' s t - test ) . C The mRNA levels of Ngn1 , GAP43 and OMP in the MOE of the NC and miR - 200b / a KD mice , as revealed by qPCR analysis ( n = 4 mice each group ; data represent the mean ± SEM ; * p < 0 . 05 ; Student ' s t - test ) . D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d . The white arrows indicate the cells positive for both GAP43 and BrdU . D ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . D : Scale bars , 20 μm ; D ' : Scale bars , 2 μm . E Quantification of the ratio of the number of BrdU + GAP43 + cells and the number of total GAP43 + cells in the MOE of the NC and miR - 200b / a KD mice injected with BrdU at 3 d ( n = 3 mice each group ; data represent the mean ± SEM ; * * * * p < 0 . 0001 ; Student ' s t - test ) . F Representative IF staining with Cas3 antibody in the MOE of the NC and miR - 200b / a KD mice . Scale bars , 20 μm . G Quantification of the number of Cas3 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A Representative IF staining for Tuj1, GAP43, and OMP in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. B Quantification of the number of Tuj1+, GAP43+, and OMP+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; *p<0.05, **p<0.01, ***p<0.001; Student's t-test). C The mRNA levels of Ngn1, GAP43 and OMP in the MOE of the NC and miR-200b/a KD mice, as revealed by qPCR analysis (n=4 mice each group; data represent the mean±SEM; *p<0.05; Student's t-test). D Representative IF costaining with GAP43 and BrdU antibodies in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d. The white arrows indicate the cells positive for both GAP43 and BrdU. D' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). D: Scale bars, 20 μm; D': Scale bars, 2 μm. E Quantification of the ratio of the number of BrdU+ GAP43+ cells and the number of total GAP43+ cells in the MOE of the NC and miR-200b/a KD mice injected with BrdU at 3 d (n=3 mice each group; data represent the mean±SEM; ****p<0.0001; Student's t-test). F Representative IF staining with Cas3 antibody in the MOE of the NC and miR-200b/a KD mice. Scale bars, 20 μm. G Quantification of the number of Cas3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).


Figure 5A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR - 200b / a KD mice . C TET 3 ' UTR - dependent expression of a luciferase reporter gene was suppressed by miR - 200a overexpression . Luciferase reporter gene under the control of the wild - type ( WT ) or mutant ( Mut ) TET3 3 ' UTR was transfected along with the negative control ( nc ) RNA or miR - 200a mimic duplexes into HeLa cells ( n = 6 from six independent experiments ; data represent the mean ± SEM ; Mut - nc vs . Mut - mimic : p = 0 . 3697 , * p < 0 . 05 ; Student ' s t - test ) . D , E The expression of mature miR - 200a ( D ) and TET3 ( E ) mRNA in the 3T3 - L1 cells treated with miR - 200a inhibitor , miR - 200a mimic or control , as revealed by qPCR analysis ( n = 4 from four independent experiments ; data represent the mean ± SEM ; D : F = 66 . 37 , E : F = 16 . 39 , NC vs . mimic : p = 0 . 2597 , * p < 0 . 05 , * * * p < 0 . 001 ; one - way ANOVA and Bonferroni pairwise comparisons ) . F Western Blot analysis of the TET3 protein level in the 3T3 - L1 cells treated with miR - 200a inhibitor , miR - 200a mimic or control ( n = 3 from three independent experiments ) . Actin served as a loading control . The molecular weight of each band is indicated at the right . Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR - 200a ( G ) NC mimic ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right . H Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR - 200b ( H ) or NC mimic ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right . I Western Blot analysis of the TET3 protein level in the MOE of the NC and miR - 200b / a KD mice ( n = 3 mice each group ) . Actin served as a loading control . The molecular weight of each band is indicated at the right . The blue number shows the ratio of the average intensity of TET3 / Actin . J Representative IF staining of TET3 in the MOE of the NC and miR - 200b / a KD mice . Scale bars : 20 μm . K Quantification of the number of TET3 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) . L Representative IF staining of 5mC in the MOE of the NC and miR - 200b / a knockdown mice . L ' is shown at higher magnification of the boxed region . L : Scale bars , 20 μm ; L ' : Scale bars , 2 μm . M Quantification of the number of 5mC + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) . N Representative IF staining of 5hmC in the MOE of the NC and miR - 200b / a knockdown mice . N ' is shown at higher magnification of the boxed region . N : Scale bars , 20 μm ; N ' : Scale bars , 2 μm . Quantification of the number of 5hmC + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' 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 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 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 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 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 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 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 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

Figure 5A The number of differentially expressed olfrs from RNA sequencing in the MOE of the NC and miR-200b/a KD mice.C TET 3' UTR-dependent expression of a luciferase reporter gene was suppressed by miR-200a overexpression. Luciferase reporter gene under the control of the wild-type (WT) or mutant (Mut) TET3 3' UTR was transfected along with the negative control (nc) RNA or miR-200a mimic duplexes into HeLa cells (n=6 from six independent experiments; data represent the mean±SEM; Mut-nc vs. Mut-mimic: p=0.3697, *p<0.05; Student's t-test).D, E The expression of mature miR-200a (D) and TET3 (E) mRNA in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control, as revealed by qPCR analysis (n=4 from four independent experiments; data represent the mean±SEM; D: F=66.37, E: F=16.39, NC vs. mimic: p=0.2597, *p<0.05, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons).F Western Blot analysis of the TET3 protein level in the 3T3-L1 cells treated with miR-200a inhibitor, miR-200a mimic or control (n=3 from three independent experiments). Actin served as a loading control. The molecular weight of each band is indicated at the right.Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200a (G) NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.H Western Blot analysis of the TET3 protein level in the MOE of the mice intranasally injected with miR-200b (H) or NC mimic (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right.I Western Blot analysis of the TET3 protein level in the MOE of the NC and miR-200b/a KD mice (n=3 mice each group). Actin served as a loading control. The molecular weight of each band is indicated at the right. The blue number shows the ratio of the average intensity of TET3/Actin.J Representative IF staining of TET3 in the MOE of the NC and miR-200b/a KD mice. Scale bars: 20 μm.K Quantification of the number of TET3+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).L Representative IF staining of 5mC in the MOE of the NC and miR-200b/a knockdown mice. L' is shown at higher magnification of the boxed region. L: Scale bars, 20 μm; L': Scale bars, 2 μm.M Quantification of the number of 5mC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test).N Representative IF staining of 5hmC in the MOE of the NC and miR-200b/a knockdown mice. N' is shown at higher magnification of the boxed region. N: Scale bars, 20 μm; N': Scale bars, 2 μm.Quantification of the number of 5hmC+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).


Figure 6A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both TET3 and Mash1 . A ' is shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . A : Scale bars , 20 μm ; A ' : Scale bars , 2 μm . B Quantification of the number of costained TET3 + and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * * p < 0 . 001 ; Student ' s t - test ) . C , D The resident / intruder experiment showed that the attack frequency ( C ) and duration ( D ) of the miR - 200b / a + TET3 DKD mice were significantly improved compared with those of the miR - 200b / a KD controls ; [ 200 KD : miR - 200b / a KD ; ( 200 + T ) DKD : ( miR - 200b / a + TET3 ) DKD ; ( NC : n = 18 mice , miR - 200b / a KD : n = 14 mice , miR - 200b / a + TET3 DKD : n = 16 mice ; data represent the mean ± SEM ; C : F = 17 . 84 , D : F = 12 . 06 , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; One - way ANOVA and Bonferroni pairwise comparisons ) ] . E , F The analysis of male - female mating behaviors showed that the frequency ( E ) and duration ( F ) of mating to females of the miR - 200b / a + TET3 DKD mice were significantly reduced compared with those of their NC controls and were not dramatically different compared with those of the miR - 200b / a KD mice ( NC : n = 18 mice , miR - 200b / a KD : n = 14 mice , miR - 200b / a + TET3 DKD : n = 16 mice ; data represent the mean ± SEM ; E : F = 15 . 62 , F : F = 15 . 56 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) . G Representative IF staining for Mash1 , Tuj1 , Cas3 and Ki67 in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice . Scale bars : 20 μm . H - K Quantification of the numbers of Mash1 + ( H ) , Tuj1 + ( I ) , Cas3 + ( J ) and Ki67 + ( K ) cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; Mash1 : F = 41 . 45 , Tuj1 : F = 113 . 9 , Cas3 : F = 92 . 51 , Ki67 : F = 61 . 35 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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 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 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 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

Figure 6A Representative IF staining for costaining with TET3 and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both TET3 and Mash1. A' is shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). A: Scale bars, 20 μm; A': Scale bars, 2 μm. B Quantification of the number of costained TET3+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; ***p<0.001; Student's t-test). C, D The resident/intruder experiment showed that the attack frequency (C) and duration (D) of the miR-200b/a+TET3 DKD mice were significantly improved compared with those of the miR-200b/a KD controls; [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean±SEM; C: F=17.84, D: F=12.06, *p<0.05, ***p<0.001, ****p<0.0001; One-way ANOVA and Bonferroni pairwise comparisons)].E, F The analysis of male-female mating behaviors showed that the frequency (E) and duration (F) of mating to females of the miR-200b/a+TET3 DKD mice were significantly reduced compared with those of their NC controls and were not dramatically different compared with those of the miR-200b/a KD mice (NC: n=18 mice, miR-200b/a KD: n=14 mice, miR-200b/a+TET3 DKD: n=16 mice; data represent the mean ±SEM; E: F=15.62, F: F=15.56, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).G Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice. Scale bars: 20 μm. H-K Quantification of the numbers of Mash1+(H), Tuj1+(I), Cas3+(J) and Ki67+(K) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=41.45, Tuj1: F=113.9, Cas3: F=92.51, Ki67: F=61.35, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


Figure 7A The REST mRNA expression in the MOE of the NC , miR - 200b / a KD and miR - 200b / a + TET3 DKD mice , as revealed by qPCR analysis [ 200 KD : miR - 200b / a KD ; ( 200 + T ) DKD : ( miR - 200b / a + TET3 ) DKD ; n = 4 mice each group ; data represent the mean ± SEM ; F = 9 . 409 , NC vs . 200 : p = 0 . 1367 , 200 vs . 200 + T : p = 0 . 1896 , * * p < 0 . 01 ; one - way ANOVA and Bonferroni pairwise comparisonsB Western Blot analysis of the REST protein level in the MOE of the NC , miR - 200b / a KD and miR - 200b / a + TET3 DKD mice ( n = 3 mice each group ) . Actin served as a loading control . C Representative IF staining for REST in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a and TET3 DKD mice . Scale bars : 20 μm . D Quantification of the number of REST + cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + TET3 DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; F = 12 . 11 , * p < 0 . 05 ; one - way ANOVA and Bonferroni pairwise comparisons ) . Representative IF costaining with REST and Mash1 antibodies in the MOE of the NC and miR - 200b / a KD mice . The white arrows indicate the cells positive for both REST and Mash1 . E ' are shown at higher magnification of the boxed region , as three channel merged images ( left image ) and the separated channel ( middle and right images ) . E : Scale bars , 20 μm ; E ' : Scale bars , 2 μm . F Quantification of the number of costained REST + and Mash1 + cells in the MOE of the NC and miR - 200b / a KD mice ( n = 4 mice each group ; data represent the mean ± SEM ; * * p < 0 . 01 ; Student ' s t - test ) . G Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against TET3 . Input protein ( 100 ug ) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies . IgG antibodies were used as the negative control . H Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against REST . Input protein ( 100 ug ) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies . IgG antibodies were used as the negative 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 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 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 O O O O O O O O O O O O O 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 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 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

Figure 7A The REST mRNA expression in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice, as revealed by qPCR analysis [200 KD: miR-200b/a KD; (200+T) DKD: (miR-200b/a+TET3) DKD; n=4 mice each group; data represent the mean±SEM; F=9.409, NC vs. 200: p=0.1367, 200 vs. 200+T: p=0.1896, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisonsB Western Blot analysis of the REST protein level in the MOE of the NC, miR-200b/a KD and miR-200b/a+TET3 DKD mice (n=3 mice each group). Actin served as a loading control.C Representative IF staining for REST in the MOE of the NC, miR-200b/a KD, and miR-200b/a and TET3 DKD mice. Scale bars: 20 μm.D Quantification of the number of REST+ cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+TET3 DKD mice (n=4 mice each group; data represent the mean±SEM; F=12.11, *p<0.05; one-way ANOVA and Bonferroni pairwise comparisons).Representative IF costaining with REST and Mash1 antibodies in the MOE of the NC and miR-200b/a KD mice. The white arrows indicate the cells positive for both REST and Mash1. E' are shown at higher magnification of the boxed region, as three channel merged images (left image) and the separated channel (middle and right images). E: Scale bars, 20 μm; E': Scale bars, 2 μm.F Quantification of the number of costained REST+ and Mash1+ cells in the MOE of the NC and miR-200b/a KD mice (n=4 mice each group; data represent the mean±SEM; **p<0.01; Student's t-test).G Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against TET3. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.H Mouse MOE proteins were immunoprecipitated with polyclonal antibodies against REST. Input protein (100 ug) and immunoprecipitates were subjected to immunoblot analysis with the indicated antibodies. IgG antibodies were used as the negative control.


Figure 8A , B The resident / intruder experiment showed that the attack frequency ( A ) and duration ( B ) of the miR - 200b / a + REST DKD mice were dramatically improved compared with those of the miR - 200b / a KD mice [ 200 KD : miR - 200b / a KD ; ( 200 + R ) DKD : ( miR - 200b / a + REST ) DKD ; NC : n = 10 mice , miR - 200b / a KD : n = 9 mice , miR - 200b / a + REST DKD : n = 13 mice ; data represent the mean ± SEM ; A : F = 10 . 62 , B : F = 10 . 54 , * * * p < 0 . 001 ; one - way ANOVA and Bonferroni pairwise comparisons ] . C , D The analysis of male - female mating behaviors showed that the frequency ( C ) and duration ( D ) of mating to females of the miR - 200b / a + REST DKD mice were significantly improved compared with those of the miR - 200b / a KD mice ( NC : n = 10 mice , miR - 200b / a KD : n = 9 mice , miR - 200b / a + REST DKD : n = 13 mice ; data represent the mean ± SEM ; C : F = 6 . 162 , D : F = 6 . 360 , * p < 0 . 05 , * * p < 0 . 01 ; one - way ANOVA and Bonferroni pairwise comparisons ) . Representative IF staining for Mash1 , Tuj1 , Cas3 and Ki67 in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice . Scale bars : 20 μm . F - I Quantification of the number of Mash1 + ( F ) , Tuj1 + ( G ) , Cas3 + ( H ) and Ki67 + ( I ) cells in the MOE of the NC , miR - 200b / a KD , and miR - 200b / a + REST DKD mice ( n = 4 mice each group ; data represent the mean ± SEM ; Mash1 : F = 35 . 53 , Tuj1 : F = 62 . 28 , Cas3 : F = 115 . 7 , Ki67 : F = 77 . 43 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 ; one - way ANOVA and Bonferroni pairwise comparisons ) .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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 8A, B The resident/intruder experiment showed that the attack frequency (A) and duration (B) of the miR-200b/a+REST DKD mice were dramatically improved compared with those of the miR-200b/a KD mice [200 KD: miR-200b/a KD; (200+R) DKD: (miR-200b/a+REST) DKD; NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; A: F=10.62, B: F=10.54, ***p<0.001; one-way ANOVA and Bonferroni pairwise comparisons].C, D The analysis of male-female mating behaviors showed that the frequency (C) and duration (D) of mating to females of the miR-200b/a+REST DKD mice were significantly improved compared with those of the miR-200b/a KD mice (NC: n=10 mice, miR-200b/a KD: n=9 mice, miR-200b/a+REST DKD: n=13 mice; data represent the mean±SEM; C: F=6.162, D: F=6.360, *p<0.05, **p<0.01; one-way ANOVA and Bonferroni pairwise comparisons).Representative IF staining for Mash1, Tuj1, Cas3 and Ki67 in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice. Scale bars: 20 μm. F-I Quantification of the number of Mash1+(F), Tuj1+(G), Cas3+(H) and Ki67+(I) cells in the MOE of the NC, miR-200b/a KD, and miR-200b/a+REST DKD mice (n=4 mice each group; data represent the mean±SEM; Mash1: F=35.53, Tuj1: F=62.28, Cas3: F=115.7, Ki67: F=77.43, **p<0.01, ***p<0.001, ****p<0.0001; one-way ANOVA and Bonferroni pairwise comparisons).


f1The rate of glucose uptake ( a , e ) , lactate released after 4 h of incubation ( b , f ) , glycolytic flux ( c , g ) and pentose - phosphate flux ( d , h ) were assessed in MEF and mouse cortical neurons in primary culture , as indicated , obtained from either WT or Pink1 KO ( Pink1 − / − ) mice . Blood concentrations of glucose ( i ) and lactate ( j ) , and the rate of 14C - lactate appearance in the blood from acutely injected [ U - 14C ] glucose in the tail vein ( k ) of WT and Pink1 KO mice were analysed . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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 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

f1The rate of glucose uptake (a,e), lactate released after 4 h of incubation (b,f), glycolytic flux (c,g) and pentose-phosphate flux (d,h) were assessed in MEF and mouse cortical neurons in primary culture, as indicated, obtained from either WT or Pink1 KO (Pink1−/−) mice.Blood concentrations of glucose (i) and lactate (j), and the rate of 14C-lactate appearance in the blood from acutely injected [U-14C]glucose in the tail vein (k) of WT and Pink1 KO mice were analysed. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


f2Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO miceMEF ( a ) ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT . Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null mice MEF ( b ) ß - Actin was used as loading control . Quantitative real - time PCR ( RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO neurons ( c ) . ß - Actin mRNA was used for normalization , and the Pink1 KO data are expressed as the change relative to WT . Western blot analysis of the protein abundances of PINK1 , GLUT1 , GLUT3 ( not detectable in MEF ) , HK2 , GAPDH and HIF1α in WT and Pink1 - null neurons ( d ) ; ß - Actin was used as loading control . ( e ) Western blot against PINK1 , GLUT1 , GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples ; α - Tubulin was used as loading control . Only representative western blots are shown per condition ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . The mean ± s . e . m . values of the mRNA data were calculated from the fold change of each ß - Actin - normalized transcript abundance in the Pink1 KO samples versus that in the WT . Thus , in all cases , the WT values were considered = 1 . 00 . * P0 . 05 versus the corresponding WT ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

f2Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO miceMEF (a) ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null mice MEF (b) ß-Actin was used as loading control.Quantitative real-time PCR (RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO neurons (c). ß-Actin mRNA was used for normalization, and the Pink1 KO data are expressed as the change relative to WT.Western blot analysis of the protein abundances of PINK1, GLUT1, GLUT3 (not detectable in MEF), HK2, GAPDH and HIF1α in WT and Pink1-null neurons (d); ß-Actin was used as loading control.(e) Western blot against PINK1, GLUT1, GLUT3 and HIF1α in WT and Pink1 KO mice skeletal muscle samples; α-Tubulin was used as loading control. Only representative western blots are shown per condition; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. The mean±s.e.m. values of the mRNA data were calculated from the fold change of each ß-Actin-normalized transcript abundance in the Pink1 KO samples versus that in the WT. Thus, in all cases, the WT values were considered=1.00. *P0.05 versus the corresponding WT (Student's t-test; n=3-4 independent experiments).


f3 ( a ) RT - qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α , or siControl ( 100 nM ) , after 72 h . ( b ) siHif1α effectively knockdowns expressed Hif1α cDNA WT , but not a Hif1α cDNA silent mutant form refractory to siHif1α ( Hif1α cDNA REFR . ) in mice MEF . ( c ) RT - qPCR analysis of the relative mRNA abundances of Glut1 , Glut3 , Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α . ß - Actin mRNA was used for normalization , and all data are expressed as the change relative to WT - siControl . ( d ) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α ; ß - actin was used as loading control . As shown , siHif1α partially prevented the increase in GLUT1 , HK2 and GAPDH protein abundances . The increased rate of lactate released after 4 h of incubation ( e ) and glycolytic flux ( f ) observed in the Pink1 KO MEF was prevented by siHif1α . siHif1α was also effective at preventing the endogenous increased HIF1α protein ( g ) , as well as the increased glycolytic flux ( h ) observed in Pink1 KO primary neurons . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information .

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 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 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 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 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 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

f3(a) RT-qPCR analysis of Hif1α mRNA abundance in MEF transfected with siHif1α, or siControl (100 nM), after 72 h.(b) siHif1α effectively knockdowns expressed Hif1α cDNA WT, but not a Hif1α cDNA silent mutant form refractory to siHif1α (Hif1α cDNA REFR.) in mice MEF.(c) RT-qPCR analysis of the relative mRNA abundances of Glut1, Glut3, Hk2 and Gapdh in WT and Pink1 KO mice MEF transfected with siControl or siHif1α. ß-Actin mRNA was used for normalization, and all data are expressed as the change relative to WT-siControl.(d) Western blot against HIF1α in WT and in Pink1 KO mice MEF transfected with siHif1α or siControl shows the efficacy of the siHif1α at knocking down endogenous HIF1α; ß-actin was used as loading control. As shown, siHif1α partially prevented the increase in GLUT1, HK2 and GAPDH protein abundances.The increased rate of lactate released after 4 h of incubation (e) and glycolytic flux (f) observed in the Pink1 KO MEF was prevented by siHif1α.siHif1α was also effective at preventing the endogenous increased HIF1α protein (g), as well as the increased glycolytic flux (h) observed in Pink1 KO primary neurons. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information.


f4 ( a ) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase ( CS ) , and ( b ) mitochondrial inner membrane potential ( Δψm ) in WT and Pink1 KO mice MEF . ( c ) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α ( or its siControl ) ; ß - actin was used as loading control . d ) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl . Rate of whole - cell H2O2 ( e ) and mitochondrial O2 · − ( f ) detection in WT and Pink1 KO MEF ( g ) Detection of mitochondrial O2 · − using the mitochondrial - specific probe MitoSox in MEF incubated in the absence ( − ) or in the presence ( + ) of the plasma membrane permeable GSH - EE , or by expressing mitochondrial - tagged forms of glutamate - cysteine ligase , ( + mitoGCL ; empty vector , pIRES2 - EGFP , denoted as − ) or catalase ( + mitoCAT ; empty vector denoted as − ) , shows effective mitochondrial ROS removal . ( h ) Western blot against HIF1α in Pink1 KO MEF incubated with ( or without ) GSH - EE , or transfected with empty vector , mitoGCL or mitoCAT , showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS ; α - TUBULIN was used as loading control . Only representative western blots are shown ; the replicates and the semi - quantitative estimation of the band intensities are shown in the Supplementary Information . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

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

f4(a) Mitochondrial respiratory chain complexes activity values normalized to citrate synthase (CS), and (b) mitochondrial inner membrane potential (Δψm) in WT and Pink1 KO mice MEF.(c) Western blot against PDK1 and HIF1α in WT and Pink1 KO MEF transfected with siHif1α (or its siControl); ß-actin was used as loading control.d) PDH complex activity in WT and Pink1 KO MEF transfected with siHif1α or siControl.Rate of whole-cell H2O2 (e) and mitochondrial O2·− (f) detection in WT and Pink1 KO MEF(g) Detection of mitochondrial O2·− using the mitochondrial-specific probe MitoSox in MEF incubated in the absence (−) or in the presence (+) of the plasma membrane permeable GSH-EE, or by expressing mitochondrial-tagged forms of glutamate-cysteine ligase, (+mitoGCL; empty vector, pIRES2-EGFP, denoted as −) or catalase (+ mitoCAT; empty vector denoted as −), shows effective mitochondrial ROS removal.(h) Western blot against HIF1α in Pink1 KO MEF incubated with (or without) GSH-EE, or transfected with empty vector, mitoGCL or mitoCAT, showing reduction in HIF1α protein abundance by the treatments that remove mitochondrial ROS; α-TUBULIN was used as loading control. Only representative western blots are shown; the replicates and the semi-quantitative estimation of the band intensities are shown in the Supplementary Information. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


f5 ( a ) Proportion of BrdU incorporation into DNA and cell cycle phases , and ( b ) cell growth in WT and Pink1 KO MEF after 72 h in the absence ( denoted as - 2DG ) or in the presence ( + 2DG ) of the glucose - metabolizing inhibitor , 2 - deoxyglucose ( 2DG , 1 mM ) . ( c ) Proportions of BrdU incorporation into DNA and cell cycle phases , and ( d ) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α ( + siHif1α ) or siControl ( denoted as - siHif1α ) . ( e ) Ki67 , Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain , and the corresponding quantification . * P0 . 05 ; # P0 . 05 versus all other conditions ( analysis of variance followed by Bonferroni test ; n = 3 - 4 independent experiments ) . All data are expressed as mean ± s . e . m . Scale bar , 100 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 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

f5(a) Proportion of BrdU incorporation into DNA and cell cycle phases, and (b) cell growth in WT and Pink1 KO MEF after 72 h in the absence (denoted as -2DG) or in the presence (+2DG) of the glucose-metabolizing inhibitor, 2-deoxyglucose (2DG, 1 mM).(c) Proportions of BrdU incorporation into DNA and cell cycle phases, and (d) cell growth in WT and Pink1 KO MEF 72 h after transfection with siHif1α (+siHif1α) or siControl (denoted as -siHif1α).(e) Ki67, Sox2 and DAPI immunostaining in the subventricular zone of WT and Pink1 KO mouse brain, and the corresponding quantification. *P0.05; #P0.05 versus all other conditions (analysis of variance followed by Bonferroni test; n=3-4 independent experiments). All data are expressed as mean±s.e.m. Scale bar, 100 μm.


f6a ) Rate of mitochondrial O2 · - detection in WT and Pink1 KO mice primary neurons . ( b ) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons . ( c ) Proportion of apoptosis in WT and Pink1 KO neurons , as assessed by the proportion of annexin V - positive / 7 - AAD - negative cells , determined by flow cytometry . Data are expressed as mean ± s . e . m . * P0 . 05 ( Student ' s t - test ; n = 3 - 4 independent experiments ) .

B-PANEL_START 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

f6a) Rate of mitochondrial O2·- detection in WT and Pink1 KO mice primary neurons.(b) Proportion of BrdU incorporation into DNA of WT and Pink1 KO neurons.(c) Proportion of apoptosis in WT and Pink1 KO neurons, as assessed by the proportion of annexin V-positive/7-AAD-negative cells, determined by flow cytometry. Data are expressed as mean±s.e.m. *P0.05 (Student's t-test; n=3-4 independent experiments).


Figure 1 ( A , B ) Airyscan confocal analysis of PC1 ( 568 , red ) co - localization with GFP - LC3 ( green ) in ( A ) MEF ( B ) Saos2 . Scale bars = 10 µm . ( C ) Airyscan confocal analysis of PC2 ( 647 , red ) co - localization with LC3 ( 488 , green ) in RCS cells . Scale bars = 10 µm . ( A - C ) The insets show higher magnification ( A = x4 . 68 ; B = x6 . 76 ; C = x7 . 33 ) and single colour channels of the boxed area . ( D ) Quantification of GFP ( A , B ) or LC3 ( C ) vesicles positive for PC1 or PC2 , expressed as % of total LC3 ( mean + / - SEM ) , n = 18 cells ( MEFs and Saos2 ) ; n = 12 ( RCS ) from 3 independent experiments . ( E - G ) Scanning confocal microscopy analysis of MEFs , Saos2 and RCS cells treated with BafA1 , immunolabeled for PC1 or PC2 and LAMP1 . Nuclei were stained with Hoechst . ( E , F ) Scale bars = 10 µm , ( G ) Scale bars = 5 µm . The insets show higher magnification ( E = x4 . 99 ; F = x6 . 49 ; G = x2 . 01 ) and single colour channels of the boxed area . ( H ) Transmission EM analysis in Saos2 cells , treated with BafA1 , showing in detail a lysosome which contains immunolabeled PC1 ( with nanogold particles ) , as indicated by arrows . ( I ) Scanning confocal microscopy analysis of Saos2 WT and CRISPR - Cas9 IDUA Saos2 at steady state , immunolabeled for PC1 and LAMP1 . Nuclei were stained with Hoechst . Scale bar = 10 µm . The insets show higher magnification ( left = x3 . 09 ; right = x3 . 12 ) and single colour channels of the boxed area . Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell ( mean + / - SEM ) . n = 31 WT cells , n = 33 CRISPR cells counted ; 3 independent experiments . Student ' s unpaired , two - tailed T - Test * * * P < 0 . 0001 . ( J ) WT and CRISPR IDUA Saos2 lysed and analysed by western blot . Data are representative of 3 independent experiments .

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 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 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 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

Figure 1(A, B) Airyscan confocal analysis of PC1 (568, red) co-localization with GFP-LC3 (green) in (A) MEF (B) Saos2. Scale bars = 10 µm. (C) Airyscan confocal analysis of PC2 (647, red) co-localization with LC3 (488, green) in RCS cells. Scale bars = 10 µm. (A-C) The insets show higher magnification (A = x4.68; B = x6.76; C = x7.33) and single colour channels of the boxed area. (D) Quantification of GFP (A,B) or LC3 (C) vesicles positive for PC1 or PC2, expressed as % of total LC3 (mean +/- SEM), n = 18 cells (MEFs and Saos2); n=12 (RCS) from 3 independent experiments. (E-G) Scanning confocal microscopy analysis of MEFs, Saos2 and RCS cells treated with BafA1, immunolabeled for PC1 or PC2 and LAMP1. Nuclei were stained with Hoechst. (E, F) Scale bars = 10 µm, (G) Scale bars = 5 µm. The insets show higher magnification (E = x4.99; F = x6.49; G = x2.01) and single colour channels of the boxed area.(H) Transmission EM analysis in Saos2 cells, treated with BafA1, showing in detail a lysosome which contains immunolabeled PC1 (with nanogold particles), as indicated by arrows.(I) Scanning confocal microscopy analysis of Saos2 WT and CRISPR-Cas9 IDUA Saos2 at steady state, immunolabeled for PC1 and LAMP1. Nuclei were stained with Hoechst. Scale bar = 10 µm. The insets show higher magnification (left = x3.09; right = x3.12) and single colour channels of the boxed area. Bar graph shows quantification of lysosomes containing PC1 expressed as % of total LAMP1 per cell (mean +/- SEM). n= 31 WT cells, n=33 CRISPR cells counted; 3 independent experiments. Student's unpaired, two-tailed T-Test *** P<0.0001.(J) WT and CRISPR IDUA Saos2 lysed and analysed by western blot. Data are representative of 3 independent experiments.


Figure 2U2OS expressing ( A ) GFP - LC3 mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification ( A = x3 . 93 from the boxed region are shown on the right . Scale bar = 10 µm . U2OS expressing ( B ) GFP - 2 - FYVE ( green ) , mCherry - PC2 ( red ) and RDEL - HALO ( blue ) were imaged live by spinning disc microscopy . Single and merge channels time - lapse stills at higher magnification B = x3 . 42 from the boxed region are shown on the right . Scale bar = 10 µm . ( C ) Airyscan analysis of Saos2 cells expressing GFP - LC3 ( green ) and immunolabeled for PC1 ( 405 , blue ) and CANX ( 647 , red ) . The insets show higher magnification ( x5 . 26 ) and single colour channels of the boxed area . Scale bar = 10 µm . ( D ) Correlative Light Electron Microscopy ( CLEM ) and Electron Tomography of Saos2 cells transfected with GFP - LC3 ( green ) and labelled for PC1 ( 568 , red and nanogold particles ) and CANX ( 647 , blue ) . Cells were first imaged by confocal microscopy ( top left panel ) and then the same region was retraced in EM ( upper middle panel ) and overlay is shown ( upper right panel ) . Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules . ( E ) Single tomography slice ( left panel , taken from boxed are in D at a magnification of x2 . 84 ) , overlay with immunofluorescence ( IF ) ( central panel ) and IF 3D rendering of AV ( green ) and the CANX positive vesicle containing gold particles of labelled collagen ( blue and white respectively ) inside an AV ( right panel ) .

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 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 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 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

Figure 2U2OS expressing (A) GFP-LC3 mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification (A = x3.93 from the boxed region are shown on the right. Scale bar = 10 µm.U2OS expressing (B) GFP-2-FYVE (green), mCherry-PC2 (red) and RDEL-HALO (blue) were imaged live by spinning disc microscopy. Single and merge channels time-lapse stills at higher magnification B = x3.42 from the boxed region are shown on the right. Scale bar = 10 µm.(C) Airyscan analysis of Saos2 cells expressing GFP-LC3 (green) and immunolabeled for PC1 (405, blue) and CANX (647, red). The insets show higher magnification (x5.26) and single colour channels of the boxed area. Scale bar = 10 µm.(D) Correlative Light Electron Microscopy (CLEM) and Electron Tomography of Saos2 cells transfected with GFP-LC3 (green) and labelled for PC1 (568, red and nanogold particles) and CANX (647, blue). Cells were first imaged by confocal microscopy (top left panel) and then the same region was retraced in EM (upper middle panel) and overlay is shown (upper right panel). Arrow indicates a LC3 positive vesicle containing CANX and PC1 molecules.(E) Single tomography slice (left panel, taken from boxed are in D at a magnification of x2.84), overlay with immunofluorescence (IF) (central panel) and IF 3D rendering of AV (green) and the CANX positive vesicle containing gold particles of labelled collagen (blue and white respectively) inside an AV (right panel).


Figure 3 ( A ) . Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of total number of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h . n = 20 cells per condition ; three independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * * P < 0 . 0001 . ( B ) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1 , fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . Scale bar = 10 µm . Insets show magnification of the boxed area . Bar graph ( on the left ) shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 12 , 10 , 12 , 10 , 7 , 10 cells per genotype respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . ns ≥ 0 . 05 , * * * 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 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

Figure 3(A). Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of total number of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes and treated with 100 nM BafA1 for 9h. n=20 cells per condition; three independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ***P<0.0001.(B) MEF cell lines lacking the expression of indicated genes were treated for 12h with 50 nM BafA1, fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). Scale bar = 10 µm. Insets show magnification of the boxed area. Bar graph (on the left) shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=12, 10, 12, 10, 7, 10 cells per genotype respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. ns ≥0.05, *** P<0.0001.


Figure 4 ( A ) WT and CRISPR - Cas9 Fam134b knockout MEFs were treated as indicated , lysed and analysed by western blot with the indicated antibodies . Western blots are representative of 4 independent experiments . ( B ) WT and CRISPR - Cas9 Fam134b MEFs were immunolabeled for PC1 ( 568 , red ) , nuclei stained with Hoechst ( blue ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . ( C ) CRISPR FAM134B MEF mock , wild type FAM134B - HA or FAM134Blir - HA transfected were immunolabeled for PC1 ( 568 , red ) , LAMP1 ( 488 , green ) and HA ( 647 , violet ) and analysed by scanning confocal microscopy . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , quantification of n = 10 cells per treatment ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test was performed . ns ≥ 0 . 05 , * * * 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 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 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(A) WT and CRISPR-Cas9 Fam134b knockout MEFs were treated as indicated, lysed and analysed by western blot with the indicated antibodies. Western blots are representative of 4 independent experiments.(B) WT and CRISPR-Cas9 Fam134b MEFs were immunolabeled for PC1 (568, red), nuclei stained with Hoechst (blue) and analysed by scanning confocal microscopy. Scale bar = 10 µm.(C) CRISPR FAM134B MEF mock, wild type FAM134B-HA or FAM134Blir-HA transfected were immunolabeled for PC1 (568, red), LAMP1 (488, green) and HA (647, violet) and analysed by scanning confocal microscopy. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), quantification of n=10 cells per treatment; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test was performed. ns ≥0.05, *** P<0.0001.


Figure 5 ( A ) Bar graph shows quantification of lysosomes ( LAMP1 + ) containing PC1 expressed as % of lysosomes ( mean + / - SEM ) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes , treated with 100 nM BafA1 for 9h . n = 18 cells / treatment ; 3 independent experiments . One - way ANOVA ( P < 0 . 0001 ) with Dunnett ' s multiple comparisons test performed , * * P < 0 . 005 , * * * P < 0 . 0001 . ( B ) WT and Canx - / - MEFs were untreated or treated with BafA1 ( 10 μM ) for 6 h , lysed and analysed by western blot with indicated antibodies . Filamin and β - actin were used as loading control . Dashed line indicates that unnecessary lanes were removed . Western blot is representative of 3 independent experiments . ( C ) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 ( 568 , red ) and LAMP1 ( 488 , green ) . CST was added where indicated . Scale bar = 10 µm . Inset panels show magnification of the boxed area . Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1 , expressed as % of total lysosomes ( mean + / - SEM ) , n = 8 , 8 , 12 , 8 , 8 , 8 cells respectively ; 3 independent experiments . One - way ANOVA with Dunnett ' s multiple comparisons test performed and P value adjusted for multiple comparisons . * * * 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 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 O O O O O O O O O O O O O O O O O

Figure 5(A) Bar graph shows quantification of lysosomes (LAMP1+) containing PC1 expressed as % of lysosomes (mean +/- SEM) in Saos2 cells mock transfected or transfected with SiRNA against the indicated genes, treated with 100 nM BafA1 for 9h. n=18 cells/treatment; 3 independent experiments. One-way ANOVA (P<0.0001) with Dunnett's multiple comparisons test performed, ** P<0.005, *** P<0.0001.(B) WT and Canx-/- MEFs were untreated or treated with BafA1 (10 μM) for 6 h, lysed and analysed by western blot with indicated antibodies. Filamin and β-actin were used as loading control. Dashed line indicates that unnecessary lanes were removed. Western blot is representative of 3 independent experiments.(C) MEF cell lines lacking the indicated genes were treated for 12h with 50 nM BafA1 fixed and immunolabeled for PC1 (568, red) and LAMP1 (488, green). CST was added where indicated. Scale bar = 10 µm. Inset panels show magnification of the boxed area. Bar graph on the right shows quantification of LAMP1 vesicles positive for PC1, expressed as % of total lysosomes (mean +/- SEM), n=8, 8, 12, 8, 8, 8 cells respectively; 3 independent experiments. One-way ANOVA with Dunnett's multiple comparisons test performed and P value adjusted for multiple comparisons. *** P<0.0001.


Figure 6 ( A , B ) Volcano plot comparing protein fold changes between WT versus Fam134b - / - ( A ) and WT versus Canx - / - MEFs ( B ) . Significantly regulated proteins are labelled in red ( log2 fold change > 1 , - log10 p > 1 . 3 ) . Red dots with blue ring indicate collagen 1 peptides . Graphs represent statistics from three separate experiments for each genotype .

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

Figure 6(A, B) Volcano plot comparing protein fold changes between WT versus Fam134b-/- (A) and WT versus Canx-/- MEFs (B). Significantly regulated proteins are labelled in red (log2 fold change > 1, -log10 p>1.3). Red dots with blue ring indicate collagen 1 peptides. Graphs represent statistics from three separate experiments for each genotype.


Figure 7 ( B ) U2OS cells were transfected with empty vector control ( pcDNA3 ) , FAM134B - HA WT or mutant constructs as indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against CANX , FAM134B 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed . U2OS cells were transfected with HALO - PC2 , FAM134B - HA or FAM134Blir - HA constructs , treated with 100 nM BafA1 for 6h and with CST where indicated . Complexes were immune - isolated with HA - magnetic beads , separated by western blot and visualised with antibodies against HALO , CANX , FAM134B , LC3 5 % of the input is shown . Western blots are representative of 3 independent experiments . Dashed line indicates that unnecessary lanes were removed .

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 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 7(B) U2OS cells were transfected with empty vector control (pcDNA3), FAM134B-HA WT or mutant constructs as indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against CANX, FAM134B 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.U2OS cells were transfected with HALO-PC2, FAM134B-HA or FAM134Blir-HA constructs, treated with 100 nM BafA1 for 6h and with CST where indicated. Complexes were immune-isolated with HA-magnetic beads, separated by western blot and visualised with antibodies against HALO, CANX, FAM134B, LC3 5% of the input is shown. Western blots are representative of 3 independent experiments. Dashed line indicates that unnecessary lanes were removed.


Figure 1 ( B ) SEC trace and SDS - PAGE of purified NHE9 * ( residues 8 to and 574 out of 644 ) as depicted by schematic . ( C ) Activity of NHE9 * co - reconstituted with ATPase into liposomes to mimic the in vivo situation ( schematic ) . Representative ACMA fluorescence traces of liposome reconstituted NHE9 * ( blue ) , NHE9 * double - mutant N243A - D244A ( red ) , and rat fructose transporter GLUT5 ( black ) . ATP - driven H + pumping establishes a ΔpH ( 0 - 3 min ) . H + efflux is initiated by the addition of 40 mM NaCl , and subsequent addition of NH4Cl ( 4 min ) collapses the proton gradient . ( D ) Michaelis - Menten kinetics for NHE9 * ( red ) , NHE9 ΔCTD ( green ) , N243A - D244A ( blue ) and rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In all experiments errors bars , s . e . m . ; n = 3 technical repeats . The apparent KM values are an average from n = 3 separate protein reconstitutions .

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 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

Figure 1(B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic.(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.(D) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.


Figure 4A ) Native mass spectrum of NHE9 * purified in the presence ( top panel ) or the absence of additional brain lipids ( bottom panel ) . In the presence of brain lipids , NHE9 * is detected as exclusively dimeric protein with poorly resolved peaks , indicating the presence of multiple lipid adducts . Without brain lipid addition , NHE9 * appears as sharp peaks , revealing several adducts of approximately 1 kDa each , as well as a minor monomer population . B ) Thermal stabilization of purified dimeric NHE9 * - GFP by lipids . Normalized mean FSEC peak fluorescence before heating ( open bars ) , and after heating and centrifugation ( grey bars ) in presence of different lipids . Data presented are mean values ± data range of n = 4 experiments ( see Materials and Methods ) . C ) Thermal shift stabilization of purified dimeric NHE9 * - GFP in the presence of PIP2 ( red ) compared to PIP2 - free ( black ) . Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations . E ) Thermal shift stabilization of the dimeric NHE9 * - K85Q - K105Q - K107Q - GFP by PIP2 ( red ) compared with thermal shift in the absence ( black ) of PIP2 ; data shown are mean values ± data range of n = 2 technical repeats ; the apparent Tm was calculated with a sigmoidal 4 - parameter logistic regression function ; the average ΔTm presented is calculated from n = 2 independent titrations . ( F ) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts ; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained .

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

Figure 4A) Native mass spectrum of NHE9* purified in the presence (top panel) or the absence of additional brain lipids (bottom panel). In the presence of brain lipids, NHE9* is detected as exclusively dimeric protein with poorly resolved peaks, indicating the presence of multiple lipid adducts. Without brain lipid addition, NHE9* appears as sharp peaks, revealing several adducts of approximately 1 kDa each, as well as a minor monomer population.B) Thermal stabilization of purified dimeric NHE9*-GFP by lipids. Normalized mean FSEC peak fluorescence before heating (open bars), and after heating and centrifugation (grey bars) in presence of different lipids. Data presented are mean values ± data range of n = 4 experiments (see Materials and Methods).C) Thermal shift stabilization of purified dimeric NHE9*-GFP in the presence of PIP2 (red) compared to PIP2-free (black). Data presented are normalized mean FSEC peak fluorescence as mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. E) Thermal shift stabilization of the dimeric NHE9*-K85Q-K105Q-K107Q-GFP by PIP2 (red) compared with thermal shift in the absence (black) of PIP2; data shown are mean values ± data range of n = 2 technical repeats; the apparent Tm was calculated with a sigmoidal 4-parameter logistic regression function; the average ΔTm presented is calculated from n = 2 independent titrations. (F) Native MS of the triple mutant reveals monomeric NHE9 with no notable lipid adducts; a peak highlighted by * was determined to be a soluble contaminant as it was also apparent at MS conditions where no NHE9 was retained.


Figure 1Verification of screen hits by mtKeima - based FACS analysis of mitophagy . Upper : representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4 . Bottom : quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs . The percentage of cells with high mitophagy level is indicated in red . Two independent sgRNAs were used for each gene . NTC : non - targeting control . Imaging analysis of mitophagy levels in the indicated HeLa cells . Left : sequencing result of WT and FBXL4 - KO cells and representative mitophagy images , white arrows point to mitolysosomes ( FBXL4 - KO cells were not labeled because of the large numbers of mitolysosomes ) ; middle : percentage of cells with mitophagy ( mitolysosome - positive ) ; right : quantification of mitolysosome / mitochondria area . n : number of cells analyzed ( middle ) ; number of imaging areas ( 20 - 30 cells / area ) analyzed ( right ) . FACS analysis of mitophagy levels in the indicated HeLa cells . Left : representative FACS results ; right : quantitative analysis of mitophagy levels .

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 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 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

Figure 1Verification of screen hits by mtKeima-based FACS analysis of mitophagy. Upper: representative FACS analysis of mitophagy in HeLa cells expressing sgNTC and sgFBXL4. Bottom: quantitative analysis of mitophagy levels in HeLa cells expressing the indicated sgRNAs. The percentage of cells with high mitophagy level is indicated in red. Two independent sgRNAs were used for each gene. NTC: non-targeting control.Imaging analysis of mitophagy levels in the indicated HeLa cells. Left: sequencing result of WT and FBXL4-KO cells and representative mitophagy images, white arrows point to mitolysosomes (FBXL4-KO cells were not labeled because of the large numbers of mitolysosomes); middle: percentage of cells with mitophagy (mitolysosome-positive); right: quantification of mitolysosome/mitochondria area. n: number of cells analyzed (middle); number of imaging areas (20-30 cells/area) analyzed (right).FACS analysis of mitophagy levels in the indicated HeLa cells. Left: representative FACS results; right: quantitative analysis of mitophagy levels.


Figure 2Immunoblot analysis of the indicated HeLa cells . B : BNIP3 ; N : NIX . Two clones were shown for each double and triple knockout cells . Representative live cell imaging ( E ) of mitophagy levels in the indicated HeLa cells . n : number of cells analyzed ( left ) ; number of imaging areas analyzed ( right ) .

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Figure 2Immunoblot analysis of the indicated HeLa cells. B: BNIP3; N: NIX. Two clones were shown for each double and triple knockout cells.Representative live cell imaging (E) of mitophagy levels in the indicated HeLa cells. n: number of cells analyzed (left); number of imaging areas analyzed (right).


Figure 3Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion . Mitochondria from HeLa FBXL4 - FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X - 100 buffer . Different mitochondrial preparations were then digested with Protease K or trypsin . Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction . Purified mitochondria from HeLa FBXL4 - FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions . S : supernatant ; P : pellet . Determination of the submitochondrial localization of FBXL4 ( 1 - 23 ) - RFP by Protease K digestion . Experiments were performed similarly as ( A ) .

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Figure 3Determination of the submitochondrial localization of FBXL4 by Protease K and trypsin digestion. Mitochondria from HeLa FBXL4-FLAG stable line were purified and stored as intact mitochondria or treated with hypotonic swelling buffer or lysed with Triton X-100 buffer. Different mitochondrial preparations were then digested with Protease K or trypsin.Analysis of the association of FBXL4 with membrane by alkaline carbonate extraction. Purified mitochondria from HeLa FBXL4-FLAG stable line were treated with alkaline carbonate buffer at the indicated pH and then centrifuged to collect the supernatant and the pellet fractions. S: supernatant; P: pellet.Determination of the submitochondrial localization of FBXL4(1-23)-RFP by Protease K digestion. Experiments were performed similarly as (A).


Figure 4Immunoprecipitation analysis of the FBXL4 - Skp1 - Cullin1 ( SCF - FBXL4 ) complex . HeLa cells were infected with lentiviruses expressing the indicated genes . ∆ F : the F - box deletion mutant of FBXL4 - FLAG ; 4A : FBXL4 - 4A mutant as shown in ( A ) . Immunoprecipitation analysis of the ubiquitination of BNIP3 . FLAG - BNIP3 ( knockin ) , FBXL4 - KO , HA - ubiquitin ( Ub ) HeLa cells were rescued with WT or ∆ F FBXL4 , and treated with DMSO or MG132 ( 20 μM ) for 8 hours . Immunoprecipitation analysis of the ubiquitination of NIX . FLAG - NIX ( knockin ) , FBXL4 - KO , HA - Ub HeLa cells were treated the same as ( F ) . Immunoblot analysis of the indicated HeLa cells . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 - FLAG , FBXL4 ( 4A ) - FLAG or FBXL4 ( ∆ F ) - FLAG .

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Figure 4Immunoprecipitation analysis of the FBXL4-Skp1-Cullin1 (SCF-FBXL4) complex. HeLa cells were infected with lentiviruses expressing the indicated genes. ∆F: the F-box deletion mutant of FBXL4-FLAG; 4A: FBXL4-4A mutant as shown in (A).Immunoprecipitation analysis of the ubiquitination of BNIP3. FLAG-BNIP3 (knockin), FBXL4-KO, HA-ubiquitin (Ub) HeLa cells were rescued with WT or ∆F FBXL4, and treated with DMSO or MG132 (20 μM) for 8 hours.   Immunoprecipitation analysis of the ubiquitination of NIX. FLAG-NIX (knockin), FBXL4-KO, HA-Ub HeLa cells were treated the same as (F).  Immunoblot analysis of the indicated HeLa cells. FBXL4-KO HeLa cells were rescued with vector, FBXL4-FLAG, FBXL4(4A)-FLAG or FBXL4(∆F)-FLAG.


Figure 5Immunoblot analysis of the indicated HeLa cells . WT and FBXL4 - KO HeLa cells were rescued with vector , WT or mutant FBXL4 - FLAG . Immunoprecipitation analysis of FBXL4 - substrate interaction . FBXL4 - KO HeLa cells were rescued with vector , FBXL4 ( ∆ F ) - FLAG or mutant FBXL4 ( ∆ F ) - FLAG . Immunoprecipitation analysis of the integrity of the SCF - FBXL4 complex . Upper : FBXL4 - KO HeLa cells expressing vector , WT or mutant FBXL4 - FLAG were crosslinked with DSP and subject to anti - FLAG immunoprecipitation ; lower : quantification of the ratio of Cullin1 / FLAG band intensities .

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Figure 5Immunoblot analysis of the indicated HeLa cells. WT and FBXL4-KO HeLa cells were rescued with vector, WT or mutant FBXL4-FLAG.Immunoprecipitation analysis of FBXL4-substrate interaction. FBXL4-KO HeLa cells were rescued with vector, FBXL4(∆F)-FLAG or mutant FBXL4(∆F)-FLAG.Immunoprecipitation analysis of the integrity of the SCF-FBXL4 complex. Upper: FBXL4-KO HeLa cells expressing vector, WT or mutant FBXL4-FLAG were crosslinked with DSP and subject to anti-FLAG immunoprecipitation; lower: quantification of the ratio of Cullin1/FLAG band intensities.


Figure 6Viability of the indicated mice . n : number of mice analyzed . Immunoblot analysis of organs from the indicated mice at P0 of age . Elevated BNIP3 and NIX levels are highlighted by red boxes ; reduced mitochondrial proteins are highlighted by blue boxes . Three mice for each genotype were analyzed . Protein samples from HeLa cells treated with DMSO or Actinomycin D ( ActD ) were used as positive control for PARP cleavage . Live cell confocal imaging of mitophagy level in the indicated mouse liver . Left : representative images . The dashed line indicates cell boundary . Right : quantitative analysis of mitophagy levels in hepatocytes . n : number of cells analyzed . Data are mean ± SD . Statistics : two - tailed unpaired Student ' s t - test ; * * * P < 0 . 001 .

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Figure 6Viability of the indicated mice. n: number of mice analyzed.   Immunoblot analysis of organs from the indicated mice at P0 of age. Elevated BNIP3 and NIX levels are highlighted by red boxes; reduced mitochondrial proteins are highlighted by blue boxes. Three mice for each genotype were analyzed. Protein samples from HeLa cells treated with DMSO or Actinomycin D (ActD) were used as positive control for PARP cleavage.  Live cell confocal imaging of mitophagy level in the indicated mouse liver. Left: representative images. The dashed line indicates cell boundary. Right: quantitative analysis of mitophagy levels in hepatocytes. n: number of cells analyzed. Data are mean ± SD. Statistics: two-tailed unpaired Student's t-test; ***P < 0.001.


Figure 7Viability of the indicated mice . n : number of mice analyzed . Immunoblot analysis of liver samples from the indicated mice at P0 of age . Left : immunoblot analysis , three mice for each genotype were analyzed ; right : quantification of the ratio of mitochondrial protein / actin band intensities . Data are mean + SD from three biological replicates . Statistics : two - tailed unpaired Student ' s t - test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; ns , not significant .

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Figure 7Viability of the indicated mice. n: number of mice analyzed.Immunoblot analysis of liver samples from the indicated mice at P0 of age. Left: immunoblot analysis, three mice for each genotype were analyzed; right: quantification of the ratio of mitochondrial protein/actin band intensities. Data are mean + SD from three biological replicates. Statistics: two-tailed unpaired Student's t-test; *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.


Figure 1GPS reporter cells expressing peptide libraries ending with a random ( X ) or Gly ( G ) residue were treated with dominant - negative ( DN ) cullins and analyzed . Data are presented as mean ± standard deviation from nine replicates . GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC - box proteins and analyzed . VHL and FEM1C serve as unrelated BC - box protein controls . Data are presented as mean ± standard deviation from nine replicates .

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Figure 1GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.


Figure 2Comparisons of protein stability among wild - type protein and the mutants indicated on the right .

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Figure 2Comparisons of protein stability among wild-type protein and the mutants indicated on the right.


Figure 3 ( A ) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations . The number on each graph denotes the left - tailed p - value of the - 1 residue . CT - inside and CT - outside represent membrane proteins with their C - termini facing the cytosol or extracellular space , respectively . ( G ) Confocal images of U2OS cells expressing MTSGLUL - GFP proteins with or without a C - terminal diGly degron . MitoTracker staining shows mitochondria . Scale bar = 20 µm . ( H ) Confocal images of U2OS cells expressing MTSGLUL - GFP - diGly degron fusion proteins co - stained with MitoTracker , and with or without DNCul2 treatment . Scale bar = 20 µm .

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Figure 3(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.