( b ) Modulation of autophagy alters Dendra - tau clearance dynamics . The fluorescence intensity of each individual cell was quantified at each timepoint ( n ≥ 31 cells , ≥ 9 larvae per treatment group ) and mean cell intensity values for each drug treatment at each timepoint were calculated . Images were taken immediately after photoconversion and at 24 , 30 and 48 h intervals thereafter . Rapamycin treatment significantly increased the rate of Dendra - tau clearance . Ammonium chloride ( NH4Cl ) treatment significantly decreases the rate of Dendra - tau clearance ( * * P0 . 01 , * * * P0 . 001 , one - way analysis of variance ( ANOVA ) ) . Error bars are ± s . e . m .

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

(b) Modulation of autophagy alters Dendra-tau clearance dynamics. The fluorescence intensity of each individual cell was quantified at each timepoint (n≥ 31 cells, ≥9 larvae per treatment group) and mean cell intensity values for each drug treatment at each timepoint were calculated. Images were taken immediately after photoconversion and at 24, 30 and 48 h intervals thereafter. Rapamycin treatment significantly increased the rate of Dendra-tau clearance. Ammonium chloride (NH4Cl) treatment significantly decreases the rate of Dendra-tau clearance (**P0.01, ***P0.001, one-way analysis of variance (ANOVA)). Error bars are ±s.e.m.


( c ) Dendra - tau clearance in the presence of CALM : representative images of CALM with mosaic expression of Dendra - tau and full - length Dendra - tau taken immediately after photoconversion and at 6 , 24 , 30 , 48 and 54 h after conversion . The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co - expressing Dendra - tau and either full - length CALM or Δ - ANTH CALM ( larvae - ANTH - HA ) constructs ( n ≥ 100 cells , ≥ 9 larvae per treatment group ) at each time point was calculated . ( i ) Expression of full - length HA significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM ( * * * P0 . 001 , one - way ANOVA ) . One graph , representative of three independent experiments , is presented . ( CALM - ANTH - HA : 132 cells , 20 fishes ; CALM - CALM : 18 cells , 8 fishes ) . Another two experiments are shown in Supplementary Fig . S5C . Error bars are mean ± s . d . ( ii ) Treatment with CALM alters the dynamics of CALM clearance . Expression of full - length CALM significantly delayed the clearance of CALM at all time points compared with Δ - ANTH CALM , as observed in i ( P0 . 001 , one - way ANOVA ) . However , treatment of Δ - ANTH CALM injected CALM with CALM slows the CALM clearance by 70 % to a level comparable to that observed in larvae injected with full - length Dendra - tau . Ammonium chloride treatment of Dendra - tau injected with full - length null results in a modest ( 22 % ) decrease in Dendra - tau clearance , suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect ( n ≥ 25 cells , n ≥ 9 Dendra - tau per treatment group ) . Error bars are mean ± s . d . Note that i and ii are distinct experiments .

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

(c) Dendra-tau clearance in the presence of CALM: representative images of CALM with mosaic expression of Dendra-tau and full-length Dendra-tau taken immediately after photoconversion and at 6, 24, 30, 48 and 54 h after conversion. The fluorescence intensity of individual cells was quantified and mean fluorescent intensity of cells co-expressing Dendra-tau and either full-length CALM or Δ-ANTH CALM (larvae-ANTH-HA) constructs (n≥ 100 cells, ≥9 larvae per treatment group) at each time point was calculated. (i) Expression of full-length HA significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM (***P0.001, one-way ANOVA). One graph, representative of three independent experiments, is presented. (CALM-ANTH-HA: 132 cells, 20 fishes; CALM-CALM: 18 cells, 8 fishes). Another two experiments are shown in Supplementary Fig. S5C. Error bars are mean ±s.d. (ii) Treatment with CALM alters the dynamics of CALM clearance. Expression of full-length CALM significantly delayed the clearance of CALM at all time points compared with Δ-ANTH CALM, as observed in i (P0.001, one-way ANOVA). However, treatment of Δ-ANTH CALM injected CALM with CALM slows the CALM clearance by 70% to a level comparable to that observed in larvae injected with full-length Dendra-tau. Ammonium chloride treatment of Dendra-tau injected with full-length null results in a modest (22%) decrease in Dendra-tau clearance, suggesting that CALM overexpression and ammonium chloride treatment have a cumulative effect (n≥25 cells, n≥9 Dendra-tau per treatment group). Error bars are mean ±s.d. Note that i and ii are distinct experiments.


( a ) rho : : GFP - tau fishes were incubated from 3 to 9 d . p . f . in either dimethyl sulphoxide ( DMSO ) or rapamycin ( i ) or from 3 to 7 d . p . f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin ( ii ) . Images through the central retina at 9 d . p . f . ( top panel ; ( i ) ) reveal rod degeneration ( arrow ) throughout the retina in control ( DMSO - treated ) larvae , whereas rod photoreceptors are present throughout the retina , particularly in the central region following treatment with rapamycin ( arrow ) . Images taken through the central retina at 7 d . p . f . ( bottom panel ; ( ii ) ) show normal photoreceptors in the marginal zones ( arrows ) and only limited numbers in the central region . NH4Cl exacerbates degeneration - photoreceptors are absent from the central retina and reduced / absent from marginal zones ( arrows ) . Sections were stained with anti - rhodopsin ( 1D1 ) antibody . GFP labels whole rod photoreceptors , whereas rhodopsin is present in the rod outer segment . GFP co - localizes with the red rhodopsin label in all experimental conditions . Scale bars , 50 μm . Quantification of rodphotoreceptor degeneration ( n = 10 larvae per group ; * * * P0 . 001 ; # # # P0 . 001 , two - tailed unpaired t - test ) . Error bars are mean ± s . 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O 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 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 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

(a) rho::GFP-tau fishes were incubated from 3 to 9 d.p.f. in either dimethyl sulphoxide (DMSO) or rapamycin (i) or from 3 to 7 d.p.f in EM alone or 10 mM ammonium chloride or 100 nM Wortmannin (ii). Images through the central retina at 9 d.p.f. (top panel; (i)) reveal rod degeneration (arrow) throughout the retina in control (DMSO-treated) larvae, whereas rod photoreceptors are present throughout the retina, particularly in the central region following treatment with rapamycin (arrow). Images taken through the central retina at 7 d.p.f. (bottom panel; (ii)) show normal photoreceptors in the marginal zones (arrows) and only limited numbers in the central region. NH4Cl exacerbates degeneration-photoreceptors are absent from the central retina and reduced/absent from marginal zones (arrows). Sections were stained with anti-rhodopsin (1D1) antibody. GFP labels whole rod photoreceptors, whereas rhodopsin is present in the rod outer segment. GFP co-localizes with the red rhodopsin label in all experimental conditions. Scale bars, 50 μm. Quantification of rodphotoreceptor degeneration (n=10 larvae per group; ***P0.001; ###P0.001, two-tailed unpaired t-test). Error bars are mean ±s.d.


( b ) Full - length CALM electroporation into rho : : GFP larvae did not cause degeneration . , while full - length CALM electroporation into rho : : GFP - tau larvae exacerbated photoreceptor degeneration . Central retina sections in the region of the optic nerve head are presented . Scale bars , 50 μm . Data represent the ratio ( in % ) of the intensity of the GFP signal between CALM - HA electroporated eye versus control eye for five individuals per transgenic line . * * * P0 . 05 ; two - tailed t - test . Error bars are mean ± s . 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 O O O O O O O O O O O O O O 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) Full-length CALM electroporation into rho::GFP larvae did not cause degeneration., while full-length CALM electroporation into rho::GFP-tau larvae exacerbated photoreceptor degeneration. Central retina sections in the region of the optic nerve head are presented. Scale bars, 50 μm. Data represent the ratio (in %) of the intensity of the GFP signal between CALM-HA electroporated eye versus control eye for five individuals per transgenic line. ***P0.05; two-tailed t-test. Error bars are mean ±s.d.


( c ) NH4Cl treatment of rho : : GFP - tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control ( non - electroporated ) side but did not alter the degeneration caused by CALM electroporation . Rapamycin treatment of Rapamycin : : GFP - tau immediately after unilateral CALM electroporation rescued photoreceptors on the control ( non - electroporated ) side but not on the CALM - electroporated side . To demonstrate that loss of GFP corresponds to loss of photoreceptors , sections were stained with anti - rhodopsin ( 1D1 ) antibody . Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish ; Mann - Whitney test was used to compare drug treatment in the right eye of different fishes . * P0 . 05 . Scale bars , 50 μm . Error bars are mean ± s . e . m .

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

(c) NH4Cl treatment of rho::GFP-tau immediately after unilateral CALM electroporation caused photoreceptor degenerationon the control (non-electroporated) side but did not alter the degeneration caused by CALM electroporation. Rapamycin treatment of Rapamycin::GFP-tau immediately after unilateral CALM electroporation rescued photoreceptors on the control (non-electroporated) side but not on the CALM-electroporated side. To demonstrate that loss of GFP corresponds to loss of photoreceptors, sections were stained with anti-rhodopsin (1D1) antibody. Wilcoxon signed rank test was used to compare the left eye versus the right eye of the same fish; Mann-Whitney test was used to compare drug treatment in the right eye of different fishes. *P0.05. Scale bars, 50 μm. Error bars are mean ±s.e.m.


( a ) Histological section to demonstrate the individual cell layers of the zebrafish retina . The photoreceptor layer ( PR , comprising rod and cone photoreceptors ) lies immediately adjacent to the retinal pigment epithelium ( RPE ) at the outermost surface of the eye . Thioflavin - S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer ( marked with yellow dotted lines ) . No labelling was observed in the retina of rho : : GFP at 8 d . p . f . , whereas distinct thioflavin - S - positive tangles ( arrows ) were observed in the photoreceptor layer of rho : : GFP - tau fish . Note , the RPE is highly autofluorescent due to the presence of silver pigment . High power regions are shown in the top right of each panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Histological section to demonstrate the individual cell layers of the zebrafish retina. The photoreceptor layer (PR, comprising rod and cone photoreceptors) lies immediately adjacent to the retinal pigment epithelium (RPE) at the outermost surface of the eye. Thioflavin-S labelling of retinal sections was used to identify neurofibrillary tangles in the photoreceptor layer (marked with yellow dotted lines). No labelling was observed in the retina of rho::GFP at 8 d.p.f., whereas distinct thioflavin-S-positive tangles (arrows) were observed in the photoreceptor layer of rho::GFP-tau fish. Note, the RPE is highly autofluorescent due to the presence of silver pigment. High power regions are shown in the top right of each panel.


( b ) Unilateral electroporation of CALM into the retina of rho : : GFP - tau zebrafish resulted in a marked increase in thioflavin - S positive tangles in the electroporated retina in the photoreceptor layer ( PR ) compared with the control side . Top panel are lower magnification images to show the retinal cell layers . Thioflavin - S labelling is restricted to the PR layer . Note the RPE is highly autofluorescent due to the presence of silver pigment . Bottom panel are higher magnification images to show individual thioflavin - S tangles the largest of which are indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Unilateral electroporation of CALM into the retina of rho::GFP-tau zebrafish resulted in a marked increase in thioflavin-S positive tangles in the electroporated retina in the photoreceptor layer (PR) compared with the control side. Top panel are lower magnification images to show the retinal cell layers. Thioflavin-S labelling is restricted to the PR layer. Note the RPE is highly autofluorescent due to the presence of silver pigment. Bottom panel are higher magnification images to show individual thioflavin-S tangles the largest of which are indicated by arrows.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA for non - acclimation , − 5 ° C , 30 min ; CA for cold acclimation , − 8 ° C , 40 min ) .

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

survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA for non-acclimation, −5°C, 30 min; CA for cold acclimation, −8°C, 40 min).


Expression of CBFs ( D ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 3 h ( D ) and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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

Expression of CBFs (D) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 3 h (D) and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


Expression of CBFs targets ( E ) in Col - 0 , atann1 , and atann1 AtANN1 ( # 10 ) plants . Twelve - day - old plants grown on MS medium at 22 ° C were placed at 4 ° C for 24 h ( E ) , and total RNA was extracted and subjected to qRT - PCR analysis . Relative transcript levels in untreated Col - 0 plants were set to 1 .

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

Expression of CBFs targets (E) in Col-0, atann1, and atann1 AtANN1 (#10) plants. Twelve-day-old plants grown on MS medium at 22°C were placed at 4°C for 24 h (E), and total RNA was extracted and subjected to qRT-PCR analysis. Relative transcript levels in untreated Col-0 plants were set to 1.


A Time - course analysis of cytosolic free calcium concentration ( [ Ca2 + ] cyt ) dynamics in 10 - d - old wild type ( Col - 0 ) , atann1 , and atann1 AtANN1 complementation line ( # 10 ) after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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

A Time-course analysis of cytosolic free calcium concentration ([Ca2+]cyt) dynamics in 10-d-old wild type (Col-0), atann1, and atann1 AtANN1 complementation line (#10) after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C GST pull - down assay showing that OST1 interacts with AtANN1 in vitro . Purified recombinant GST - OST1 , GST - OST1G33R , or GST proteins from E . coli were immunoprecipitated with GST beads and then incubated with MBP - His - AtANN1 . Precipitated proteins were detected with anti - GST and anti - His 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

C GST pull-down assay showing that OST1 interacts with AtANN1 in vitro. Purified recombinant GST-OST1, GST-OST1G33R, or GST proteins from E. coli were immunoprecipitated with GST beads and then incubated with MBP-His-AtANN1. Precipitated proteins were detected with anti-GST and anti-His antibodies.


D Interaction of OST1 and AtANN1 detected by bimolecular fluorescence complementation ( BiFC ) assays . The construct combinations were co - transformed into N . benthamiana leaves and expressed for 2 d . The signal was detected by confocal microscopy . Scale bar , 25 µm . The fluorescence intensity was scanned using the ImageJ plot profile tool . The y - axes indicate relative pixel intensity .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 OST1 and AtANN1 detected by bimolecular fluorescence complementation (BiFC) assays. The construct combinations were co-transformed into N. benthamiana leaves and expressed for 2 d. The signal was detected by confocal microscopy. Scale bar, 25 µm. The fluorescence intensity was scanned using the ImageJ plot profile tool. The y-axes indicate relative pixel intensity.


E , F Interaction of OST1 and AtANN1 detected by Co - IP assays in Arabidopsis . Twelve - day - old OST1 - Myc overexpressing plants grown on MS medium at 22 ° C were placed at 4 ° C for 0 , 0 . 5 , 2 h , and total proteins were extracted and immunoprecipitated with anti - Myc agarose beads . The wild type ( Col - 0 ) treated with 4 ° C for 2 h was used as control . The OST1 - Myc protein was detected with anti - Myc antibody and the AtANN1 protein was detected with anti - AtANN1 antibody . Representative pictures are shown in ( E ) and relative protein level in ( F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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,F Interaction of OST1 and AtANN1 detected by Co-IP assays in Arabidopsis. Twelve-day-old OST1-Myc overexpressing plants grown on MS medium at 22°C were placed at 4°C for 0, 0.5, 2 h, and total proteins were extracted and immunoprecipitated with anti-Myc agarose beads. The wild type (Col-0) treated with 4°C for 2 h was used as control. The OST1-Myc protein was detected with anti-Myc antibody and the AtANN1 protein was detected with anti-AtANN1 antibody. Representative pictures are shown in (E) and relative protein level in (F).


G Co - IP assay showing the interaction between OST1 and AtANN1 in vivo . The construct combinations were expressed in N . benthamiana leaves . Total proteins were extracted after the N . benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti - Myc agarose beads . The proteins were detected with anti - Myc and anti - GFP 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

G Co-IP assay showing the interaction between OST1 and AtANN1 in vivo. The construct combinations were expressed in N. benthamiana leaves. Total proteins were extracted after the N. benthamiana leaves treated with 4oC or 22oC and immunoprecipitated with anti-Myc agarose beads. The proteins were detected with anti-Myc and anti-GFP antibodies.


Freezing phenotype ( A ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

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

Freezing phenotype (A) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) and ion leakage ( C ) of Col - 0 , ost1 - 3 , atann1 , and atann1 ost1 - 3 . Twelve - day - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

survival rate (B) and ion leakage (C) of Col-0, ost1-3, atann1, and atann1 ost1-3. Twelve-day-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


OST1 gene expression D ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 .

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

OST1 gene expression D) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1.


protein levels of OST1 and AtANN1 ( E ) in Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . OST1 and AtANN1 proteins were detected with anti - Myc and anti - AtANN1 antibodies . HSP90 was used as a 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 O O O O O O

protein levels of OST1 and AtANN1 (E) in Col-0, OST1-Myc, atann1 and OST1-Myc atann1. OST1 and AtANN1 proteins were detected with anti-Myc and anti-AtANN1 antibodies. HSP90 was used as a control.


Freezing phenotype ( F ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) . Representative pictures are shown in ( F ) .

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

Freezing phenotype (F) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min). Representative pictures are shown in (F).


survival rate ( G ) , and ion leakage ( H ) of Col - 0 , OST1 - Myc , atann1 and OST1 - Myc atann1 . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , - 5 ° C , 30 min ; CA , - 8 ° C , 40 min ) .

survival rate (G), and ion leakage (H) of Col-0, OST1-Myc, atann1 and OST1-Myc atann1. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, -5°C, 30 min; CA, -8°C, 40 min).


A , B OST1 phosphorylates AtANN1 ( A ) and AtANN4 ( B ) in vitro . Purified recombinant MBP - His - OST1 was incubated with GST - AtANN1 , GST - AtANN4 or GST in kinase reaction buffer with 1 µCi [ γ - 32P ] ATP for 30 min at 30 ° C , followed by separation with SDS - PAGE . Phosphorylated AtANN1 and AtANN4 were detected by autoradiography . Recombinant OST1 , AtANN1 and AtANN4 were stained by Coomassie brilliant blue ( CBB ) .

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A,B OST1 phosphorylates AtANN1 (A) and AtANN4 (B) in vitro. Purified recombinant MBP-His-OST1 was incubated with GST-AtANN1, GST-AtANN4 or GST in kinase reaction buffer with 1 µCi [γ-32P] ATP for 30 min at 30°C, followed by separation with SDS-PAGE. Phosphorylated AtANN1 and AtANN4 were detected by autoradiography. Recombinant OST1, AtANN1 and AtANN4 were stained by Coomassie brilliant blue (CBB).


C In - gel kinase assays of OST1 in Col - 0 and ost1 - 3 mutant under cold stress . Twelve - day - old Col - 0 and ost1 - 3 mutant were treated at 4 ° C for 2 h . Total protein extracts were prepared and separated on a SDS - PAGE gel containing 0 . 2 mg / mL GST - AtANN1 as a substrate , and incubated with 70 µCi [ γ - 32P ] ATP . Top , autoradiograph ; bottom , CBB staining .

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C In-gel kinase assays of OST1 in Col-0 and ost1-3 mutant under cold stress. Twelve-day-old Col-0 and ost1-3 mutant were treated at 4°C for 2 h. Total protein extracts were prepared and separated on a SDS-PAGE gel containing 0.2 mg/mL GST-AtANN1 as a substrate, and incubated with 70 µCi [γ-32P] ATP. Top, autoradiograph; bottom, CBB staining.


D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1 . After phosphorylation , the proteins were separated by SDS - PAGE and subjected to autoradiography . Top , autoradiograph ; bottom , CBB staining .

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D In vitro kinase analysis of the indicated mutant forms of AtANN1 by OST1. After phosphorylation, the proteins were separated by SDS-PAGE and subjected to autoradiography. Top, autoradiograph; bottom, CBB staining.


E OST1 phosphorylates AtANN1 at S289 in vivo in LC / MS analysis . Total proteins were extracted from 12 - d - old AtANN1 - Myc overexpressing plants treated at 4 ° C for 0 , 10 , 30 and 120 min , followed by trypsin digestion . Phosphopeptides were enriched for mass spectrometry analysis . Representative picture for 10 min was shown .

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E OST1 phosphorylates AtANN1 at S289 in vivo in LC/MS analysis. Total proteins were extracted from 12-d-old AtANN1-Myc overexpressing plants treated at 4°C for 0, 10, 30 and 120 min, followed by trypsin digestion. Phosphopeptides were enriched for mass spectrometry analysis. Representative picture for 10 min was shown.


Freezing phenotype ( A ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( A ) .

Freezing phenotype (A) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (A).


survival rate ( B ) , and ion leakage ( C ) of Col - 0 , atann1 , and atann1 AtANN1S289E ( # 5 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

survival rate (B), and ion leakage (C) of Col-0, atann1, and atann1 AtANN1S289E (#5) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


Freezing phenotype ( D ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) . Representative pictures are shown in ( D ) .

Freezing phenotype (D) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min). Representative pictures are shown in (D).


survival rate ( E ) , and ion leakage ( F ) of Col - 0 , Atann1 , and atann1 AtANN1S289A ( # 16 ) plants . 12 - d - old plants grown on MS medium at 22 ° C were exposed to freezing temperatures ( NA , − 5 ° C , 30 min ; CA , − 8 ° C , 40 min ) .

survival rate (E), and ion leakage (F) of Col-0, Atann1, and atann1 AtANN1S289A (#16) plants. 12-d-old plants grown on MS medium at 22°C were exposed to freezing temperatures (NA, −5°C, 30 min; CA, −8°C, 40 min).


A AtANN1 current recordings in Xenopus oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , OST1 , or AtANN1 + OST1 cRNA . Bath solution was described in Materials and Methods . The voltage protocol as well as time and current scale bars for the recordings are shown . B Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( A ) . Negative current is influx of cations into the oocytes .

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A AtANN1 current recordings in Xenopus oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, OST1, or AtANN1 + OST1 cRNA. Bath solution was described in Materials and Methods. The voltage protocol as well as time and current scale bars for the recordings are shown. B Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (A). Negative current is influx of cations into the oocytes.


C Percentage of total Xenophus oocytes in different nA range according to ( A ) .

C Percentage of total Xenophus oocytes in different nA range according to (A).


D AtANN1 current recordings in oocytes . Whole - cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1 , AtANN1S289A , OST1 , AtANN1 + OST1 , and AtANN1S289A + OST1 cRNA . E Current - voltage ( I - V ) relationship of the steady - state whole - cell currents in Xenopus oocytes described in ( D ) .

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D AtANN1 current recordings in oocytes. Whole-cell currents were recorded in Xenopus oocytes injected with ddH2O or with AtANN1, AtANN1S289A, OST1, AtANN1 + OST1, and AtANN1S289A + OST1 cRNA. E Current-voltage (I-V) relationship of the steady-state whole-cell currents in Xenopus oocytes described in (D).


F Percentage of total Xenophus oocytes in different nA range according to ( D ) .

F Percentage of total Xenophus oocytes in different nA range according to (D).


A Time - course analysis of [ Ca2 + ] cyt dynamics between 10 - day - old wild type ( Col - 0 ) and ost1 - 3 mutant after treatment with ice - cold water or ice - cold water containing 10 mM LaCl3 ( arrow shows the time point of treatment ) . Luminescence was recorded at 1 - s intervals . B Quantification of the cold - induced [ Ca2 + ] cyt changes shown in ( A ) . Peak [ Ca2 + ] cyt indicates the highest [ Ca2 + ] cyt after treatment .

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A Time-course analysis of [Ca2+]cyt dynamics between 10-day-old wild type (Col-0) and ost1-3 mutant after treatment with ice-cold water or ice-cold water containing 10 mM LaCl3 (arrow shows the time point of treatment). Luminescence was recorded at 1-s intervals. B Quantification of the cold-induced [Ca2+]cyt changes shown in (A). Peak [Ca2+]cyt indicates the highest [Ca2+]cyt after treatment.


C MST assays of the calcium - binding affinity of GST - AtANN1 , GST - AtANN1S289A , and GST - AtANN1S289E . GST was used as a control .

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C MST assays of the calcium-binding affinity of GST-AtANN1, GST-AtANN1S289A, and GST-AtANN1S289E. GST was used as a control.


3 ‐ month ‐ old wild ‐ type ( C57BL / 6 ) , Ltbr − / − , Lta − / − , LIGHT − / − , and Ltbr − / − Hox11 − / − mice ; percentage of mice with at least one ANA .

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3‐month‐old wild‐type (C57BL/6), Ltbr−/−, Lta−/−, LIGHT−/−, and Ltbr−/−Hox11−/− mice; percentage of mice with at least one ANA.


6 ‐ month ‐ old Ltbr − / − mice ( n = 15 ) ; titers of individual mice .

6‐month‐old Ltbr−/− mice (n = 15); titers of individual mice.


LIA of 6 ‐ month ‐ old Ltbr − / − mice , wild ‐ type mice , and 3 ‐ month ‐ old Rorγt ‐ Ltb − / − mice .

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LIA of 6‐month‐old Ltbr−/− mice, wild‐type mice, and 3‐month‐old Rorγt‐Ltb−/− mice.


LTβR ‐ Fc or control immunoglobulin ( Ig ) ‐ treated wild ‐ type mice ; percentage of mice with at least one ANA . E denotes gestational day . * ANA were determined with LIA at the age of 3 months . * * ANA were determined with LIA 3 months after the start of treatment .

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LTβR‐Fc or control immunoglobulin (Ig)‐treated wild‐type mice; percentage of mice with at least one ANA. E denotes gestational day. *ANA were determined with LIA at the age of 3 months. **ANA were determined with LIA 3 months after the start of treatment.


3 ‐ month ‐ old cell ‐ specific LT knockout mice ; percentage of mice with at least one ANA .

3‐month‐old cell‐specific LT knockout mice; percentage of mice with at least one ANA.


Bone marrow cells from wild ‐ type or Ltbr − / − mice were transferred at neonatal age into lethally irradiated wild ‐ type or Ltbr − / − mice ; percentage of mice with at least one ANA . ANA were determined with LIA at the age of 3 months .

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Bone marrow cells from wild‐type or Ltbr−/− mice were transferred at neonatal age into lethally irradiated wild‐type or Ltbr−/− mice; percentage of mice with at least one ANA. ANA were determined with LIA at the age of 3 months.


Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages . Numbers represent the percentages within the indicated regions ( left panel ) . No significant differences were found . Data represent means ± s . e . m .

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Thymic reconstitution was analyzed by counting total cell number and flow cytometry for cell percentages. Numbers represent the percentages within the indicated regions (left panel). No significant differences were found. Data represent means ± s.e.m.


T ‐ cell reconstitution of spleen was assessed by flow cytometry . Numbers represent the percentages within the indicated regions .

T‐cell reconstitution of spleen was assessed by flow cytometry. Numbers represent the percentages within the indicated regions.


Total IgG from nude mice engrafted with Ltbr − / − or wild ‐ type thymi was determined by ELISA . No significant differences were found . Data represent means ± s . e . m .

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Total IgG from nude mice engrafted with Ltbr−/− or wild‐type thymi was determined by ELISA. No significant differences were found. Data represent means ± s.e.m.


Sera were collected 3 months after transplantation and tested for ANA ; percentage of mice with at least one ANA reactivity .

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Sera were collected 3 months after transplantation and tested for ANA; percentage of mice with at least one ANA reactivity.


Ltbr − / − mice were treated or not with antibiotics from birth ( upper panel ) , conventionalized or germfree wild ‐ type mice were treated with the LTβR ‐ Fc fusion protein from gestational day 18 until 6 weeks after birth ( lower panel ) ; percentage of mice with at least one ANA . ANA were tested with LIA at the age of 3 months .

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Ltbr−/− mice were treated or not with antibiotics from birth (upper panel), conventionalized or germfree wild‐type mice were treated with the LTβR‐Fc fusion protein from gestational day 18 until 6 weeks after birth (lower panel); percentage of mice with at least one ANA. ANA were tested with LIA at the age of 3 months.


Real ‐ time PCR for SFB in luminal , mucosal , and fecal samples of multiple ANA ‐ positive and ANA ‐ negative Ltbr − / − mice . Analysis with ANOVA , P = 0 . 02 for differences between groups , n = 4 mice per group . Data represent means ± s . e . m .

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Real‐time PCR for SFB in luminal, mucosal, and fecal samples of multiple ANA‐positive and ANA‐negative Ltbr−/− mice. Analysis with ANOVA, P = 0.02 for differences between groups, n = 4 mice per group. Data represent means ± s.e.m.


C3H / HeN germfree pregnant mice were treated with the LTβR ‐ Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring . Pups were also treated in the neonatal period to prevent the development of ILF . Mice were colonized at 8 weeks of age with SFB or E . coli and were sacrificed on d20 or on d60 postcolonization ; percentage of mice with at least one ANA . E denotes gestational day .

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C3H/HeN germfree pregnant mice were treated with the LTβR‐Fc fusion protein on day 16 and 18 of gestation to prevent the development of PP in their offspring. Pups were also treated in the neonatal period to prevent the development of ILF. Mice were colonized at 8 weeks of age with SFB or E. coli and were sacrificed on d20 or on d60 postcolonization; percentage of mice with at least one ANA. E denotes gestational day.


LTβR ‐ Fc ‐ treated wild ‐ type or IL17R − / − mice ; percentage of mice with at least one ANA . E denotes gestational day . ANA were determined with LIA at the age of 3

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LTβR‐Fc‐treated wild‐type or IL17R−/− mice; percentage of mice with at least one ANA. E denotes gestational day. ANA were determined with LIA at the age of 3 months


Effects of ActA and BMP4 on the expression of selected pluripotency , PGC , and mesodermal genes during PGC ‐ precursor induction . DM : dorsomorphin , SB : SB431542 . Samples were calibrated with iPSC values , and iPSC values depict 1 . Y ‐ axes are in log2 scale .

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Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale.


Gene expression dynamics of selected pluripotency , PGC , and mesodermal genes during prolonged PGC ‐ precursor induction of up to day 7 . Y ‐ axes are in log2 scale .

Gene expression dynamics of selected pluripotency, PGC, and mesodermal genes during prolonged PGC‐precursor induction of up to day 7. Y‐axes are in log2 scale.


Immunofluorescence analysis for OCT4 ( red ) , BLIMP1 ( green ) , and T ( white ) on days 0 , 1 , and 2 . Nuclei were stained with Hoechst ( blue ) . The culture contained BMP4 ( 5 ng / ml ) , Activin A ( 50 ng / ml ) , and bFGF ( 20 ng / ml ) . Scale bar : 100 μm .

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Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm.


FACS analysis of the concentration ‐ dependent effect of BMP4 on TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 4 . B25 : 25 ng / ml BMP4 ; B50 : 50 ng / ml BMP4 ; B100 : 100 ng / ml BMP4 .

FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4.


FACS analysis of TRA ‐ 1 ‐ 81 and cKIT during PGC induction of up to day 8 .

FACS analysis of TRA‐1‐81 and cKIT during PGC induction of up to day 8.


Gene expression analysis of selected PGC markers in TRA ‐ 1 ‐ 81 + / cKIT + and SSEA1 + / cKIT + FACS fractions of d4 PGCLCs . Samples were calibrated with iPSC values , and iPSC values depict 1 .

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Gene expression analysis of selected PGC markers in TRA‐1‐81+/cKIT+ and SSEA1+/cKIT+ FACS fractions of d4 PGCLCs. Samples were calibrated with iPSC values, and iPSC values depict 1.


Gene expression dynamics of selected pluripotency and PGC genes in FACS ‐ sorted , specified cells during PGC induction . The value for iPSCs is set as 1 , and values are on log2 scale .

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Gene expression dynamics of selected pluripotency and PGC genes in FACS‐sorted, specified cells during PGC induction. The value for iPSCs is set as 1, and values are on log2 scale.


Immunofluorescence analysis for OCT4 ( green ) , BLIMP1 ( red ) , and STELLA ( yellow ) in TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 6 . Nuclei were stained with Hoechst ( blue ) . Scale bar : 15 μm .

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Immunofluorescence analysis for OCT4 (green), BLIMP1 (red), and STELLA (yellow) in TRA‐1‐81+/cKIT+ PGCLCs on day 6. Nuclei were stained with Hoechst (blue). Scale bar: 15 μm.


Bisulfite sequence analysis of DMRs of the imprinted genes ( PEG1 , KvDMR1 , PEG10 , and NESP55 ) in iPSCs ( top ) and d6 TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs ( bottom ) . White and black circles represent unmethylated and methylated CpG sequences , respectively .

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Bisulfite sequence analysis of DMRs of the imprinted genes (PEG1, KvDMR1, PEG10, and NESP55) in iPSCs (top) and d6 TRA‐1‐81+/cKIT+ PGCLCs (bottom). White and black circles represent unmethylated and methylated CpG sequences, respectively.


Immunofluorescence analysis for 5mC in d6 PGCLCs . Nuclei were stained with Hoechst ( blue ) . Scale bar : 50 μm .

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Immunofluorescence analysis for 5mC in d6 PGCLCs.Nuclei were stained with Hoechst (blue). Scale bar: 50 μm.


Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice . ( i ) left ovary : control ovary without transplantation ; right ovary : teratoma from iPSC ‐ reconstituted ovary ; ( ii ) PGCLC ‐ reconstituted ovaries . Note that no teratomas had formed . Bottom panels : Immunohistochemical analysis for NUMA in sections of iPSC ‐ and PGCLC ‐ reconstituted ovaries . ( iii ) iPSC ‐ induced teratoma shown in ( i ) ; ( iv ) ovary containing NUMA + cells shown in ( ii ) . Insert : higher magnification of selected area . Scale bars : ( i ) and ( ii ) , 0 . 5 cm ; ( iii ) and ( iv ) , 50 μm .

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Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice. (i) left ovary: control ovary without transplantation; right ovary: teratoma from iPSC‐reconstituted ovary; (ii) PGCLC‐reconstituted ovaries. Note that no teratomas had formed. Bottom panels: Immunohistochemical analysis for NUMA in sections of iPSC‐ and PGCLC‐reconstituted ovaries. (iii) iPSC‐induced teratoma shown in (i); (iv) ovary containing NUMA+ cells shown in (ii). Insert: higher magnification of selected area. Scale bars: (i) and (ii), 0.5 cm; (iii) and (iv), 50 μm.


Effects of KSR and WNT3A during PGC ‐ precursor induction on the expression of selected pluripotency , PGC , and a mesodermal gene of d2 cultures . The value for iPSCs is set as 1 , and values are on log2 scale . Data are presented as means ± SD ( n = 3 ) .

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Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3).


Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA ‐ 1 ‐ 81 and cKIT .

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Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT.


Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0 % ( control ) or 20 % KSR ( KSR ) condition during PGC ‐ precursor induction . Samples were calibrated with iPSC values , and iPSC values depict 1 . Data are presented as means ± SD ( n = 3 ) .

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Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3).


Immunofluorescence analysis for OCT4 ( red ) , BLIMP1 ( green ) , and T ( yellow ) in d2 precursors cultured in increasing KSR concentrations . Nuclei were stained with Hoechst ( blue ) . Scale bar : 100 μm .

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Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.


Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction . Knockdown of BLIMP1 ( F ) and PRDM14 ( G ) was done using the lentiviral system with two individual shRNA vectors for each gene . The knockdown efficiencies were assessed by qPCR ( left panel , each ) . The induction of d4 PGCLCs analyzed by FACS , gated for TRA ‐ 1 ‐ 81 and cKIT ( right panel , each ) . Data are presented as means ± SD ( n = 3 ) .

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Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction. Knockdown of BLIMP1 (F) and PRDM14 (G) was done using the lentiviral system with two individual shRNA vectors for each gene. The knockdown efficiencies were assessed by qPCR (left panel, each). The induction of d4 PGCLCs analyzed by FACS, gated for TRA‐1‐81 and cKIT (right panel, each). Data are presented as means ± SD (n = 3).


( A ) NSCLC expressing single site mutants of EGFR , PC9 ( 3X106 ) or H3255 ( 8X106 ) , were seeded in 10 - cm dishes . On the next day , complete media were replaced with media containing serum ( 1 % ) and the cells were treated for 24 hours with different EGFR - specific TKIs ( erlotinib , 50 nM ; osimertinib , 50 nM , or afatinib , 10 nM ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , 5 μg / ml each ) . Thereafter , cells were washed with cold saline and extracted . Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes . After blocking , membranes were incubated overnight with the indicated primary antibodies , followed by incubation with horseradish peroxidase - conjugated secondary antibodies ( 60 minutes ) , and treatment with Clarity ™ Western ECL Blotting Substrates ( Bio - Rad ) . ECL signals were detected using the ChemiDoc ™ Imaging System ( Bio - Rad ) and images were acquired using the ImageLab software . Signals ( relative to Control ) were quantified and normalized to the signals of GAPDH ( numbers shown below each lane ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NSCLC expressing single site mutants of EGFR, PC9 (3X106) or H3255 (8X106), were seeded in 10-cm dishes. On the next day, complete media were replaced with media containing serum (1%) and the cells were treated for 24 hours with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). Thereafter, cells were washed with cold saline and extracted. Proteins were separated using gel electrophoresis and transferred onto nitrocellulose membranes. After blocking, membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (60 minutes), and treatment with Clarity™ Western ECL Blotting Substrates (Bio-Rad). ECL signals were detected using the ChemiDoc™ Imaging System (Bio-Rad) and images were acquired using the ImageLab software. Signals (relative to Control) were quantified and normalized to the signals of GAPDH (numbers shown below each lane).


with different EGFR - specific TKIs ( erlotinib , 50 nM ; osimertinib , 50 nM , or afatinib , 10 nM ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , 5 μg / ml each ) . ( C ) PC9 cells were seeded on coverslips and treated for 24 hours Cells were washed in acidic buffer , fixed in paraformaldehyde ( 4 % ) and incubated with specific primary antibodies , followed by an Alexa Fluor 555 - conjugated secondary antibody ( pseudo colored in green ) . DAPI ( blue ) was used to stain nuclei . Images were captured using a confocal microscope ( 40X magnification ) .

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with different EGFR-specific TKIs (erlotinib, 50 nM; osimertinib, 50 nM, or afatinib, 10 nM), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, 5 μg/ml each). (C) PC9 cells were seeded on coverslips and treated for 24 hours Cells were washed in acidic buffer, fixed in paraformaldehyde (4%) and incubated with specific primary antibodies, followed by an Alexa Fluor 555-conjugated secondary antibody (pseudo colored in green). DAPI (blue) was used to stain nuclei. Images were captured using a confocal microscope (40X magnification).


PC9 cells ( 3X106 / mouse ) were subcutaneously implanted in the flanks of CD1 - nu / nu mice . When tumors became palpable , mice were randomized in groups of 5 - 9 animals and treated for 90 days ( grey areas ) with 2XmAbs ( cetuximab plus trastuzumab , 0 . 2 mg / mouse / injection ) , once every three days , or with erlotinib ( 50 mg / kg / day ) , once per day . Alternatively , mice were treated with combinations of erlotinib ( 50 , 20 , 10 , 5 or 1 mg / kg ) and the two monoclonal antibodies . Following 60 days of treatment reduced the frequency of erlotinib administration to once every other day ( underneath dotted line ) . animal survival ( B ) are shown . Mice were euthanized when tumor size reached 1 , 500 mm3 .

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PC9 cells (3X106/mouse) were subcutaneously implanted in the flanks of CD1-nu/nu mice. When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, 0.2 mg/mouse/injection), once every three days, or with erlotinib (50 mg/kg/day), once per day. Alternatively, mice were treated with combinations of erlotinib (50, 20, 10, 5 or 1 mg/kg) and the two monoclonal antibodies. Following 60 days of treatment reduced the frequency of erlotinib administration to once every other day (underneath dotted line). animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.


PC9 cells ( exon 19 deletion ) were subcutaneously implanted in the flank of CD1 - nu / nu mice ( 3X106 / mouse ) . When tumors became palpable , mice were randomized in groups of 5 - 9 animals and treated for 90 days ( grey areas ) with 2XmAbs ( cetuximab plus trastuzumab , each at 0 . 1 mg / mouse / injection ) once every three days , or daily with different TKIs : osimertinib ( 5 mg / kg ) , afatinib ( 5 mg / kg ) or imatinib ( 100 mg / kg ) , either alone or in combination with the two antibodies . Following 60 days of treatment , the frequency of TKI administration was reduced to once every other day ( underneath dotted lines ) , while mAb treatment remained unaltered . animal survival ( B ) are shown . Mice were euthanized when tumor size reached 1 , 500 mm3 .

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PC9 cells (exon 19 deletion) were subcutaneously implanted in the flank of CD1-nu/nu mice (3X106/mouse). When tumors became palpable, mice were randomized in groups of 5-9 animals and treated for 90 days (grey areas) with 2XmAbs (cetuximab plus trastuzumab, each at 0.1 mg/mouse/injection) once every three days, or daily with different TKIs: osimertinib (5 mg/kg), afatinib (5 mg/kg) or imatinib (100 mg/kg), either alone or in combination with the two antibodies. Following 60 days of treatment, the frequency of TKI administration was reduced to once every other day (underneath dotted lines), while mAb treatment remained unaltered. animal survival (B) are shown. Mice were euthanized when tumor size reached 1,500 mm3.


CD1 - nu / nu mice carrying PC9 xenografts were divided in groups ( 2 - 3 mice / group ) and treated for seven days with different TKIs : erlotinib ( 50 mg / kg or 20 mg / kg ) , osimertinib ( 5 mg / kg ) or afatinib ( 5 mg / kg ) , either alone or in combination with 2XmAbs ( cetuximab and trastuzumab , each at 0 . 1 mg / mouse / injection ) . when singly applied used erlotinib at 50 mg / kg . ( B ) After treatment , all mice were sacrificed and tumors extracted . Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes , which were later incubated overnight with the indicated antibodies . This was followed by incubation with peroxidase - conjugated secondary antibodies . Signals were detected using ChemidocTM ( from Bio - Rad ) , quantified and normalized to the signals of GAPDH or tubulin ( numbers shown below each lane ) .

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

CD1-nu/nu mice carrying PC9 xenografts were divided in groups (2-3 mice/group) and treated for seven days with different TKIs: erlotinib (50 mg/kg or 20 mg/kg), osimertinib (5 mg/kg) or afatinib (5 mg/kg), either alone or in combination with 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection). when singly applied used erlotinib at 50 mg/kg. (B) After treatment, all mice were sacrificed and tumors extracted. Protein extracts were resolved by means of electrophoresis and transfer to nitrocellulose membranes, which were later incubated overnight with the indicated antibodies. This was followed by incubation with peroxidase-conjugated secondary antibodies. Signals were detected using ChemidocTM (from Bio-Rad), quantified and normalized to the signals of GAPDH or tubulin (numbers shown below each lane).


A , B PC9 cells ( 3X106 / mouse ) were subcutaneously injected in the flanks of CD1 - nu / nu mice . When tumors reached a volume of approximately 500 mm3 , mice were treated daily with erlotinib ( 50 mg / kg , blue area ; panel A ) or with osimertinib ( 5 mg / kg , grey area ; panel B ) using oral gavage . Initially , all tumors displayed stable disease or they regressed , but eventually all started relapsing . Once relapsing tumors reached 800 mm3 , while under erlotinib or osimertinib treatment , we supplemented the treatment with a combination of 2XmAbs ( cetuximab and trastuzumab , each at 0 . 1 mg / mouse / injection ; twice a week ) plus either erlotinib ( 50 mg / kg ; A , red area ) or osimertinib ( 5 mg / kg ; B , orange area ) . Tumor volumes of individual mice are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of CD1-nu/nu mice. When tumors reached a volume of approximately 500 mm3, mice were treated daily with erlotinib (50 mg/kg, blue area; panel A) or with osimertinib (5 mg/kg, grey area; panel B) using oral gavage. Initially, all tumors displayed stable disease or they regressed, but eventually all started relapsing. Once relapsing tumors reached 800 mm3, while under erlotinib or osimertinib treatment, we supplemented the treatment with a combination of 2XmAbs (cetuximab and trastuzumab, each at 0.1 mg/mouse/injection; twice a week) plus either erlotinib (50 mg/kg; A, red area) or osimertinib (5 mg/kg; B, orange area). Tumor volumes of individual mice are shown.


A , B NSG mice were pre - implanted with tumor fragments derived from two different PDX models : ( A ) TM00199 ( L858R - EGFR ; from The Jackson Laboratory ) or ( B ) TM00193 ( E746 _ A750 del - EGFR ; from The Jackson Laboratory ) . Once tumors reached approximately 300 mm3 , mice were treated with monoclonal antibodies ( 2XmAbs , cetuximab plus trastuzumab , 200μg / injection ) twice a week , or with EGFR TKIs , erlotinib ( 50mg / kg ) or osimertinib ( 10mg / kg ) daily . Alternatively , mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib , for either 32 days ( A ) or 42 days ( B ) . Tumor growth was monitored twice a week . the antibodies were injected into the peritoneum and the TKIs were delivered orally . The figure shows tumor volumes of individual mice and the respective survival curves ( right hand panels ) . The grey areas mark time windows of animal treatment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B NSG mice were pre-implanted with tumor fragments derived from two different PDX models: (A) TM00199 (L858R-EGFR; from The Jackson Laboratory) or (B) TM00193 (E746_A750 del-EGFR; from The Jackson Laboratory). Once tumors reached approximately 300 mm3, mice were treated with monoclonal antibodies (2XmAbs, cetuximab plus trastuzumab, 200μg/injection) twice a week, or with EGFR TKIs, erlotinib (50mg/kg) or osimertinib (10mg/kg) daily. Alternatively, mice were treated with a combination of 2XmAbs and either erlotinib or osimertinib, for either 32 days (A) or 42 days (B). Tumor growth was monitored twice a week. the antibodies were injected into the peritoneum and the TKIs were delivered orally. The figure shows tumor volumes of individual mice and the respective survival curves (right hand panels). The grey areas mark time windows of animal treatment.


( B ) For RNAseq analyses , PC9 cells ( 3X106 / mouse ) were subcutaneously injected in the flanks of 13 CD1 - nu / nu mice . When tumors reached 500 mm3 , mice were treated daily with erlotinib ( 50 mg / kg ) . Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3 , we switched to a combination of 2XmAbs plus erlotinib ( 50 mg / kg ) . The respective tumor volumes were sacrificed when tumor volumes reached approximately 500 mm3 Mice responding to erlotinib ( N = 3 ) were sacrificed after one week of treatment Mice resistant to erlotinib ( N = 3 ) were sacrificed when tumor volumes reached 800 mm3 whereas mice responding to erlotinib + 2XmAbs ( N = 2 ) were sacrificed after one week of treatment RNA was extracted from all 13 tumors and utilized for RNA - seq analysis . Differentially expressed ( DE ) genes in the Erlotinib ( Er ) + 2XmAbs arm compared to the Control group are presented in the heatmap .

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(B) For RNAseq analyses, PC9 cells (3X106/mouse) were subcutaneously injected in the flanks of 13 CD1-nu/nu mice. When tumors reached 500 mm3, mice were treated daily with erlotinib (50 mg/kg). Once tumors that initially responded to erlotinib monotherapy started relapsing and reached 800 mm3, we switched to a combination of 2XmAbs plus erlotinib (50 mg/kg). The respective tumor volumes were sacrificed when tumor volumes reached approximately 500 mm3 Mice responding to erlotinib (N=3) were sacrificed after one week of treatment Mice resistant to erlotinib (N=3) were sacrificed when tumor volumes reached 800 mm3 whereas mice responding to erlotinib+2XmAbs (N=2) were sacrificed after one week of treatment RNA was extracted from all 13 tumors and utilized for RNA-seq analysis. Differentially expressed (DE) genes in the Erlotinib (Er)+2XmAbs arm compared to the Control group are presented in the heatmap.


( C ) Protein extracts were prepared from all tumors and analyzed using immunoblotting , as indicated . Signals were quantified and normalized to the signals of GAPDH ( numbers shown below each lane ) .

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(C) Protein extracts were prepared from all tumors and analyzed using immunoblotting, as indicated. Signals were quantified and normalized to the signals of GAPDH (numbers shown below each lane).


B ) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre - and postsynaptic markers , but not glia marker ( GFAP ) in the SN fraction . Cytosolic markers were depleted , while nuclear markers were barely detected in the SN ( n = 4 biological replicates , * * * p < 0 . 001 , * * p < 0 . 01 , * p < 0 . 05 ; one sample t - test ; error bars indicate SEM ) .

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B) Western blot on fractions obtained during SN preparation revealed the enrichment of both pre- and postsynaptic markers, but not glia marker (GFAP) in the SN fraction. Cytosolic markers were depleted, while nuclear markers were barely detected in the SN (n=4 biological replicates, *** p<0.001, ** p<0.01, * p<0.05; one sample t-test; error bars indicate SEM).


C ) Synaptoneurosomes were NMDAR - stimulated and incubated with radioactive 35S - methionine / cysteine mix for 15 - 120 min . After labeling , proteins were separated on SDS - PAGE and newly synthesized proteins in SN labeled with [ 35S ] were visualized by autoradiography ( left panel ) . To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol ( CAP , 50 μg / ml ) or with puromycin ( PURO , 3mM ) respectively ( right panel ) . After 1 hour after the stimulation , a significant increase in 35S - labelled proteins was observed ( n = 6 biological replicates , * * * p < 0 . 001 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) and the CAP treatment did not affect the overall levels of de novo synthetized proteins ( n = 6 biological replicates , ns ; p = 0 . 220 ) . In contrast , PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes ( n = 6 biological replicates , * * p = 0 . 0091 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Error bars indicate SEM .

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

C) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 15-120 min. After labeling, proteins were separated on SDS-PAGE and newly synthesized proteins in SN labeled with [35S] were visualized by autoradiography (left panel). To determine the contribution of mitochondrial and cytosolic translation the labeling was preceded by treatment with chloramphenicol (CAP, 50 μg/ml) or with puromycin (PURO, 3mM) respectively (right panel). After 1 hour after the stimulation, a significant increase in 35S-labelled proteins was observed (n=6 biological replicates, *** p<0.001; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test) and the CAP treatment did not affect the overall levels of de novo synthetized proteins (n=6 biological replicates, ns; p=0.220). In contrast, PURO treatment significantly inhibited de novo protein synthesis in synaptoneurosomes (n=6 biological replicates, ** p=0.0091, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( D - F ) Results of mass spectrometry ( label free quantification - LFQ ) analysis identifying proteins with increased abundance in NMDAR - stimulated ( 20 min ) synaptoneurosomes . ( D ) Volcano plot ( Whitney - Mann test , n = 4 per group ) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated . Mitochondrial proteins are depicted in orange . The vertical line defines the p - value statistical significance cut - off . ( E ) Biological functions of identified proteins annotated using DAVID . Third top functional category identified as mitochondrial ones . ( F ) Pie - chart showing percentage share of mitochondrial proteins among all identified upregulated 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

(D-F) Results of mass spectrometry (label free quantification - LFQ) analysis identifying proteins with increased abundance in NMDAR-stimulated (20 min) synaptoneurosomes. (D) Volcano plot (Whitney-Mann test, n=4 per group) showing abundance of identified proteins in stimulated synaptoneurosomes as compared to unstimulated. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. (E) Biological functions of identified proteins annotated using DAVID. Third top functional category identified as mitochondrial ones. (F) Pie-chart showing percentage share of mitochondrial proteins among all identified upregulated proteins.


( A - D ) Results of high - resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes . Volcano plots ( Whitney - Mann test , Benjamini Hochberg correction , n = 5 per group ) showing abundance of identified proteins in NMDAR - stimulated ( 20 min ) synaptoneurosomes as compared to unstimulated using iTRAQ8 - plex ( A ) and TMT10 - plex ( B ) labelling methods . Mitochondrial proteins are depicted in orange . The vertical line defines the p - value statistical significance cut - off . For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified , the data is presented on Figure EV2 . ( C ) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation ( assembled from all three proteomic analyzes : LFQ , iTRAQ8 ,

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Results of high-resolution quantitative mass spectrometry analysis identifying proteins with increased abundance in stimulated synaptoneurosomes. Volcano plots (Whitney-Mann test, Benjamini Hochberg correction, n=5 per group) showing abundance of identified proteins in NMDAR-stimulated (20 min) synaptoneurosomes as compared to unstimulated using iTRAQ8-plex (A) and TMT10-plex (B) labelling methods. Mitochondrial proteins are depicted in orange. The vertical line defines the p-value statistical significance cut-off. For nine mitochondrial proteins their mRNA abundance on polyribosomes was quantified, the data is presented on Figure EV2. (C) Biological functions of proteins identified as significantly upregulated after NMDAR stimulation (assembled from all three proteomic analyzes: LFQ, iTRAQ8, TMT10


( E ) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR - stimulated ( 20 min ) synaptoneurosomes . Upon NMDAR stimulation , a fraction of mRNAs is shifted towards heavy - polyribosomal fraction , which represents actively translating ribosomes . The fraction of polyribosomes used for RNA - seq experiment is depicted .

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(E) RNA absorbance profiles from polyribosomal fractionation of unstimulated and NMDAR-stimulated (20 min) synaptoneurosomes. Upon NMDAR stimulation, a fraction of mRNAs is shifted towards heavy-polyribosomal fraction, which represents actively translating ribosomes. The fraction of polyribosomes used for RNA-seq experiment is depicted.


( F ) Comparison of proteomic data with RNA - seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples . Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA - seq values of polyribosome associated mRNA with total mRNA ( y axis ) . Green dots represent mRNAs encoding proteins upregulated upon stimulation ( identified in MS experiments ) while orange dots represent mRNAs encoding mitochondrial proteins . Density curves for transcripts encoding mitochondrial proteins ( orange ) and upregulated proteins ( green ) detected by the mass spectrometry are present on the right side of the plot . Please note that both classes represent high abundant mRNAs with good ribosome association values . Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red .

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(F) Comparison of proteomic data with RNA-seq experiments on mRNAs associated with polyribosomes from synaptoneurosomal samples. Ribosome association of synaptoneurosomal mRNAs determined by normalizing RNA-seq values of polyribosome associated mRNA with total mRNA (y axis). Green dots represent mRNAs encoding proteins upregulated upon stimulation (identified in MS experiments) while orange dots represent mRNAs encoding mitochondrial proteins. Density curves for transcripts encoding mitochondrial proteins (orange) and upregulated proteins (green) detected by the mass spectrometry are present on the right side of the plot. Please note that both classes represent high abundant mRNAs with good ribosome association values. Transcripts with statistically significant increased or decreased abundance on the polyribosomes in response to the stimulation are depicted in red.


( A ) Respiration activity of permeabilized synaptoneurosomes ( upper panel ) as compared to permeabilized HEK293T ( lower panel ) . Oxygen concentration ( blue line ) and oxygen consumption ( red line ) measured using O2k oxygraph are shown .

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(A) Respiration activity of permeabilized synaptoneurosomes (upper panel) as compared to permeabilized HEK293T (lower panel). Oxygen concentration (blue line) and oxygen consumption (red line) measured using O2k oxygraph are shown.


( B ) Blue native gel electrophoresis ( BN - PAGE ) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria .

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(B) Blue native gel electrophoresis (BN-PAGE) analysis of mitochondrial respiratory chain complexes from synaptoneurosomes as compared to HEK293T mitochondria.


( C ) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN - PAGE followed by Western blot using antibodies specific for : complex I ( Ndufb8 ) , complex II ( Sdha ) , complex III ( Uqcrc2 ) , complex IV ( Cox6a ) and complex V ( Atp5a ) . Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria .

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(C) Detection of respiratory chain complexes and supercomplexes in SN and HEK293T mitochondria using BN-PAGE followed by Western blot using antibodies specific for: complex I (Ndufb8), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox6a) and complex V (Atp5a). Note similar composition of OXPHOS supercomplexes and complexes in synaptoneurosomes as in cellular mitochondria.


( D ) Selected bands ( 1 - 8 ) visible in blue - native gel were excised and analyzed using mass spectrometry ( LC - MS ) . Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands .

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(D) Selected bands (1-8) visible in blue-native gel were excised and analyzed using mass spectrometry (LC-MS). Proteomic analysis confirms the presence of protein subunits of different respiratory chain complexes in indicated bands.


( A ) Synaptoneurosomes were NMDAR - stimulated and incubated with radioactive 35S - methionine / cysteine mix for 1h . Samples were separated using blue - native electrophoresis ( BN - PAGE ) . Protein complexes were visualized by Coomassie staining ( left ) and autoradiography ( right ) . Autoradiography of the BN - PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes .

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(A) Synaptoneurosomes were NMDAR-stimulated and incubated with radioactive 35S-methionine/cysteine mix for 1h. Samples were separated using blue-native electrophoresis (BN-PAGE). Protein complexes were visualized by Coomassie staining (left) and autoradiography (right). Autoradiography of the BN-PAGE gel shows de novo synthetized mitochondrial proteins that are incorporated into respiratory chain complexes.


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m - chlorophenyl hydrazone ( 10 μM CCCP ) ( B ) Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed ( B , n = 4 biological replicates , * , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of CCCP ( B ; n = 4 biological replicates , * p = 0 . 02 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test )

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

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with carbonyl cyanide m-chlorophenyl hydrazone (10 μM CCCP) (B) Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed (B, n=4 biological replicates, * , ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of CCCP (B; n=4 biological replicates, * p=0.02, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test)


To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture ( containing 1 μM valinomycin , 20 μM oligomycin , 8 μM antimycin ) ( C ) . Additionaly , in order to block protein synthesis , SN were pretreated with puromycin ( PURO , 3mM ) ( C ) . Protein complexes were separated on BN - PAGE and visualized by Coomassie staining and autoradiography . 1 hour after the stimulation , significant increase in 35S - labelled proteins was observed C , n = 5 biological replicates , * * p = 0 . 0023 ; repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S - methionine into respiratory chain complexes when samples were incubated in the presence of VOA ( C ; n = 5 biological replicates , * * p = 0 . 0044 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Also , significant inhibition of 35S - methionine / cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin ( C , n = 5 biological replicates , * p = 0 . 0236 , repeated measures one - way ANOVA , post - hoc Sidak ' s multiple comparisons test ) . Error bars indicate SEM .

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

To block protein import and incorporation of newly synthesized proteins into respiratory complexes the mitochondrial electrochemical potential was abolished by treatment with VOA mixture (containing 1 μM valinomycin, 20 μM oligomycin, 8 μM antimycin) (C). Additionaly, in order to block protein synthesis, SN were pretreated with puromycin (PURO, 3mM) (C). Protein complexes were separated on BN-PAGE and visualized by Coomassie staining and autoradiography. 1 hour after the stimulation, significant increase in 35S-labelled proteins was observed C, n=5 biological replicates, ** p=0.0023; repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Blocking protein import into the mitochondria significantly inhibited the incorporation of 35S-methionine into respiratory chain complexes when samples were incubated in the presence of VOA (C; n=5 biological replicates, ** p=0.0044, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Also, significant inhibition of 35S-methionine/cysteine incorporation into mitochondrial protein complexes in synaptoneurosomes was observed when samples were stimulated in the presence of puromycin (C, n=5 biological replicates, * p=0.0236, repeated measures one-way ANOVA, post-hoc Sidak's multiple comparisons test). Error bars indicate SEM.


( B ) Equal amounts of synaptoneurosomal samples isolated from mouse brains ( Fmr1 KO and wild - type littermates ) were permeabilized by saponin and assayed on MitoPlates for 2 hours . Results are presented as the average rate / min / µg of protein , + / - SEM ( n = 3 per genotype ; two - way ANOVA , post - hoc Sidak ' s multiple comparisons test ; * * * p < 0 . 0001 ) .

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(B) Equal amounts of synaptoneurosomal samples isolated from mouse brains (Fmr1 KO and wild-type littermates) were permeabilized by saponin and assayed on MitoPlates for 2 hours. Results are presented as the average rate/min/µg of protein, +/- SEM (n=3 per genotype; two-way ANOVA, post-hoc Sidak's multiple comparisons test; *** p< 0.0001).


( C ) Mitochondrial O2 consumption rates in SN were measured by high - resolution respirometry in the presence of following respiratory substrates : malate , pyruvate , and glutamate for Complexes I - IV , succinate for Complexes II - IV , glycerol 3 - phosphate for mGpDH - IV and ascorbate and TMPD for complex IV , and normalized to protein content . Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD ( * p = 0 . 0483 and * p = 0 . 0360 respectively , n = 9 per genotype , paired two - tailed t - test ) . Data are presented as a box - and - whiskers graph ( the box extends from 25th to 75th percentiles , central horizontal line is plotted at the median , whiskers show 5th - 95th percentile ) .

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(C) Mitochondrial O2 consumption rates in SN were measured by high-resolution respirometry in the presence of following respiratory substrates: malate, pyruvate, and glutamate for Complexes I-IV, succinate for Complexes II-IV, glycerol 3-phosphate for mGpDH-IV and ascorbate and TMPD for complex IV, and normalized to protein content. Fmr1 KO mitochondria show elevated levels of respiration in the presence of succinate as well as ascorbate and TMPD (*p=0.0483 and *p=0.0360 respectively, n=9 per genotype, paired two-tailed t-test). Data are presented as a box-and-whiskers graph (the box extends from 25th to 75th percentiles, central horizontal line is plotted at the median, whiskers show 5th - 95th percentile).


( D ) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild - types . Mitochondria are indicated by arrows ; presynaptic part in blue , postsynaptic part in green . 3 mice per genotype were analyzed . Scale bars , 0 . 5 µm .

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(D) Ultrastructure of Fmr1 KO brain sections revealed important differences in mitochondria morphology when compared to wild-types. Mitochondria are indicated by arrows; presynaptic part in blue, postsynaptic part in green. 3 mice per genotype were analyzed. Scale bars, 0.5 µm.


Cell number in the bone marrow of wild - type and Hdac6 knockout mice ( n = 12 - 13 mice per group ) . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( Student ' s t test ) .

Cell number in the bone marrow of wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).


Flow cytometry profiles for the LK and LSK subsets in wild - type and Hdac6 knockout mice .

Flow cytometry profiles for the LK and LSK subsets in wild-type and Hdac6 knockout mice.


Frequency of the LK subset in wild - type and Hdac6 knockout mice ( n = 12 - 13 mice per group ) . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( Student ' s t test ) .

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Frequency of the LK subset in wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).


L . number of the LSK subset in wild - type and Hdac6 knockout mice ( n = 12 - 13 mice per group ) . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( Student ' s t test ) .

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L. number of the LSK subset in wild-type and Hdac6 knockout mice (n = 12-13 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).


Flow cytometry profiles for the proportion of cycling HSPCs examined with EdU incorporation experiments .

Flow cytometry profiles for the proportion of cycling HSPCs examined with EdU incorporation experiments.


Representative images of colonies formed by bone marrow cells from wild - type and Hdac6 knockout mice . Scale bar , 200 µm . Number of colonies formed by bone marrow cells from wild - type and Hdac6 knockout mice ( n = 10 mice per group ) . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of colonies formed by bone marrow cells from wild-type and Hdac6 knockout mice. Scale bar, 200 µm. Number of colonies formed by bone marrow cells from wild-type and Hdac6 knockout mice (n = 10 mice per group). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).


Immunoprecipitation and immunoblotting showing the interaction of HA - HDAC6 with GFP - IDH1 in HEK293T cells .

Immunoprecipitation and immunoblotting showing the interaction of HA-HDAC6 with GFP-IDH1 in HEK293T cells.


Immunoprecipitation and immunoblotting showing the interaction of HA - HDAC6 with GFP - IDH1 in HEK293T cells . D . Immunoprecipitation and immunoblotting showing the interaction of GFP - IDH1 with endogenous HDAC6 in HEK293T cells .

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Immunoprecipitation and immunoblotting showing the interaction of HA-HDAC6 with GFP-IDH1 in HEK293T cells. D. Immunoprecipitation and immunoblotting showing the interaction of GFP-IDH1 with endogenous HDAC6 in HEK293T cells.


J . Immunoprecipitation and immunoblotting with various truncated mutants of HA - HDAC6 to identify the domains that mediate its interaction with GFP - IDH1 . K . Immunoprecipitation and immunoblotting with various truncated mutants of GFP - IDH1 to identify the domains that mediate its interaction with HA - HDAC6 .

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J. Immunoprecipitation and immunoblotting with various truncated mutants of HA-HDAC6 to identify the domains that mediate its interaction with GFP-IDH1. K. Immunoprecipitation and immunoblotting with various truncated mutants of GFP-IDH1 to identify the domains that mediate its interaction with HA-HDAC6.


Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the acetylated lysine ( Ace - lysine ) antibody , followed by immunoblotting with the IDH1 antibody . Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the IDH1 antibody , followed by immunoblotting with the Ace - lysine antibody .

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Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the acetylated lysine (Ace-lysine) antibody, followed by immunoblotting with the IDH1 antibody. Analysis of IDH1 acetylation via immunoprecipitation of the bone marrow cell lysate with the IDH1 antibody, followed by immunoblotting with the Ace-lysine antibody.


E . Analysis of GFP - IDH1 acetylation by immunoprecipitation with the GFP antibody followed by immunoblotting with the Ace - lysine antibody . HEK293T cells were treated with tubacin , tubastatin A ( TubA ) , trichostatin A ( TSA ) , or sodium butyrate ( NaB ) ( E ) .

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

E. Analysis of GFP-IDH1 acetylation by immunoprecipitation with the GFP antibody followed by immunoblotting with the Ace-lysine antibody. HEK293T cells were treated with tubacin, tubastatin A (TubA), trichostatin A (TSA), or sodium butyrate (NaB) (E).


Immunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild - type GFP - IDH1 or various mutants , These cells were treated with the HDAC6 inhibitor ,

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Immunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild-type GFP-IDH1 or various mutants, These cells were treated with the HDAC6 inhibitor, tubacin


Immunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild - type GFP - IDH1 or various mutants , These cells were transfected with the HDAC6

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Immunoprecipitation and immunoblotting showing the level of IDH1 acetylation in HEK293T cells transfected with wild-type GFP-IDH1 or various mutants, These cells were transfected with the HDAC6 siRNA


O - P . In vitro deacetylation analysis using GST - IDH1 and GST - IDH1 K233R proteins as substrates . The black arrowhead indicates HA - HDAC6 , while the white arrowhead marks GST - IDH1 and GST - IDH1 K233R . P is the corresponding statistical result ( n = 3 technical replicates ) . Data information : All values are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , ns , not significant ( Student ' s t test ) .

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O-P. In vitro deacetylation analysis using GST-IDH1 and GST-IDH1 K233R proteins as substrates. The black arrowhead indicates HA-HDAC6, while the white arrowhead marks GST-IDH1 and GST-IDH1 K233R. P is the corresponding statistical result (n = 3 technical replicates). Data information: All values are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (Student's t test).