( D ) Dose response of Aβ40 ( mean ± SD ) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing . ( D ) CNP520 was administered daily for 14 days ; CSF was sampled via a lumbar catheter 2 hours before dosing , and 24 hours after the last dose ( mean ± SD , n = 8 in treatment groups , n = 16 in placebo group ) .

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

(D) Dose response of Aβ40 (mean ± SD) to CNP520 relative to baseline Aβ40 levels after 2 weeks of dosing. (D) CNP520 was administered daily for 14 days; CSF was sampled via a lumbar catheter 2 hours before dosing, and 24 hours after the last dose (mean ± SD, n = 8 in treatment groups, n = 16 in placebo group).


( A ) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing . Shown are means ± SD , n = 21 - 25 for pre - dose and day 91 , and n = 2 - 7 for the intermediate time points .

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

(A) Changes of Aβ40 relative from baseline values up to 3 months of daily dosing. Shown are means ± SD, n = 21-25 for pre-dose and day 91, and n = 2 - 7 for the intermediate time points.


( B ) Dose - response for CSF Aβ40 at day 91 ( 24 hours after last dose ) and individual percent change from baseline , shown as mean and 95 % confidence interval , n = 21 - 25 per treatment group .

O O O O O O O O O O O O O O O O O O O O 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) Dose-response for CSF Aβ40 at day 91 (24 hours after last dose) and individual percent change from baseline, shown as mean and 95% confidence interval, n = 21-25 per treatment group.


( A ) Mouse forebrain Aβ40 levels in homozygous male and female APOE4 - TR mice with and without CNP520 treatment , 4 hours after dosing , shown are mean ± SEM , n = 6 / group .

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

(A) Mouse forebrain Aβ40 levels in homozygous male and female APOE4-TR mice with and without CNP520 treatment, 4 hours after dosing, shown are mean ± SEM, n = 6/group.


( B ) Distribution of APOE4 genotypes in the Phase IIa study , vs their baseline Aβ42 / 40 ratios . The dashed line at Aβ42 / 40 ratio of 0 . 09 represents a cutoff , which separates a normal ratio from an abnormally low ratio .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Distribution of APOE4 genotypes in the Phase IIa study, vs their baseline Aβ42/40 ratios. The dashed line at Aβ42/40 ratio of 0.09 represents a cutoff, which separates a normal ratio from an abnormally low ratio.


( C ) Comparison of CSF Aβ40 reduction in APOE4 non - carriers and APOE4 carriers .

(C) Comparison of CSF Aβ40 reduction in APOE4 non-carriers and APOE4 carriers.


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

(D) Comparison of CSFAβ40 reduction in participants with Aβ42/40 ≥ 0.09 and Aβ42/40 < 0.09.


( E ) Comparison of Aβ42 / 40 ratio at baseline and at day 91 for all participants in the placebo , 35 mg , and 85 mg dose group .

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

(E) Comparison of Aβ42/40 ratio at baseline and at day 91 for all participants in the placebo, 35 mg, and 85 mg dose group.


( F ) Change of Aβ42 / 40 ratio for all participants with Aβ42 / 40 ≤ 0 . 09 at baseline ; bars represent mean ± SD . Full circles : APOE4 carrier , open circles : APOE4 non - carrier .

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

(F) Change of Aβ42/40 ratio for all participants with Aβ42/40 ≤ 0.09 at baseline; bars represent mean ± SD. Full circles: APOE4 carrier, open circles: APOE4 non-carrier.


C ) , the percentage of pups gathered in the nest location is significantly reduced in Shank2 - / - mice . Mann - Whitney test , * * * P < 0 . 001 . Data information : All data are presented as mean ± SEM .

C), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM.


( H ) Shank2 + / + and Shank2 - / - mice displayed a significant preference for the novel object versus the familiar one in the test session , two - way mixed ANOVA , effect of object : * * * P < 0 . 001 , effect of genotype : P = 0 . 167 , object x genotype interaction : P = 0 . 667 , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : All graphs are presented as mean ± SEM , NS : not significant .

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

(H) Shank2+/+ and Shank2-/- mice displayed a significant preference for the novel object versus the familiar one in the test session, two-way mixed ANOVA, effect of object: ***P<0.001, effect of genotype: P=0.167, object x genotype interaction: P=0.667, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All graphs are presented as mean ± SEM, NS: not significant.


( J ) Left panel : Electron micrographs of the posterior pituitary gland from Shank2 + / + and Shank2 - / - mice in lower ( left panel ) and higher magnification . Scale bar ( 500 nm ) . Right diagram : No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2 + / + and Shank2 - / - mice , Mann - Whitney test , P = 0 . 507 , Shank2 + / + n = 3 , Shank2 - / - n = 3 . ( K - M ) Additionally , Shank2 - / - mice display no significant difference in : ( K ) Estradiol - , Mann - Whitney test , P = 0 . 475 , Shank2 + / + n = 6 , Shank2 - / - n = 7 , ( L ) Progesterone , unpaired , two - tailed Student ' s t - test , P = 0 . 570 , Shank2 + / + n = 5 , Shank2 - / - n = 5 or ( M ) Prolactin - plasma concentrations , unpaired , two - tailed Student ' s t - test , P = 0 . 980 ; Shank2 + / + n = 3 , Shank2 - / - n = 3 . Data information : All data are presented as mean ± SEM , NS : not significant . E , endothelium ; CL , capillary lumen ; M , mitochondria ; NG , neurosecretory granule ; AE , axonal endings , HYP , Hypothalamus ; PIT , Pituitary gland .

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

(J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.


( A - G ) qRT - PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house - keeping gene HMBS of 9 - 10 week old naive and postnatal day 1 Shank2 + / + and Shank2 - / - mice : ( A ) Oxytocin gene expression did not differ between Shank2 + / + and Shank2 - / - mice , one - way ANOVA , P = 0 . 335 . ( B ) Vasopressin gene expression did not differ between Shank2 + / + and Shank2 - / - mice , one - way ANOVA , P = 0 . 945 . ( C ) Prolactin gene expression did not differ between Shank2 + / + and Shank2 - / - mice , one - way ANOVA , P = 0 . 202 . Additionally , no significant difference was evident in ( D ) oxytocin receptor , one - way ANOVA , P = 0 . 675 , ( E ) prolactin receptor , one - way ANOVA , P = 0 . 912 , ( F ) vasopressin receptor , one - way ANOVA , P = 0 . 418 and ( G ) estrogen receptor alpha expression , one - way ANOVA , P = 0 . 890 in comparison to Shank2 + / + mice . All data are presented as mean ± SEM , NS : not significant . Pituitary gland , ( n = 3 pituitary glands pooled per value , n = 3 hypothalamus ( Oxytocin , Vasopressin , Oxytocin receptor , Vasopressin receptor , estrogen alpha ) n = 2 - 3 hypothalamus ( Prolactin receptor ) ) . HYP , hypothalamus ; PIT , pituitary gland .

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

(A-G) qRT-PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house-keeping gene HMBS of 9-10 week old naive and postnatal day 1 Shank2+/+ and Shank2-/- mice: (A) Oxytocin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.335. (B) Vasopressin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.945. (C) Prolactin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.202. Additionally, no significant difference was evident in (D) oxytocin receptor, one-way ANOVA, P=0.675, (E) prolactin receptor, one-way ANOVA, P=0.912, (F) vasopressin receptor, one-way ANOVA, P=0.418 and (G) estrogen receptor alpha expression, one-way ANOVA, P=0.890 in comparison to Shank2+/+ mice. All data are presented as mean ± SEM, NS: not significant. Pituitary gland, (n=3 pituitary glands pooled per value, n=3 hypothalamus (Oxytocin, Vasopressin, Oxytocin receptor, Vasopressin receptor, estrogen alpha) n=2-3 hypothalamus (Prolactin receptor)). HYP, hypothalamus; PIT, pituitary gland.


( E - H ) Repeated pup exposure does not induce major components of social attachment behavior in Shank2 - / - females : ( E ) pup grooming , unpaired , two - tailed Student ' s t - test , * * * P < 0 . 001 ( F ) crouching , Mann - Whitney test , * * * P < 0 . 001 and ( G ) nest building , Mann - Whitney test , * * * P < 0 . 001 , ( H ) maternal interaction , unpaired , two - tailed Student ' s t - test , * * * P < 0 . 001 ( trial 5 ) , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : All data are presented as mean ± SEM , NS : not significant .

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

(E-H) Repeated pup exposure does not induce major components of social attachment behavior in Shank2-/- females: (E) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (F) crouching, Mann-Whitney test, ***P<0.001 and (G) nest building, Mann-Whitney test, ***P<0.001, (H) maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001 (trial 5), Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant.


( L ) Levels of synaptic proteins in the crude synaptosomes fraction ( P2 ) from the hypothalamus of Shank2 + / + and Shank2 - / - mice . Shank2 + / + n = 5 ( black bars ) , Shank2 - / - n = 5 ( blue bars ) . Unpaired , two - tailed Student ' s t - test .

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

(L) Levels of synaptic proteins in the crude synaptosomes fraction (P2) from the hypothalamus of Shank2+/+ and Shank2-/- mice. Shank2+/+ n = 5 (black bars), Shank2-/- n = 5 (blue bars). Unpaired, two-tailed Student's t-test.


( D - G ) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2 - / - mice : ( D ) grooming , two - way ANOVA , effect of genotype : * P = 0 . 018 , effect of treatment : * * P = 0 . 002 , genotype x treatment interaction : * * * P = 0 . 001 . ( E ) Crouching , two - way ANOVA , effect of genotype : P = 0 . 091 , effect of treatment : P = 0 . 869 , genotype x treatment interaction : P = 0 . 663 , ( F ) nest building , two - way ANOVA , effect of genotype : P = 0 . 415 , effect of treatment : P = 0 . 341 genotype x treatment interaction : P = 0 . 093 , ( G ) Maternal interaction , two - way ANOVA , effect of genotype : * P = 0 . 034 , effect of treatment : P = 0 . 149 , genotype x treatment interaction : * P = 0 . 045 . Shank2 + / + vehicle injected n = 6 , Shank2 - / - vehicle injected n = 6 , Shank2 + / + CNO injected n = 6 , Shank2 - / - CNO injected n = 7 . Data information : All data are presented as mean ± SEM , NS : not significant .

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

(D-G) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2-/- mice: (D) grooming, two-way ANOVA, effect of genotype: *P=0.018, effect of treatment: **P=0.002, genotype x treatment interaction: ***P=0.001. (E) Crouching, two-way ANOVA, effect of genotype: P=0.091, effect of treatment: P=0.869, genotype x treatment interaction: P=0.663, (F) nest building, two-way ANOVA, effect of genotype: P=0.415, effect of treatment: P=0.341 genotype x treatment interaction: P=0.093, (G) Maternal interaction, two-way ANOVA, effect of genotype: *P=0.034, effect of treatment: P=0.149, genotype x treatment interaction: *P=0.045. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7. Data information: All data are presented as mean ± SEM, NS: not significant.


In vitro pull - down assays . GST or GST - PSKR1JK fusion proteins coupled with glutathione beads were used to pull down His - CPK28 . The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue ( CBB ) staining . Asterisks indicate the specific bands of fusion proteins .

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

In vitro pull-down assays. GST or GST-PSKR1JK fusion proteins coupled with glutathione beads were used to pull down His-CPK28. The amount of fusion proteins loaded on the gel was indicated by Coomassie brilliant blue (CBB) staining. Asterisks indicate the specific bands of fusion proteins.


Co - IP assays . PSKR1 - HA was co - expressed with CPK28 - GFP or GFP in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . The protein was precipitated using GFP - Trap . Both immunoprecipitated and input samples were subjected to western blotting using an anti - HA or anti - GFP antibody .

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

Co-IP assays. PSKR1-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated using GFP-Trap. Both immunoprecipitated and input samples were subjected to western blotting using an anti-HA or anti-GFP antibody.


A . Co - IP assay showing that PSK promotes CPK28 - GS2 interaction . GS2 - HA was co - expressed with CPK28 - GFP or GFP in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . The protein was precipitated by GFP - Trap . Immunoprecipitated and input samples were subjected to western blotting using an anti - HA and anti - GFP antibody . Data information : Experiments were repeated twice with similar results .

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

A. Co-IP assay showing that PSK promotes CPK28-GS2 interaction. GS2-HA was co-expressed with CPK28-GFP or GFP in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA and anti-GFP antibody. Data information: Experiments were repeated twice with similar results.


B . Co - IP assay showing that CPK28 - GS2 interaction was enhanced in the presence of PSKR1 . GS2 - HA and CPK28 - GFP were co - expressed with or without PSKR1 - FLAG in N . benthamiana leaves . The protein was precipitated by GFP - Trap . Immunoprecipitated and input samples were subjected to western blotting using an anti - HA , anti - GFP or anti - FLAG antibody . Data information : Experiments were repeated twice with similar results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-IP assay showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. GS2-HA and CPK28-GFP were co-expressed with or without PSKR1-FLAG in N. benthamiana leaves. The protein was precipitated by GFP-Trap. Immunoprecipitated and input samples were subjected to western blotting using an anti-HA, anti-GFP or anti-FLAG antibody. Data information: Experiments were repeated twice with similar results.


C . In vitro pull - down assays showing that CPK28 - GS2 interaction was enhanced in the presence of PSKR1 . Fixed amounts of His - CPK28 and GST - GS2 fusion proteins were incubated with an increasing amount of MBP - PSKR1 fusion protein and MBP protein . Asterisks indicate the specific bands of fusion proteins . Data information : Experiments were repeated twice , with similar results .

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

C. In vitro pull-down assays showing that CPK28-GS2 interaction was enhanced in the presence of PSKR1. Fixed amounts of His-CPK28 and GST-GS2 fusion proteins were incubated with an increasing amount of MBP-PSKR1 fusion protein and MBP protein. Asterisks indicate the specific bands of fusion proteins. Data information: Experiments were repeated twice, with similar results.


A . PSK induces the phosphorylation of GS2 by CPK28 in vivo . GS2 - GFP was co - expressed with CPK28 - HA ( empty vector as a negative control ) in N . benthamiana leaves . PSK ( 10 μM ) or H2O was injected 2 hours before leaves sampling . The phosphorylated GS2 protein was precipitated by GFP - Trap , and then immunoblotted with anti - pSer / pThr and anti - pSer antibodies .

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

A. PSK induces the phosphorylation of GS2 by CPK28 in vivo. GS2-GFP was co-expressed with CPK28-HA (empty vector as a negative control) in N. benthamiana leaves. PSK (10 μM) or H2O was injected 2 hours before leaves sampling. The phosphorylated GS2 protein was precipitated by GFP-Trap, and then immunoblotted with anti-pSer/pThr and anti-pSer antibodies.


C . Site - directed mutation of S334 and S360 residues in GS2 inhibited its phosphorylation by CPK28 .

C. Site-directed mutation of S334 and S360 residues in GS2 inhibited its phosphorylation by CPK28.


D . Identification of OE - GS2 transgenic lines expressing phospho - mutated versions of GS2 in the gs2 background by western blotting using an anti - GLN1 / GLN2 antibody . Ponceau S staining was used as the loading control .

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

D. Identification of OE-GS2 transgenic lines expressing phospho-mutated versions of GS2 in the gs2 background by western blotting using an anti-GLN1/GLN2 antibody. Ponceau S staining was used as the loading control.


The refined models of four crystal structures of the ribosome bound to aminoacyl - or peptidyl - tRNA analogs are displayed in their respective unbiased electron density Fo - Fc maps ( contoured at 2 . 5σ ) . The maps were calculated using phases produced by rigid body refinement of the ligand - free test structure put into the Fo data set . A , B . A - site bound substrates ACC - Puromycin ( A ) and ACCA - Pro ( B ) .

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

The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.A, B. A-site bound substrates ACC-Puromycin (A) and ACCA-Pro (B).


The refined models of four crystal structures of the ribosome bound to aminoacyl - or peptidyl - tRNA analogs are displayed in their respective unbiased electron density Fo - Fc maps ( contoured at 2 . 5σ ) . The maps were calculated using phases produced by rigid body refinement of the ligand - free test structure put into the Fo data set . C , D . P - site bound substrates ACCA - Leu - Phe ( C ) and ACCA - Pro - Pro ( D ) .

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

The refined models of four crystal structures of the ribosome bound to aminoacyl- or peptidyl-tRNA analogs are displayed in their respective unbiased electron density Fo-Fc maps (contoured at 2.5σ). The maps were calculated using phases produced by rigid body refinement of the ligand-free test structure put into the Fo data set.C, D. P-site bound substrates ACCA-Leu-Phe (C) and ACCA-Pro-Pro (D).


This figure compares conformation of aminoacyl residues of the two A - site substrates : A . ACC - PuromycinB . ACCA - Pro . To show position of the A - site substrates relative to the P - site , the model P - site substrate tRNA - Phe is shown as in the pre - attack complex of the ribosome ( pdb id 1vy4 ) . In both illustrations , the A site is viewed as through the peptide exit tunnel . In addition to S . cerevisiae residues numbering , E . coli numbering is shown in parenthesis . Comparison of the two structures shows that the side chain of the methyl - tyrosine is bound to the conserved hydrophobic A - site cleft , whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α - amine .

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

This figure compares conformation of aminoacyl residues of the two A-site substrates:A. ACC-PuromycinB. ACCA-Pro.To show position of the A-site substrates relative to the P-site, the model P-site substrate tRNA-Phe is shown as in the pre-attack complex of the ribosome (pdb id 1vy4). In both illustrations, the A site is viewed as through the peptide exit tunnel. In addition to S. cerevisiae residues numbering, E. coli numbering is shown in parenthesis. Comparison of the two structures shows that the side chain of the methyl-tyrosine is bound to the conserved hydrophobic A-site cleft, whereas the side chain of the proline residue penetrates the actual site of peptide bond formation where it may interfere with alignment of the reacting groups and proper positioning of the attacking electron pair of the α-amine.


This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel : A . Phe - Leu peptideB . Pro - Pro peptide .

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

This figure compares conformation of two dipeptides in the ribosome nascent peptide tunnel:A. Phe-Leu peptideB. Pro-Pro peptide.


B . Growth comparisons of selected kinases with the kinase ‑ dead mutant match . 24h growth curves ( OD595 ) from liquid culture of yeast strains ( W303 ) expressing human kinase in the presence or absence of 3 . 2 % ( v / v ) DMSO . Upper panel : BUB1 : BUB1 wild type , BUB1 - KD : BUB1 ( K821M ) ; upper middle panel : NEK6 : NEK6 wild type , NEK6 - KD : NEK6 ( K74M & K75M ) ; lower middle panel : PKCα - ac : PKCα ( A25E ) , PKCα - KD : PKCα ( K368R ) ; lower panel : Control with empty plasmid . Error bars represent SD from three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.


C . Human protein kinases phosphorylate large sets of yeast proteins . Upper panel : In total , 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho - motif antibodies on yeast proteins in comparison to other human kinases , kinase ‑ dead mutant versions or vector control samples . Commercially available pS / T - antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases . Lower panel : Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded . When developed with the indicated phospho - motif recognizing antibodies , immunoreactive bands indicated phosphorylation of yeast proteins .

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

C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.


C . Y2H interaction experiment with human AAR2 ( FL ) as bait and PRP8 ( aa1755 - 2335 ) as prey . Growth on selective media is abolished when AAR2 carries a S284E phospho - mimicry amino acid substitution . Mutation of second candidate phospho - site , AAR2 ( T356 ) , does not influence the AAR2 - PRP8 protein interaction .

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

C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.


D . Interaction of AAR2 and PRP8 in human cells . HA ‑ tagged PRP8 ( aa1755 - 2335 ) was co - expressed in HEK293 cells with either wildtype AAR2 , AAR2 ( S284A ) or AAR2 ( S284E ) mutant versions . Immunoprecipitation of PRP8 led to a co - precipitation of wild typeAAR2 and phospho - null AAR2 ( S284A ) . In contrast , the phospho - mimicry AAR2 ( S284E ) version did not bind PRP8 .

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

D. Interaction of AAR2 and PRP8 in human cells. HA‑tagged PRP8 (aa1755-2335) was co-expressed in HEK293 cells with either wildtype AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a co-precipitation of wild typeAAR2 and phospho-null AAR2(S284A). In contrast, the phospho-mimicry AAR2(S284E) version did not bind PRP8.


E . Results of the phospho - Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8 . Growth on selective media ( SD5 ) , of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co - expression of either SGK2 or CK2α1 ( CSNK2A1 ) . Growth reduction is visible in a 1 : 10 dilution series on selective agar ( SD5 ) in comparison to the control media ( SD3 ) . Ctrl : non - related kinases .

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

E. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.


F . Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases . Experiment as in D . Co - expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co - immunoprecipitated with RPR8 .

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

F. Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases. Experiment as in D. Co-expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co-immunoprecipitated with RPR8.


B . Results of the phospho - Y2H screen to identify kinases modulating the interaction of the ERα with 14 ‑ 3 ‑ 3β . The interaction of ERα ( prey ) with 14 ‑ 3 ‑ 3β ( bait ) is promoted by the co - expression of wildtype kinases of PAK5 , CDK17 , BUB1 and NEK6 , visible by the yeast growth on selective media in comparison to the control ( three biological replicas are shown ) . The coexpression of the kinase ‑ dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14 ‑ 3 ‑ 3β . In contrast the kinase ‑ dead mutant versions of BUB1 ( K821M ) and NEK6 ( K74M & K75M ) did not promote the Y2H interaction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction.


C . Phosphorylation of ERα in mammalian cells . ERα was co - expressed with the indicated wildtype kinases or kinase ‑ dead versions in HEK293 cells and immunoprecipitated . Phosphorylation status of ERα was analyzed with an in - gel phosphostain ( Pro - Q ® Diamond phosphoprotein gel stain , Invitrogen ) , relative to the whole protein stain ( SYPRO ® Ruby protein gel stain , Invitrogen ) .

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

C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).


D . Interaction of ERα and 14 ‑ 3 ‑ 3β in a GST pull - down assay . GST ‑ tagged 14 ‑ 3 ‑ 3β was expressed in E . coli and immobilized on beads . PA ‑ tagged ERα was co - expressed with either wildtype PAK5 or NEK6 , the kinase ‑ dead ( KD ) version or without a kinase in HEK293 cells . Immobilized 14 ‑ 3 ‑ 3β was used to pull PA ‑ tagged ERα from the HEK lysates . Co - precipitation of ERα was successful in the presence of wild kinases , but not in their absence or with kinase ‑ dead mutant versions .

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

D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions.


E . Effect of NEK6 co - expression on ERα gene activation . Relative luciferase activity of ERα in the presence and absence of 1 nM E2 ( 17β - estradiol ) was measured . A dose ‑ dependent reduction ( ng plasmid transfected ) in E2 - Erα ‑ dependent luciferase expression was observed in HEK293T , MCF - 7 and US - OS cancer cell lines . This effect was not observed with the NEK6 ( K74M & K75M ) kinase ‑ dead version . Graphs represent ≥ 5 biological replicates , each derived from averaging 6 technical replicates . Error bars indicate SD across the biological replicates .

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

E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.


D . Representative time - lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP - TMD ( ER ) ( green ) and SiR - lysosome ( orange ) . Zoom insets highlight regions of interest containing a static ( upper panels ) or mobile endosome ( lower panels ) as denoted by the white arrows . First row shows ER ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Scale bars : full size 10µm , zoom inset 3µm . Images relate to movies EV2 and 3 . E . Kymograph of the mobile vesicle in panel ( D ) . F . MDA quantification of ( D ) . Mobility of static and mobile endosomes was quantified and normalized to the static endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 4Hz . Significance two - tailed student t - test , * * * * P < 0 . 0001 .

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

D. Representative time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (ER) (green) and SiR-lysosome (orange). Zoom insets highlight regions of interest containing a static (upper panels) or mobile endosome (lower panels) as denoted by the white arrows. First row shows ER (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Scale bars: full size 10µm, zoom inset 3µm. Images relate to movies EV2 and 3. E. Kymograph of the mobile vesicle in panel (D). F. MDA quantification of (D). Mobility of static and mobile endosomes was quantified and normalized to the static endosomes. Graph represents mean ± s.d. of analyses at multiple locations within the same cell(n=20 cells per condition from 3 independent experiments). All images acquired for 90 frames at 4Hz. Significance two-tailed student t-test, **** P<0.0001.


C . Representative time - lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP - TMD ( green ) marked ER and SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Samples were treated with either DMSO or 2µm paclitaxel . Scale bar : 30µm and zoom insets 3µm . All images acquired for 90 frames at 1Hz . Images related to movie EV7 and 8 D . Kymograph of panel ( C ) . E . MDA quantification of panel ( C ) normalized to control cells , showing the average ER movement surrounding LE / Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . Significance two - tailed student t - test * * * * P < 0 . 0001 ; ns = not significant .

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

C. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) marked ER and SiR-lysosome-stained LE/Lys (orange) . Zoom insets highlight regions of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either DMSO or 2µm paclitaxel. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz.Images related to movie EV7 and 8 D. Kymograph of panel (C). E. MDA quantification of panel (C) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n = 20 cells per condition from 3 independent experiments). Significance two-tailed student t-test **** P<0.0001; ns = not significant.


F . Representative images of n = 3 of COS7 cells stably expressing GFP - TMD ( green ) to mark the ER and SiR - lysosome stained LE / Lys ( orange ) merged . Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and LE / Lys ( orange ) . Samples were treated with either siControl or siKTN1 . Scale bar : 30µm and zoom insets 3µm . All images acquired for 90 frames at 1Hz . Images related to movie EV9 and 10 G . Kymograph of panel ( F ) . H . MDA quantification of panel ( F ) normalized to control cells , showing the average ER movement surrounding LE / Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions , respectively . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 20 cells per condition from 3 independent experiments ) . Significance two - tailed student t - test . * * * * P < 0 . 0001 ; ns = not significant

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

F. Representative images of n = 3 of COS7 cells stably expressing GFP-TMD (green) to mark the ER and SiR-lysosome stained LE/Lys (orange) merged. Zoom insets highlight region of interest containing a mobile endosome as denoted by the white arrow. First row represents ER (GFP-TMD) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and LE/Lys (orange). Samples were treated with either siControl or siKTN1. Scale bar: 30µm and zoom insets 3µm. All images acquired for 90 frames at 1Hz. Images related to movie EV9 and 10 G. Kymograph of panel (F). H. MDA quantification of panel (F) normalized to control cells, showing the average ER movement surrounding LE/Lys under control or treated conditions and the overall average ER mobility under control or treatment conditions, respectively. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=20 cells per condition from 3 independent experiments). Significance two-tailed student t-test. **** P<0.0001; ns = not significant


A . Representative time - lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry - KDEL ( green ) and transiently expressing GFP - RAB7 ( orange ) . Zoom insets shows a region of interest containing a mobile RAB7 - positive endosome as denoted by the white arrows . First row represents the ER ( mCherry - KDEL ) ( green ) , the second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and GFP - RAB7 ( orange ) . Scale bar : 10µm , zoom insets 3µm . Images related to movie EV11 . Images were acquired for 90 frames at 1Hz .

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

A. Representative time-lapse spinning disk confocal images of n = 3 live COS7 cells stably expressing mCherry-KDEL (green) and transiently expressing GFP-RAB7 (orange). Zoom insets shows a region of interest containing a mobile RAB7-positive endosome as denoted by the white arrows. First row represents the ER (mCherry-KDEL) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and GFP-RAB7 (orange). Scale bar: 10µm, zoom insets 3µm. Images related to movie EV11. Images were acquired for 90 frames at 1Hz.


B . Kymograph of panel ( A ) . C . MDA quantification of panel ( A ) and figures EV2A - G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 15 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 1Hz . Significance two - tailed student t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kymograph of panel (A). C. MDA quantification of panel (A) and figures EV2A-G normalized to 1 showing average ER movement surrounding static endosomes and mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. *P<0.05, ** P<0.01, **** P<0.0001 ns = not significant.


E . Representative time - lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP - TMD ( green ) and in lower panels transiently expressing mScarlet - RILP ( magenta ) and SiR - lysosome - stained LE / Lys ( orange ) . Zoom insets shows region of interest containing ER ( GFP - TMD ) . First row represents ER ( GFP - TMD ) ( green ) , the second row shows MDA - generated ER movement ( magenta ) , third row shows a merge of the two preceding channels . Scale bar : 10µm , zoom insets 3µm . Images related to movie EV19 and movie EV20 . Images were acquired for 90 frames at 1Hz . F . MDA quantification of panel ( E ) and figure EV3A normalized to 1 showing total average ER movement . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 15 cells per condition from 3 independent experiments ) . All images acquired 90 frames at 1Hz . Significance two - tailed student t - test . * * * * P < 0 . 0001 ns = not significant .

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

E. Representative time-lapse spinning disk confocal images of n = 3 of live COS7 cells stably expressing GFP-TMD (green) and in lower panels transiently expressing mScarlet-RILP (magenta) and SiR-lysosome-stained LE/Lys (orange). Zoom insets shows region of interest containing ER (GFP-TMD). First row represents ER (GFP-TMD) (green), the second row shows MDA-generated ER movement (magenta), third row shows a merge of the two preceding channels. Scale bar: 10µm, zoom insets 3µm. Images related to movie EV19 and movie EV20. Images were acquired for 90 frames at 1Hz. F. MDA quantification of panel (E) and figure EV3A normalized to 1 showing total average ER movement. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=15 cells per condition from 3 independent experiments). All images acquired 90 frames at 1Hz. Significance two-tailed student t-test. **** P<0.0001 ns = not significant.


B . Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD - GFP ( green ) and SiR - lysosome - stained endosomes / lysosomes ( orange ) . COS7 cells in the middle panels overexpress mCherry - RILP ( magenta ) . The COS7 cells in the lower panels overexpress mCherry - TMEM55B ( magenta ) . Zoom insets show region of interest containing the ER ( TMD - GFP ) ( green ) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average . Scale bars : 15µm . zoom insets : 3µm . C . Quantification of the number of ER junctions per µm2 from panel ( B ) as resulted from ER junction analysis . Graph represents mean ± s . d . of analyses at multiple locations within one cell ( n = 15 from 3 independent experiments ) . Significance two - tailed student t - test . * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange). COS7 cells in the middle panels overexpress mCherry-RILP (magenta). The COS7 cells in the lower panels overexpress mCherry-TMEM55B (magenta). Zoom insets show region of interest containing the ER (TMD-GFP) (green) and junction analysis of the skeletonized ER where the number of ER junctions representing the quantified average. Scale bars: 15µm. zoom insets: 3µm. C. Quantification of the number of ER junctions per µm2 from panel (B) as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell (n=15 from 3 independent experiments). Significance two-tailed student t-test. ***P<0.001.


A . Representative images of time - lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP - TMD ( green ) and SiR - lysosome - stained LE / Lys ( orange ) treated with either siControl or co - treated with siVAPA , siVAPB and siMOSPD2 . Zoom insets show regions of interest containing mobile LE / Lys as denoted by the white arrow . First row represents ER ( TMD - GFP ) ( green ) , the second row shows MDA generated ER movement ( magenta ) , third row shows a merge of the two preceding channels and the fourth row shows a merge of ER ( green ) and SiR - lysosome stained LE / Lys ( orange ) . Scale bar : 5µm , zoom insets 3µm . Images related to movie EV40 and 47 . Images were acquired for 90 frames at 1Hz .

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

A. Representative images of time-lapse spinning disk confocal images of n = 3 of COS7 cells stably expressing GFP-TMD (green) and SiR-lysosome-stained LE/Lys (orange) treated with either siControl or co-treated with siVAPA, siVAPB and siMOSPD2. Zoom insets show regions of interest containing mobile LE/Lys as denoted by the white arrow. First row represents ER (TMD-GFP) (green), the second row shows MDA generated ER movement (magenta), third row shows a merge of the two preceding channels and the fourth row shows a merge of ER (green) and SiR-lysosome stained LE/Lys (orange). Scale bar: 5µm, zoom insets 3µm. Images related to movie EV40 and 47. Images were acquired for 90 frames at 1Hz.


B . Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD - GFP ( green ) and SiR - lysosome - stained endosomes / lysosomes ( orange ) treated with siControl or with siVAPA , siVAPB and siMOSPD2 . Zoom insets show region of interest containing ER ( TMD - GFP ) ( green ) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average . Scale bars : 10µm . zoom insets : 3µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative stills of spinning disk confocal images of n = 3 of COS7 cells stably expressing TMD-GFP (green) and SiR-lysosome-stained endosomes/lysosomes (orange) treated with siControl or with siVAPA, siVAPB and siMOSPD2. Zoom insets show region of interest containing ER (TMD-GFP) (green) and junction analysis of skeletonized ER where number of ER junctions representing the quantified average. Scale bars: 10µm. zoom insets: 3µm.


C . MDA quantification of ER of panels ( A , B ) and fig . EV4B - G normalized to control cells showing average ER movement surrounding mobile endosomes . Graph represents mean ± s . d . of analyses at multiple locations within the same cell ( n = 30 cells per condition from 3 independent experiments ) . All images acquired for 90 frames at 1Hz . Significance two - tailed student t - test . * * P < 0 . 01 , * * * * P < 0 . 0001 , ns = not significant D . MDA quantification of SiR - lysosome - stained LE / Lys of ( A , B ) and fig . EV4B - G normalized to 1 showing total average endosomal movement . Graph represents mean ± s . d . of n = 15 cells per condition from 3 independent experiments . Significance two tailed student t - test . ns = not significant . E . Quantification of number of ER junctions per µm2 from ( B ) and fig . H - M as resulted from ER junction analysis . Graph represents mean ± s . d . of analyses at multiple locations within one cell of n = 15 from 3 independent experiments . Significance two - tailed student t - test . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns = not significant .

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

C. MDA quantification of ER of panels (A, B) and fig. EV4B-G normalized to control cells showing average ER movement surrounding mobile endosomes. Graph represents mean ±s.d. of analyses at multiple locations within the same cell (n=30 cells per condition from 3 independent experiments). All images acquired for 90 frames at 1Hz. Significance two-tailed student t-test. **P<0.01, ****P< 0.0001, ns=not significant D. MDA quantification of SiR-lysosome-stained LE/Lys of (A, B) and fig. EV4B - G normalized to 1 showing total average endosomal movement. Graph represents mean ±s.d. of n = 15 cells per condition from 3 independent experiments. Significance two tailed student t-test. ns= not significant. E. Quantification of number of ER junctions per µm2 from (B) and fig. H - M as resulted from ER junction analysis. Graph represents mean ±s.d. of analyses at multiple locations within one cell of n = 15 from 3 independent experiments. Significance two-tailed student t-test. *P<0.05, **P<0.01 ***P<0.001, ****P< 0.0001, ns=not significant.


NIX and BNIP3 are required for mitophagy induced by MLN4924 and other HIF1α stabilisers . Parental HeLa cells and NIX / BNIP3 double knockout ( NIX / BNIP3 DKO ) HeLa cells expressing mt - Keima were treated with DFP ( 1 mM ) , MLN4924 ( 0 . 5 μM ) , or DMOG ( 10 nM ) for 24 hours and analysed by live - cell confocal microscopy . The emission signals obtained after excitation with the 458 nm laser ( neutral pH ) or 561 nm laser ( acidic pH ) are shown in green and magenta , respectively . Quantification of mitophagy shown in panel A . Mitophagy is represented as the ratio of mt - Keima 561 nm fluorescence intensity to mt - Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated condition . In B , the measurements from individual cells are represented as translucent grey dots , and the mean ratio from each independent experiment is represented by coloured circles . The centre lines and bars depict the mean of the averaged independent replicates + / - standard deviation . N = 4 . A minimum of 50 cells were analysed for each condition within each individual replicate experiment , and over 300 cells were analysed for each condition in total . P values were calculated based on the mean ratio values from independent experiments using one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) .

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

NIX and BNIP3 are required for mitophagy induced by MLN4924 and other HIF1α stabilisers. Parental HeLa cells and NIX/BNIP3 double knockout (NIX/BNIP3 DKO) HeLa cells expressing mt-Keima were treated with DFP (1 mM), MLN4924 (0.5 μM), or DMOG (10 nM) for 24 hours and analysed by live-cell confocal microscopy. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively. Quantification of mitophagy shown in panel A. Mitophagy is represented as the ratio of mt-Keima 561 nm fluorescence intensity to mt-Keima 458 nm fluorescence intensity for individual cells normalised to the mean of the untreated condition. In B , the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).


Analysis of NIX and BNIP3 protein levels after MLN4924 , DMOG , or DFP treatments . HeLa cells or HeLa NIX / BNIP3 DKO cells were treated with MLN4924 , DMOG , or DFP for 24 hours . Total - cell lysates corresponding to conditions in panel A were subject to immunoblotting .

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

Analysis of NIX and BNIP3 protein levels after MLN4924, DMOG, or DFP treatments. HeLa cells or HeLa NIX/BNIP3 DKO cells were treated with MLN4924, DMOG, or DFP for 24 hours. Total-cell lysates corresponding to conditions in panel A were subject to immunoblotting.


Inhibition of HIF1α with echinomycin prevents DFP - induced mitophagy but not MLN4924 - induced mitophagy . U2OS cells expressing mt - Keima were treated with the indicated drugs for 24 hours and analysed by live - cell confocal microscopy . Quantification of mitophagy shown in panel D . In F , the measurements from individual cells are represented as translucent grey dots , and the mean ratio from each independent experiment is represented by coloured circles . The centre lines and bars depict the mean of the averaged independent replicates + / - standard deviation . N = 4 . A minimum of 50 cells were analysed for each condition within each individual replicate experiment , and over 300 cells were analysed for each condition in total . P values were calculated based on the mean ratio values from independent experiments using one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) .

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

Inhibition of HIF1α with echinomycin prevents DFP-induced mitophagy but not MLN4924-induced mitophagy. U2OS cells expressing mt-Keima were treated with the indicated drugs for 24 hours and analysed by live-cell confocal microscopy. Quantification of mitophagy shown in panel D. In F, the measurements from individual cells are represented as translucent grey dots, and the mean ratio from each independent experiment is represented by coloured circles. The centre lines and bars depict the mean of the averaged independent replicates +/- standard deviation. N=4. A minimum of 50 cells were analysed for each condition within each individual replicate experiment, and over 300 cells were analysed for each condition in total. P values were calculated based on the mean ratio values from independent experiments using one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001).


Inhibition of HIF1α with echinomycin prevents the increase of NIX and BNIP3 in response to DFP , but not MLN4924 . U2OS cells were treated with DFP or MLN4924 over a time course . Where indicated , cells were also treated for 24 hours with echinomycin . Total - cell lysates were subject to immunoblotting as shown .

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

Inhibition of HIF1α with echinomycin prevents the increase of NIX and BNIP3 in response to DFP, but not MLN4924. U2OS cells were treated with DFP or MLN4924 over a time course. Where indicated, cells were also treated for 24 hours with echinomycin. Total-cell lysates were subject to immunoblotting as shown.


Re - expression of FBXL4 into FBXL4 - defective CRISPR lines reduces the levels of NIX and BNIP3 in FBLX4 deficient clones . FBXL4 - deficient 2G10 and FBXL4 - deficient 1D4 cell lines were stably transduced with a doxycycline - inducible FBXL4 - HA construct . Cells were treated with doxycycline for the indicated times prior to immunoblotting with the specified antibodies .

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

Re-expression of FBXL4 into FBXL4-defective CRISPR lines reduces the levels of NIX and BNIP3 in FBLX4 deficient clones. FBXL4-deficient 2G10 and FBXL4-deficient 1D4 cell lines were stably transduced with a doxycycline-inducible FBXL4-HA construct. Cells were treated with doxycycline for the indicated times prior to immunoblotting with the specified antibodies.


FBXL4 localises to the mitochondrial outer membrane . Cells transiently transfected with FBXL4 - HA - C were treated with DFP for 24 h . Cells were stained with an anti - HA antibody ( to recognise FBXL4 ) and either TOM20 ( an outer mitochondrial membrane protein ) or TIM50 ( an inner mitochondrial membrane protein ) . The line scan intensity profiles for FBXL4 ( green ) and TOM20 / TIM50 ( magenta ) represent fluorescence intensity ( y - axis ) plotted against distance ( x - axis ) . Scale bars = 5 μm .

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

FBXL4 localises to the mitochondrial outer membrane. Cells transiently transfected with FBXL4-HA-C were treated with DFP for 24 h. Cells were stained with an anti-HA antibody (to recognise FBXL4) and either TOM20 (an outer mitochondrial membrane protein) or TIM50 (an inner mitochondrial membrane protein). The line scan intensity profiles for FBXL4 (green) and TOM20/TIM50 (magenta) represent fluorescence intensity (y-axis) plotted against distance (x-axis). Scale bars = 5 μm.


FBXL4 is a proximity interactor of NIX and BNIP3 . Cells expressing inducible BirA - BNIP3 , BirA - NIX and BirA control were transduced with a lentiviral vector expressing FBXL4 , as indicated . Cells were treated with doxycycline for 48 hours ( to induce BirA - bait protein expression ) , biotin for 24 hours ( for the biotinylation reaction ) , and , where indicated , MLN4924 for 24 hours ( to stabilise NIX and BNIP3 ) . Streptavidin - coupled beads were used to capture the biotinylated proteins . FBXL4 was specifically detected in the eluate from BirA - BNIP3 and BirA - NIX compared with BirA - alone .

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

FBXL4 is a proximity interactor of NIX and BNIP3. Cells expressing inducible BirA-BNIP3, BirA-NIX and BirA control were transduced with a lentiviral vector expressing FBXL4, as indicated. Cells were treated with doxycycline for 48 hours (to induce BirA-bait protein expression), biotin for 24 hours (for the biotinylation reaction), and, where indicated, MLN4924 for 24 hours (to stabilise NIX and BNIP3). Streptavidin-coupled beads were used to capture the biotinylated proteins. FBXL4 was specifically detected in the eluate from BirA-BNIP3 and BirA-NIX compared with BirA-alone.


NIX and BNIP3 polyubiquitylation depends on FBXL4 . U2OS or U2OS - FBXL4 KO cells were co - transfected with TR - TUBE and either myc - BNIP3 or myc - NIX , as indicated . Cell lysates obtained 48 hours post - transfection were immunoprecipitated with anti - FLAG beads , and the immunoprecipitates were analysed by immunoblotting using anti - myc antibody ( to detect ubiquitylated NIX or BNIP3 ) . The line on the right marks a ladder of bands corresponding to polyubiquitylated myc - BNIP3 or myc -

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

NIX and BNIP3 polyubiquitylation depends on FBXL4. U2OS or U2OS-FBXL4 KO cells were co-transfected with TR-TUBE and either myc-BNIP3 or myc-NIX, as indicated. Cell lysates obtained 48 hours post-transfection were immunoprecipitated with anti-FLAG beads, and the immunoprecipitates were analysed by immunoblotting using anti-myc antibody (to detect ubiquitylated NIX or BNIP3). The line on the right marks a ladder of bands corresponding to polyubiquitylated myc-BNIP3 or myc-NIX


Loss of FBXL4 leads to an increase in mitophagy , which can be reduced by re - expression of FBXL4 . U2OS mt - Keima FBXL4 KO clones ( 2G10 and 1D4 ) expressing doxycycline - inducible wild - type FBXL4 - HA were treated with doxycycline for 72 hours . The emission signals obtained after excitation with the 458 nm laser ( neutral pH ) or 561 nm laser ( acidic pH ) are shown in green and magenta , respectively .

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

Loss of FBXL4 leads to an increase in mitophagy, which can be reduced by re-expression of FBXL4. U2OS mt-Keima FBXL4 KO clones (2G10 and 1D4) expressing doxycycline-inducible wild-type FBXL4-HA were treated with doxycycline for 72 hours. The emission signals obtained after excitation with the 458 nm laser (neutral pH) or 561 nm laser (acidic pH) are shown in green and magenta, respectively.


Depleting NIX / BNIP3 via siRNA reduces the increased mitophagy caused by FBXL4 deficiency . U2OS mt - Keima cells and U2OS mt - Keima FBXL4 KO 2G10 cells were transfected with siRNAs targeting both NIX and BNIP3 ( NIX / BNIP3 si ) or non - targeting siRNA ( NT si ) . UT = untransfected .

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

Depleting NIX/BNIP3 via siRNA reduces the increased mitophagy caused by FBXL4 deficiency. U2OS mt-Keima cells and U2OS mt-Keima FBXL4 KO 2G10 cells were transfected with siRNAs targeting both NIX and BNIP3 (NIX/BNIP3 si) or non-targeting siRNA (NT si). UT=untransfected.


Corresponding cells from D - E were harvested for immunoblotting to analyse the extent of NIX and BNIP3 reduction after siRNA transfection .

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

Corresponding cells from D-E were harvested for immunoblotting to analyse the extent of NIX and BNIP3 reduction after siRNA transfection.


Hyperstable NIX deletion mutants increase mitophagy compared with wild - type NIX . Hela Flp - in NIX KO mt - Keima cells stably expressing inducible NIX ( WT ) , FLAG - NIX ∆ 151 - 170 , or FLAG - NIX ∆ 171 - 184 were treated with doxycycline for 48 hours Mitophagy was visualised as in A and quantified as in B . Hyperstable BNIP3 deletion mutants increase mitophagy compared with wild - type NIX . Hela Flp - in NIX / BNIP3 DKO mt - Keima cells stably expressing BNIP3 deletion mutants were treated with doxycycline for 48 hours translucent grey dots represent measurements from individual cells . Coloured circles represent the mean ratio from independent experiments . The centre lines and bars represent the mean of the independent replicate means + / - standard deviation . N = 3 . At least 200 cells were analysed per condition . P values were calculated based on the mean values using a one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Scale bars = 20 μm .

Hyperstable NIX deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX KO mt-Keima cells stably expressing inducible NIX(WT), FLAG-NIX∆151-170, or FLAG-NIX∆171-184 were treated with doxycycline for 48 hours Mitophagy was visualised as in A and quantified as in B. Hyperstable BNIP3 deletion mutants increase mitophagy compared with wild-type NIX. Hela Flp-in NIX/BNIP3 DKO mt-Keima cells stably expressing BNIP3 deletion mutants were treated with doxycycline for 48 hours translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicate means +/- standard deviation. N=3. At least 200 cells were analysed per condition. P values were calculated based on the mean values using a one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001). Scale bars = 20 μm.


FBXL4 patient - derived variants exhibit reduced efficiency compared to wild - type FBXL4 in mediating the downregulation and destabilization of NIX and BNIP3 . U2OS FBXL4 KO ( 2G10 ) cells were rescued with constructs expressing wild - type FBXL4 - HA , FBXL4 ( F - box mut ) , FBXL4 ( ∆ MTS ) , or specified patient variants . Cells were treated with cycloheximide for 3 hours prior to harvesting . Samples were lysed , and immunoblotting was performed . GFP serves as a marker of transduction efficiency / transgene expression . EV = empty vector .

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

FBXL4 patient-derived variants exhibit reduced efficiency compared to wild-type FBXL4 in mediating the downregulation and destabilization of NIX and BNIP3. U2OS FBXL4 KO (2G10) cells were rescued with constructs expressing wild-type FBXL4-HA, FBXL4(F-box mut), FBXL4(∆MTS), or specified patient variants. Cells were treated with cycloheximide for 3 hours prior to harvesting. Samples were lysed, and immunoblotting was performed. GFP serves as a marker of transduction efficiency/transgene expression. EV = empty vector.


FBXL4 - Arg482Trp and FBXL4 - Gly568Ala patient variants are less efficient than FBXL4 - wild - type at assembling into a complex with SKP1 and CUL1 . FBXL4 - KO cells expressing wild - type FBXL4 - HA or FBXL4 variants were harvested and lysed . Whole - cell extracts were subjected to immunoprecipitation ( IP ) with anti - HA agarose beads and immunoblotting , as indicated .

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

FBXL4-Arg482Trp and FBXL4-Gly568Ala patient variants are less efficient than FBXL4-wild-type at assembling into a complex with SKP1 and CUL1. FBXL4-KO cells expressing wild-type FBXL4-HA or FBXL4 variants were harvested and lysed. Whole-cell extracts were subjected to immunoprecipitation (IP) with anti-HA agarose beads and immunoblotting, as indicated.


NIX / BNIP3 depletion mediated by siRNA reduces mitophagy in FBXL4 - deficient patient fibroblast cells . FBXL4 - deficient patient fibroblasts were transfected with siRNAs targeting both NIX and BNIP3 ( NIX / BNIP3 si ) or non - targeting siRNA ( NT si ) ( left ) . Live - cell confocal microscopy was performed to visualise mitophagy and quantification was performed as in D ( middle ) . The siRNA - mediated depletion of NIX and BNIP3 was evaluated by Western blotting ( right ) . translucent grey dots represent measurements from individual cells . Coloured circles represent the mean ratio from independent experiments . The centre lines and bars represent the mean of the independent replicates + / - standard deviation . in H , N = 3 . Over 100 cells were analysed per condition . P values were calculated based on the mean values using a one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 005 , * * * P < 0 . 001 , * * * * P < 0 . 0001 ) . Scale bar = 20 μm .

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

NIX/BNIP3 depletion mediated by siRNA reduces mitophagy in FBXL4-deficient patient fibroblast cells. FBXL4-deficient patient fibroblasts were transfected with siRNAs targeting both NIX and BNIP3 (NIX/BNIP3 si) or non-targeting siRNA (NT si) (left). Live-cell confocal microscopy was performed to visualise mitophagy and quantification was performed as in D (middle). The siRNA-mediated depletion of NIX and BNIP3 was evaluated by Western blotting (right). translucent grey dots represent measurements from individual cells. Coloured circles represent the mean ratio from independent experiments. The centre lines and bars represent the mean of the independent replicates +/- standard deviation. in H, N=3. Over 100 cells were analysed per condition. P values were calculated based on the mean values using a one-way ANOVA (*P<0.05, **P<0.005, ***P<0.001, ****P<0.0001). Scale bar = 20 μm.


Ubiquitylation of endogenous NIX is restored upon expression of FBXL4 in patient - derived cells . Patient derived fibroblasts were transfected with FLAG - tagged TR - TUBE , as indicated . FLAG - tagged TUBE protein was affinity purified from the corresponding lysates using FLAG - beads , and the precipitates were analysed by immunoblotting using antibodies to endogenous NIX .

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Ubiquitylation of endogenous NIX is restored upon expression of FBXL4 in patient-derived cells. Patient derived fibroblasts were transfected with FLAG-tagged TR-TUBE, as indicated. FLAG-tagged TUBE protein was affinity purified from the corresponding lysates using FLAG-beads, and the precipitates were analysed by immunoblotting using antibodies to endogenous NIX.


E The representative images of IF - IHC staining of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 200 μm . F Quantification of numbers of Iba - 1 + cells on the meningeal regions . Mock n = 5 ; ZIKV n = 7 embryos . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM .

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E The representative images of IF-IHC staining of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 200 μm. F Quantification of numbers of Iba-1+ cells on the meningeal regions. Mock n=5; ZIKV n=7 embryos. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


G The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 50 μm . H Quantification of total number of Iba - 1 + cells per ChP area . I Percentage of Iba - 1 + cells on the surface of ChP . J Percentage of round - shaped Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM . Mock n = 5 ; ZIKV n = 6 biological

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G The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm. H Quantification of total number of Iba-1+ cells per ChP area. I Percentage of Iba-1+ cells on the surface of ChP. J Percentage of round-shaped Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicates


K The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) , CD68 ( red ) and DAPI ( blue ) . Scale bar , 50 μm . L Percentage of CD68 + ; Iba - 1 + cells out of total Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM .

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K The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm. L Percentage of CD68+; Iba-1+ cells out of total Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


B Representative animal tracks in the sociability test . C Percentage of time the subject mice spent in each chamber in the sociability session of three - chamber test . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . empty ( E ) chamber ; ZIKV ( n = 16 ) stranger 1 chamber vs . empty chamber ; one - way ANOVA with Bonferroni post hoc test . D Quantification of time spent investigating the stranger mouse and empty cage during sociability session . Mock ( n = 11 ) sniffing time at stranger 1 mouse vs . empty cup , ZIKV ( n = 16 ) sniffing time at stranger 1 mouse vs . empty cup . E Preference indices in sociability . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . F Representative animal tracks in the social memory test . G Percentage of time the subject mice spent in each in the social memory session . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . stranger 2 ( S2 ) chamber , p < 0 . 0001 ; ZIKV ( n = 16 ) stranger 1 chamber vs . stranger 2 chamber ; one - way ANOVA with Bonferroni post hoc test . H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session . Mock ( n = 11 ) sniffing time at stranger 1 vs . stranger 2 mouse ; ZIKV ( n = 16 ) sniffing time at stranger 1 vs . stranger 2 mouse . I Preference indices in the social novelty session of the three - chamber test . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test unless described otherwise ; n represent biological replicates .

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B Representative animal tracks in the sociability test. C Percentage of time the subject mice spent in each chamber in the sociability session of three-chamber test. Mock (n=11) stranger 1 (S1) chamber vs. empty (E) chamber; ZIKV (n=16) stranger 1 chamber vs. empty chamber; one-way ANOVA with Bonferroni post hoc test. D Quantification of time spent investigating the stranger mouse and empty cage during sociability session. Mock (n=11) sniffing time at stranger 1 mouse vs. empty cup, ZIKV (n=16) sniffing time at stranger 1 mouse vs. empty cup. E Preference indices in sociability. Mock (n=11) vs. ZIKV (n=16). F Representative animal tracks in the social memory test. G Percentage of time the subject mice spent in each in the social memory session. Mock (n=11) stranger 1 (S1) chamber vs. stranger 2 (S2) chamber, p< 0.0001; ZIKV (n=16) stranger 1 chamber vs. stranger 2 chamber; one-way ANOVA with Bonferroni post hoc test. H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session. Mock (n=11) sniffing time at stranger 1 vs. stranger 2 mouse; ZIKV (n=16) sniffing time at stranger 1 vs. stranger 2 mouse. I Preference indices in the social novelty session of the three-chamber test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates.


B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC . C Quantitation of sEPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 4 mice ; ZIKV , n = 15 neurons from 5 mice in B and C . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

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B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC. C Quantitation of sEPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 4 mice; ZIKV, n= 15 neurons from 5 mice in B and C. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


E Quantifications of sIPSC frequency from pyramidal neurons in mPFC . F Quantifications of sIPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 5 mice ; ZIKV , n = 18 neurons from 6 mice in E and F . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

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E Quantifications of sIPSC frequency from pyramidal neurons in mPFC. F Quantifications of sIPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 5 mice; ZIKV, n= 18 neurons from 6 mice in E and F. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


A Representative images of PV + , SST + and CAL + cells in the prelimbic ( PL , upper panel ) and infralimbic ( IL , lower panel ) cortex . Scale bars : 200 μm .

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A Representative images of PV+, SST+ and CAL+ cells in the prelimbic (PL, upper panel) and infralimbic (IL, lower panel) cortex. Scale bars: 200 μm.


H IHC staining of mPFC stained with antibodies against vGLUT1 . Scale bars , 5 μm . I Quantification of vGLUT1 + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

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H IHC staining of mPFC stained with antibodies against vGLUT1. Scale bars, 5 μm. I Quantification of vGLUT1+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


J IHC staining of mPFC stained with antibodies against vGAT . Scale bars , 5 μm . K Quantification of vGAT + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

J IHC staining of mPFC stained with antibodies against vGAT. Scale bars, 5 μm. K Quantification of vGAT+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


B Representative confocal images of the vHIP stained with mCherry ( red ) and c - Fos ( green ) . C Quantification of mCherry and c - Fos double positive cells out of total mCherry - positive cells in the vHIP CA1 regions . Scale bar , 50 μm . Mock ( n = 5 mice ) vs . ZIKV ( n = 5 mice ) , p = 0 . 0013 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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B Representative confocal images of the vHIP stained with mCherry (red) and c-Fos (green). C Quantification of mCherry and c-Fos double positive cells out of total mCherry-positive cells in the vHIP CA1 regions. Scale bar, 50 μm. Mock (n= 5 mice) vs. ZIKV (n= 5 mice), p=0.0013. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


F Representative EPSC traces of the pyramidal neurons in the mPFC . G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC ( PL and IL ) . Mock ( n = 17 neurons from 5 mice ) vs . ZIKV ( n = 19 neurons from 5 mice ) , p = 0 . 0122 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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F Representative EPSC traces of the pyramidal neurons in the mPFC. G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC (PL and IL). Mock (n=17 neurons from 5 mice) vs. ZIKV (n=19 neurons from 5 mice), p=0.0122. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


B Representative examples of GFP - labeled presynaptic boutons . Scale bars : 2 . 5 μm . C Quantification of presynaptic bouton density and weighted size in the mPFC ( PL and IL ) . Mock : n = 57 axons from 6 mice ; ZIKV : n = 63 axons from 7 mice . D Quantification of presynaptic bouton weighted size in the mPFC ( PL and IL ) . Mock : n = 2974 boutons from 6 mice ; ZIKV : n = 3486 boutons from 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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B Representative examples of GFP-labeled presynaptic boutons. Scale bars: 2.5 μm. C Quantification of presynaptic bouton density and weighted size in the mPFC (PL and IL). Mock: n= 57 axons from 6 mice; ZIKV: n= 63 axons from 7 mice. D Quantification of presynaptic bouton weighted size in the mPFC (PL and IL). Mock: n= 2974 boutons from 6 mice; ZIKV: n= 3486 boutons from 7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


H Comparisons of percentages of vHIP - innervated mPFC ( PL and IL ) neuron subtypes . Mock , n = 4 mice ; ZIKV , n = 7 mice . P = 0 . 5343 in CaMKIIa , 0 . 0117 in PV , 0 . 7491 in CAL , 0 . 4484 in SST , 0 . 1519 in other uncharacterized neuron subtypes . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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H Comparisons of percentages of vHIP-innervated mPFC (PL and IL) neuron subtypes. Mock, n=4 mice; ZIKV, n=7 mice. P= 0.5343 in CaMKIIa, 0.0117 in PV, 0.7491 in CAL, 0.4484 in SST, 0.1519 in other uncharacterized neuron subtypes. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


J Representative confocal images of CTB488 - labeled tdTomato - positive neurons in the vHIP . White arrowheads indicate CTB488 single positive neurons . Scale bars : 200 μm . K Quantification of the percentage of tdTomato ; CTB488 double - positive cells out of total CTB488 - positive cells . Mock , n = 7 mice ; ZIKV , n = 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

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J Representative confocal images of CTB488-labeled tdTomato-positive neurons in the vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm. K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


C Representative animal tracks in three - chamber test . D Discrimination indices of the social memory session of three - chamber test . mCherry mock , n = 8 mice ; mCherry ZIKV , n = 10 mice ; hM4Di ZIKV n = 10 mice , mCherry mock vs . mCherry ZIKV p = 0 . 0041 ; mCherry ZIKV vs . hM4Di ZIKV p = 0 . 0115 ; and mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

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C Representative animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


E Representative confocal images of mCherry ( red ) and c - Fos ( green ) double - labeled cells in the vHIP CA1 regions . Scale bars , 50 μm . F . Quantification of the percentage of c - fos ; mCherry double positive cells out of total mCherry cells . mCherry mock , n = 6 mice ; mCherry ZIKV , n = 4 mice ; hM4Di ZIKV n = 6 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

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E Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


( C ) Co - immunoprecipitation of Cdc4 using either unphosphorylated or Clb5 - Cdk1 - phosphorylated Far1 ( 85 - 150 ) as bait . The experiment was repeated twice , a representative example is shown .

(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.


( D ) Time - lapse fluorescence images showing the expression and degradation of wild - type and S87A S91A mutant Far1 ( 1 - 150 ) - GFP and nuclear - cytoplasmic shuttling of Whi5 - mCherry . Nuclear export of Whi5 - mCherry takes place at 0 min ( Start point ) and was used to synchronize the cells for quantitative analysis .

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(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.


( E , F ) Mean ± SEM fluorescence intensities of the indicated Far1 ( 1 - 150 ) - GFP mutants over the cell cycle .

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(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle.