Figure 2A . Amyloid staining ( 6E10 ) combined with miR - 132FISH in ant - 132 - and scramble - injected mice at six months of age . Scale bar , 500 μm . B . Quantification of amyloid burden in hippocampus and cortex of ant - 132 - injected and control animals ( see Materials and Methods ) . Sample size , n = 4 per group . C , D . ELISA of soluble ( TBS ) and insoluble ( Formic acid ) Aβ40 and Aβ42 levels in the hippocampus of ant - 132 - ( C ) and miR - 132 - injected ( D ) animals at six and three months of age , respectively . Sample size , n = 6 per group .

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Figure 2A. Amyloid staining (6E10) combined with miR-132FISH in ant-132- and scramble-injected mice at six months of age. Scale bar, 500 μm. B. Quantification of amyloid burden in hippocampus and cortex of ant-132-injected and control animals (see Materials and Methods). Sample size, n=4 per group.C, D. ELISA of soluble (TBS) and insoluble (Formic acid) Aβ40 and Aβ42 levels in the hippocampus of ant-132- (C) and miR-132-injected (D) animals at six and three months of age, respectively. Sample size, n=6 per group.


Figure 3Western blot analysis of total TAU and pTAU ( AT8 , AT270 ) levels upon miR - 132 knockdown ( A ) or overexpression ( B ) at six and three months of age , respectively . Sample size , n = 9 per group ( A ) and n = 6 per group ( B ) . Values were normalized to the respective control groups and presented as mean ± SEM . Student ' s t - test was used .

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Figure 3Western blot analysis of total TAU and pTAU (AT8, AT270) levels upon miR-132 knockdown (A) or overexpression (B) at six and three months of age, respectively. Sample size, n=9 per group (A) and n=6 per group (B). Values were normalized to the respective control groups and presented as mean ± SEM. Student's t-test was used.


Figure 4B . Positive hits were validated by semi - quantitative qPCR in ant - 132 - injected mousehippocampus . Black line indicates mean of scramble control set to 1 . Dashed line indicates chosen threshold of 1 . 3 - fold . Sample size , n = 9 per group . Values were normalized to scramble - injected group and presented as mean ± SEM .

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Figure 4B. Positive hits were validated by semi-quantitative qPCR in ant-132-injected mousehippocampus. Black line indicates mean of scramble control set to 1. Dashed line indicates chosen threshold of 1.3-fold. Sample size, n=9 per group. Values were normalized to scramble-injected group and presented as mean ± SEM.


Figure 5A . Luciferasereporter assay of wild - type ( wt ) and mutant ( mut ) ITPKB 3 ' UTR in HEK293 cells co - transfected with a synthetic miR - 132 ( miR - 132 ) or a negative control ( Neg Ctr ) oligonucleotide . B . ELISA of Aβ40 and Aβ42 levels in HEK293 - APPswecells transfected with a miR - 132 antisense oligonucleotide ( miR - 132 inh ) , an siRNA against ITPKB ( ITPKB siRNA ) or both . The assays in A and B were performed in three independent experiments , each in triplicates . C . Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB . Sample size , n = 6 per group . Values were normalized to control - injected group ( Ctr ) and presented as mean ± SEM . D . ELISA of insoluble ( Formic acid - soluble ) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA - and control - injected animals at three months of age . Sample size , n = 6 per group . Values were normalized to control group and presented as mean ± SEM . E , F . Western blot analysis of ITPKB levels upon miR - 132 down - ( E ) or up - regulation ( F ) at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to control groups and presented as mean ± SEM .

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Figure 5A. Luciferasereporter assay of wild-type (wt) and mutant (mut) ITPKB 3'UTR in HEK293 cells co-transfected with a synthetic miR-132 (miR-132) or a negative control (Neg Ctr) oligonucleotide.B. ELISA of Aβ40 and Aβ42 levels in HEK293-APPswecells transfected with a miR-132 antisense oligonucleotide (miR-132 inh), an siRNA against ITPKB (ITPKB siRNA) or both. The assays in A and B were performed in three independent experiments, each in triplicates.C. Western blot analysis of ITPKB knock down in APPPS1hippocampus using an siRNA oligonucleotide against ITPKB. Sample size, n=6 per group. Values were normalized to control-injected group (Ctr) and presented as mean ± SEM.D. ELISA of insoluble (Formic acid-soluble) Aβ40 and Aβ42 levels in the hippocampus of ITPKB siRNA- and control-injected animals at three months of age. Sample size, n=6 per group. Values were normalized to control group and presented as mean ± SEM.E, F. Western blot analysis of ITPKB levels upon miR-132 down- (E) or up-regulation (F) at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to control groups and presented as mean ± SEM.


Figure 6Western blot analysis of CTFβ ( A ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used . Western blot analysis of sAPPβ - swe ( B ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used . Western blot analysis of phosphorylated ERK1 / 2 ( pERK1 / 2 ) ( C ) levels upon miR - 132 down - ( ant - 132 ) ( left panel ) or upregulation ( miR - 132 ) ( right panel ) in APPPS1hippocampus at six and three months of age , respectively . Sample size , n = 9 per group for miR - 132 down regulation and n = 6 per group for miR - 132 overexpression . Values were normalized to respective control groups and presented as mean ± SEM . The Student ' s t - test was used .

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Figure 6Western blot analysis of CTFβ (A) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.Western blot analysis of sAPPβ-swe (B) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.Western blot analysis of phosphorylated ERK1/2 (pERK1/2) (C) levels upon miR-132 down- (ant-132) (left panel) or upregulation (miR-132) (right panel) in APPPS1hippocampus at six and three months of age, respectively. Sample size, n=9 per group for miR-132 down regulation and n=6 per group for miR-132 overexpression. Values were normalized to respective control groups and presented as mean ± SEM. The Student's t-test was used.


Figure 7A . Double immunofluorescence staining of amyloid plaques ( 6E10 ) and ITPKB in AD prefrontal cortex . Scale bar , 50 μm . Arrowheads indicate ITPKB immunopositivity . B . miR - 132FISH coupled with double immunofluorescence against hyperphosphorylated TAU ( AT8 ) - containing neurofibrillary tangles ( NFTs ) and ITPKB in AD prefrontal cortex . miR - 124 was used as a control . Scale bar , 50 μm . C . Quantification of miR - 132 , ITPKB and hyperphosphorylated TAU ( pTAU ) signal in single neurons . Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed ( see Materials and Methods ) . Sample size ( AD patients ) , n = 3 . Values are presented as mean ± SEM . Two - way Anova was used . Quantifications are summarized in the table provided . Arrow directions refer to the comparison of each normalized signal to the same signal in the other group ( in the " low miR - 132 " group comparisons are made to the " high miR - 132 " group and vice versa ) .

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Figure 7A. Double immunofluorescence staining of amyloid plaques (6E10) and ITPKB in AD prefrontal cortex. Scale bar, 50 μm. Arrowheads indicate ITPKB immunopositivity.B. miR-132FISH coupled with double immunofluorescence against hyperphosphorylated TAU (AT8)-containing neurofibrillary tangles (NFTs) and ITPKB in AD prefrontal cortex. miR-124 was used as a control. Scale bar, 50 μm.C. Quantification of miR-132, ITPKB and hyperphosphorylated TAU (pTAU) signal in single neurons. Integrated intensity values of each signal per cell were normalized to the mean integrated density of each signal across all the cells analyzed (see Materials and Methods). Sample size (AD patients), n=3. Values are presented as mean ± SEM. Two-way Anova was used. Quantifications are summarized in the table provided. Arrow directions refer to the comparison of each normalized signal to the same signal in the other group (in the "low miR-132" group comparisons are made to the "high miR-132" group and vice versa).


Figure 8Semi - quantitative PCR of miR - 132 and western blot analysis of ITPKB , phosphorylated ( pERK1 / 2 ) and total ERK1 / 2 , phosphorylated ( AT8 , AT270 ) and total TAU , phosphorylated sphingosine kinase 1 ( pSphK1 ) , BACE1 , full length APP ( flAPP ) , APP CTFs and sAPPβ in human AD hippocampi ( AD ) compared to non demented control samples ( ND ) . Sample size , n = 39 for AD and n = 15 for ND . Values were normalized to ND and presented as mean ± SEM . The Student ' s t - test was used .

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Figure 8Semi-quantitative PCR of miR-132 and western blot analysis of ITPKB, phosphorylated (pERK1/2) and total ERK1/2, phosphorylated (AT8 , AT270) and total TAU, phosphorylated sphingosine kinase 1 (pSphK1), BACE1, full length APP (flAPP), APP CTFs and sAPPβ in human AD hippocampi (AD) compared to non demented control samples (ND). Sample size, n=39 for AD and n=15 for ND. Values were normalized to ND and presented as mean ± SEM. The Student's t-test was used.


Figure 1 ( B ) Phenotypes of affected individuals . Camptodactyly , absent flexion crease , and limited forearm supination were observed . ( C - E ) T1 weighted MRI scan on upper limbs of subject IV : 7 . ( C ) The pronator quadratus absence of affected side was indicated by a red arrow . ( D ) No difference was found in palmar muscles . ( E ) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow . ( F - H ) T1 weighted MRI scan on upper limbs of subject IV : 8 . ( F ) Increased epimysial fat was indicated by red arrow . ( G ) Completely loss of thenar eminences of both hands were indicated by red arrows . ( H ) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows . ( I ) The MET variants by Sanger sequencing was indicated by red arrow . ( J ) 293T cells were transfected with FLAG - tagged MET / METMut / Vector plasmids , and 48 h post transfection , cells were treated with 10 ng / mL recombinant human HGF for 1 h . Then MET / METMut protein purification and tyrosine kinase assay were conducted . Western Blot pictures representative of n = 3 experiments . METMut means p . Y1234C mutant MET . EGFR means epidermal growth factor receptor and serves as a positive control . NC means negative control .

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Figure 1(B) Phenotypes of affected individuals. Camptodactyly, absent flexion crease, and limited forearm supination were observed.(C-E) T1 weighted MRI scan on upper limbs of subject IV:7. (C) The pronator quadratus absence of affected side was indicated by a red arrow. (D) No difference was found in palmar muscles. (E) Loss of lumbrcalis and interosseus muscles of fifth finger of affected side was indicated by a red arrow. (F-H) T1 weighted MRI scan on upper limbs of subject IV:8. (F) Increased epimysial fat was indicated by red arrow. (G) Completely loss of thenar eminences of both hands were indicated by red arrows. (H) Loss of radial lumbrcalis and interosseus muscles of both hands were indicated by red arrows.(I) The MET variants by Sanger sequencing was indicated by red arrow.(J) 293T cells were transfected with FLAG-tagged MET/METMut/Vector plasmids, and 48 h post transfection, cells were treated with 10 ng/mL recombinant human HGF for 1 h. Then MET/METMut protein purification and tyrosine kinase assay were conducted. Western Blot pictures representative of n = 3 experiments. METMut means p.Y1234C mutant MET. EGFR means epidermal growth factor receptor and serves as a positive control. NC means negative control.


Figure 2 ( A ) Gross appearance of wild - type and heterozygous newborns . ( B ) Graph showing weight of P0 mice . Sample size : WT ( n = 4 ) and Hetero ( n = 5 ) . * P < 0 . 05 , by one - way ANOVA and follow by Dunnett ' s post hoc test . Sections of spine from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm . Sections of forelimbs , hindlimbs , hand , and foot from wild types and heterozygotes at P0 , were conducted with HE staining and immunofluorescence staining using anti - myosin heavy chain antibody . Scale bars , 100 μm . ( H ) The mean number of myofibers in the muscles of the spine , forelimb , hindlimb , hand and foot was qualified . NS means no statistic significance . * P < 0 . 05 , * * P < 0 . 01 , by two - tailed independent student ' s t test . In ( C - H ) , n = 3 . ( I ) Transmission electron microscope analysis of gastrocnemius from wild - type and heterozygous newborns . Black arrows denote mitochodria . Scale bars , 2 μm . WT means wild types , Hetero means heterozygotes .

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Figure 2(A) Gross appearance of wild-type and heterozygous newborns. (B) Graph showing weight of P0 mice. Sample size: WT (n = 4) and Hetero (n = 5). *P < 0.05, by one-way ANOVA and follow by Dunnett's post hoc test.Sections of spine from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.Sections of forelimbs, hindlimbs, hand, and foot from wild types and heterozygotes at P0, were conducted with HE staining and immunofluorescence staining using anti-myosin heavy chain antibody. Scale bars, 100 μm.(H) The mean number of myofibers in the muscles of the spine, forelimb, hindlimb, hand and foot was qualified. NS means no statistic significance. *P < 0.05, **P < 0.01, by two-tailed independent student's t test. In (C-H), n = 3.(I) Transmission electron microscope analysis of gastrocnemius from wild-type and heterozygous newborns. Black arrows denote mitochodria. Scale bars, 2 μm. WT means wild types, Hetero means heterozygotes.


Figure 3 ( A ) In situ hybridization of E10 . 5 embryos using Pax3 probe . Pax3 expression ( brown signal ) was observed in limb bud and demomyotome ( DM ) , respectively . Cells labeled with Pax3 were indicated by red arrow . Scale bars , 200 μm . ( B ) Gross appearance of E14 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm . ( C ) HE staining of forelimb , hindlimb , and paraspinal muscle of E14 . 5 embryos . Scale bars , 100 μm . ( D ) Gross appearance of E16 . 5 embryos of indicated genotype . Scale bars , 2 . 5 mm . ( E ) HE staining of forelimb , hindlimb , and paraspinal muscle of E16 . 5 embryos . n = 3 , scale bars , 200 μm . ( F ) Anti - Ki67 antibody was used to label proliferative myoblasts ( red fluorescence ) with DAPI labeled nucleuses ( blue fluorescence ) in paraspinal muscle . Scale bar , 25 μm . ( G ) Bar graph showing statistical analysis of positive rate of Ki67 - labeled nuclei , n = 3 with more than 150 cells analyzed per n , * * P < 0 . 01 , by chi - square test ( χ2 test ) . WT means wild types , Hetero means heterozygotes , and Homo means homozygotes .

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Figure 3(A) In situ hybridization of E10.5 embryos using Pax3 probe. Pax3 expression (brown signal) was observed in limb bud and demomyotome (DM), respectively. Cells labeled with Pax3 were indicated by red arrow. Scale bars, 200 μm.(B) Gross appearance of E14.5 embryos of indicated genotype. Scale bars, 2.5 mm.(C) HE staining of forelimb, hindlimb, and paraspinal muscle of E14.5 embryos. Scale bars, 100 μm.(D) Gross appearance of E16.5 embryos of indicated genotype. Scale bars, 2.5 mm.(E) HE staining of forelimb, hindlimb, and paraspinal muscle of E16.5 embryos. n = 3, scale bars, 200 μm.(F) Anti-Ki67 antibody was used to label proliferative myoblasts (red fluorescence) with DAPI labeled nucleuses (blue fluorescence) in paraspinal muscle. Scale bar, 25 μm. (G) Bar graph showing statistical analysis of positive rate of Ki67-labeled nuclei, n = 3 with more than 150 cells analyzed per n, **P < 0.01, by chi-square test (χ2 test). WT means wild types, Hetero means heterozygotes, and Homo means homozygotes.


Figure 1LDH assay was performed after 24 h of cold stress on ice . Data are presented as mean ± SEM ; n = 3 , biological replicates . * * * * p < 0 . 0001 , * * * p < 0 . 001 by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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Figure 1LDH assay was performed after 24 h of cold stress on ice. Data are presented as mean ± SEM; n = 3, biological replicates. ****p < 0.0001, ***p < 0.001 by one-way ANOVA followed by Dunnett's multiple comparison test.


Figure 2Immunoblots of MICU1 and its mutants rescued by the lentivirus . Arrows indicate MICU1 or the dimer . * indicates the potential DTT - resistant dimer .

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Figure 2Immunoblots of MICU1 and its mutants rescued by the lentivirus. Arrows indicate MICU1 or the dimer. * indicates the potential DTT-resistant dimer.


Figure 3MMP was monitored by TMRE fluorescence at every 1 h . FCCP ( 200 µM ) and antimycin A ( 50 µM ) were used as pretreatments for 30 min . Data were normalized by basal state of WT A549 cells ( B - D ) Data at 1 h ( C ) , 5 h ( D are presented as mean ± SEM ; n = 4 ( B - D ) biological replicates , * * * * p < 0 . 0001 , * * p < 0 . 01 , * p < 0 . 05 by paired T - test ( C ) , or one - way ANOVA followed by Dunnett ' s multiple comparison test ( DLDH release was measured after cold stress for 24 h . FCCP ( 200 µM ) , antimycin A ( 50 µM ) , mitoQ and decylQ ( 500 nM ) were used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 4 ( G ) , biological replicates , * * * * p < 0 . 0001 , * * * p < 0 . 001 , * * p < 0 . 01 , by one - way ANOVA followed by Dunnett ' s or Bonferroni ' s multiple comparison test . whose raw value were normalized by the C11 - BODIPY 581 / 591 without cold stress of non - treatment , are presented as mean ± SEM ; n = 5 , biological replicates . * * p < 0 . 01 , * p < 0 . 05 , by one - way ANOVA followed by Dunnett ' s multiple comparison test .

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Figure 3MMP was monitored by TMRE fluorescence at every 1 h. FCCP (200 µM) and antimycin A (50 µM) were used as pretreatments for 30 min. Data were normalized by basal state of WT A549 cells (B-D) Data at 1 h (C), 5 h (D are presented as mean ± SEM; n = 4 (B-D) biological replicates, ****p < 0.0001, **p < 0.01, *p < 0.05 by paired T-test (C), or one-way ANOVA followed by Dunnett's multiple comparison test (DLDH release was measured after cold stress for 24 h. FCCP (200 µM), antimycin A (50 µM), mitoQ and decylQ (500 nM) were used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 4 (G) , biological replicates, ****p < 0.0001, ***p < 0.001, **p < 0.01, by one-way ANOVA followed by Dunnett's or Bonferroni's multiple comparison test. whose raw value were normalized by the C11-BODIPY 581/591 without cold stress of non-treatment, are presented as mean ± SEM; n = 5, biological replicates. **p < 0.01, *p < 0.05, by one-way ANOVA followed by Dunnett's multiple comparison test.


Figure 4LDH release was measured after cold stress for 24 h . BAPTA - AM ( 10 µM ) was used as pretreatments for 30 min . Data are presented as mean ± SEM ; n = 3 , biological replicates , * * * * p < 0 . 0001 , by unpaired T - test . LDH release was measured after cold stress for 16 h . Data are presented as mean ± SEM ; n = 5 , biological replicates , * p < 0 . 05 , by unpaired T - test .

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Figure 4LDH release was measured after cold stress for 24 h. BAPTA-AM (10 µM) was used as pretreatments for 30 min. Data are presented as mean ± SEM; n = 3, biological replicates, ****p < 0.0001, by unpaired T-test.LDH release was measured after cold stress for 16 h. Data are presented as mean ± SEM; n = 5, biological replicates, *p < 0.05, by unpaired T-test.


Figure 1A - B ) Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin ( Rapa ) for the indicated time periods ( A ) ( n = 7 ) or for 5 h with the indicated concentrations ( B ) ( n = 6 ) . One hour before harvesting , 100 nM bafilomycin A1 was added . Upper panels : representative Western blots ; lower panels : quantification of the LC3 - II / GAPDH ratio . C ) GFP - LC3 - punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin . Left : representative pictures . The scale bar represents 10 µm . Concentrations are mentioned in the right lower corner . Right : Quantification of the number of punctae per cell ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA .

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

Figure 1A-B) Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated with DMSO or 1 µM rapamycin (Rapa) for the indicated time periods (A) (n = 7) or for 5 h with the indicated concentrations (B) (n = 6). One hour before harvesting, 100 nM bafilomycin A1 was added. Upper panels: representative Western blots; lower panels: quantification of the LC3-II/GAPDH ratio.C) GFP-LC3-punctae quantification in HeLa cells treated for 5 h with different concentrations of rapamycin. Left: representative pictures. The scale bar represents 10 µm. Concentrations are mentioned in the right lower corner. Right: Quantification of the number of punctae per cell (n = 3). * p<0.05, repeated measurements ANOVA.


Figure 2A ) Representative measurements ( n = 4 ) of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 0 . 3 µM and 100 µM ATP , 1 µM thapsigargin ( Tg ) or 10 µM ionomycin ( Iono ) in intact HeLa cells treated with different concentrations of rapamycin ( Rapa ) for 5 h . 45 s prior to the addition of ATP , Tg or Iono , EGTA ( 3 mM ) was given to buffer extracellular Ca2 + as indicated . B ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 4 ) . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA . C ) Mean resting cytosolic [ Ca2 + ] , measured in Fura2 - loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h , as well as in the absence ( Ctrl ) or presence of DMSO ( n = 3 ) . * p < 0 . 05 , repeated measurements ANOVA . D ) Unidirectional 45Ca2 + - flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO ( Ctrl ) . Mean fractional 45Ca2 + release ( % / 2 min ) is shown as a function of time with the effect of 0 . 7 µM IP3 ( circles ) or no addition ( squares ) . The horizontal bar indicates the presence of IP3 . E ) Quantitative analysis of the IP3 - induced 45Ca2 + release in cells pretreated for 5 h with 1 µM rapamycin or DMSO ( Ctrl ) ( n = 8 ) . * * * p < 0 . 001 , paired Student ' s t - test .

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Figure 2A) Representative measurements (n = 4) of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 0.3 µM and 100 µM ATP, 1 µM thapsigargin (Tg) or 10 µM ionomycin (Iono) in intact HeLa cells treated with different concentrations of rapamycin (Rapa) for 5 h. 45 s prior to the addition of ATP, Tg or Iono, EGTA (3 mM) was given to buffer extracellular Ca2+ as indicated.B) Quantification of the average amplitude of the response (F−F0) (n = 4). * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.C) Mean resting cytosolic [Ca2+], measured in Fura2-loaded HeLa cells treated with the indicated concentrations of rapamycin for 5 h, as well as in the absence (Ctrl) or presence of DMSO (n = 3). * p<0.05, repeated measurements ANOVA.D) Unidirectional 45Ca2+-flux experiments in permeabilized cells pretreated with 1 µM rapamycin for 5 h or with DMSO (Ctrl). Mean fractional 45Ca2+ release (%/2 min) is shown as a function of time with the effect of 0.7 µM IP3 (circles) or no addition (squares). The horizontal bar indicates the presence of IP3.E) Quantitative analysis of the IP3-induced 45Ca2+ release in cells pretreated for 5 h with 1 µM rapamycin or DMSO (Ctrl) (n = 8). *** p<0.001, paired Student's t-test.


Figure 3A - B ) Western - blot analysis for luminal Ca2 + - binding proteins in HeLa cells treated with the indicated concentrations of rapamycin ( Rapa ) for 5 h : calreticulin ( CRT ) ( A ) and BiP / Grp78 ( BiP ) ( B ) . Upper panels : representative Western blots ; lower panels : quantification of the protein / GAPDH ratio ( n = 4 ) . C ) Western - blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h . Upper panels : representative Western blots ; lower panel : quantification of the SERCA2 / GAPDH ratio ( n = 4 ) . D ) Representative plot showing the decrease in ER 45Ca2 + content ( logarithmic scale ) in a Ca2 + - free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO . The passively bound Ca2 + was determined by loading the cells with 45Ca2 + in the presence of 10 µM of the Ca2 + ionophore A23187 and then subtracted from the stored 45Ca2 + . The ER Ca2 + - leak rate can be estimated as the rate of decline of the ER 45Ca2 + - store content as a function of time . E ) Quantification of the mean 45Ca2 + - store content at the beginning of the measurement ( t0 ) ( n = 5 ) . F ) Quantification of the mean slope of the curve in D after transformation to a linear scale , which is a measure of the 45Ca2 + - leak rate ( n = 5 ) . * p < 0 . 05 ; * * p < 0 . 01 , paired Student ' s t - test .

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

Figure 3A-B) Western-blot analysis for luminal Ca2+-binding proteins in HeLa cells treated with the indicated concentrations of rapamycin (Rapa) for 5 h: calreticulin (CRT) (A) and BiP/Grp78 (BiP) (B). Upper panels: representative Western blots; lower panels: quantification of the protein/GAPDH ratio (n = 4).C) Western-blot analysis for SERCA2 in HeLa cells treated with the indicated concentrations of rapamycin for 5 h. Upper panels: representative Western blots; lower panel: quantification of the SERCA2/GAPDH ratio (n = 4).D) Representative plot showing the decrease in ER 45Ca2+ content (logarithmic scale) in a Ca2+-free efflux medium without ATP as a function of time in permeabilized HeLa cells pretreated for 5 h with 1 µM rapamycin or with DMSO. The passively bound Ca2+ was determined by loading the cells with 45Ca2+ in the presence of 10 µM of the Ca2+ ionophore A23187 and then subtracted from the stored 45Ca2+. The ER Ca2+-leak rate can be estimated as the rate of decline of the ER 45Ca2+-store content as a function of time.E) Quantification of the mean 45Ca2+-store content at the beginning of the measurement (t0) (n = 5).F) Quantification of the mean slope of the curve in D after transformation to a linear scale, which is a measure of the 45Ca2+-leak rate (n = 5). * p<0.05; ** p<0.01, paired Student's t-test.


Figure 4A ) Representative Western - blot analysis for Atg12 ( showing the autophagic Atg12 - Atg5 complex ) , GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with ( + Dox ) or without ( - Dox ) doxycycline and treated with DMSO or 0 . 1 , 1 or 5 µM rapamycin ( Rapa ) for 5 h ( n = 3 ) . B - C ) Representative measurements of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h . Prior to the addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate the extracellular Ca2 + as indicated . B - C ) Representative measurements of cytosolic Ca2 + signals , displayed as Fura2 ratio ( F340 / F380 ) , showing the effect of 1 mM ATP ( B ) or 10 µM ionomycin ( Iono ) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h . Prior to the addition of ATP or Iono , EGTA ( 3 mM ) was added to chelate the extracellular Ca2 + as indicated . D ) Quantification of the average amplitude of the response ( F − F0 ) ( n = 3 , 4 , 5 and 6 for ATP - Dox , ATP + Dox , Iono - Dox and Iono + Dox , resp . ) * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 , repeated measurements ANOVA .

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

Figure 4A) Representative Western-blot analysis for Atg12 (showing the autophagic Atg12-Atg5 complex), GAPDH and LC3 of protein lysates obtained from MEF cells pretreated with (+Dox) or without (-Dox) doxycycline and treated with DMSO or 0.1, 1 or 5 µM rapamycin (Rapa) for 5 h (n = 3).B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.B-C) Representative measurements of cytosolic Ca2+ signals, displayed as Fura2 ratio (F340/F380), showing the effect of 1 mM ATP (B) or 10 µM ionomycin (Iono) in intact MEF cells pretreated with or without doxycycline and treated with different concentrations of rapamycin for 5 h. Prior to the addition of ATP or Iono, EGTA (3 mM) was added to chelate the extracellular Ca2+ as indicated.D) Quantification of the average amplitude of the response (F−F0) (n = 3, 4, 5 and 6 for ATP-Dox, ATP+Dox, Iono-Dox and Iono+Dox, resp.) * p<0.05; ** p<0.01; *** p<0.001, repeated measurements ANOVA.


Figure 52 + - dependent . Western - blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO , 1 µM rapamycin ( Rapa ) , 10 µM BAPTA - AM or both . One hour before harvesting , 100 nM bafilomycin A1 was added . Left : representative Western blots ; right : quantification of the LC3 - II / GAPDH ratio ( n = 6 ) . * p < 0 . 05 , repeated measurements ANOVA .

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Figure 52+-dependent. Western-blot analysis for GAPDH and LC3 of protein lysates obtained from HeLa cells treated for 5 h with DMSO, 1 µM rapamycin (Rapa), 10 µM BAPTA-AM or both. One hour before harvesting, 100 nM bafilomycin A1 was added. Left: representative Western blots; right: quantification of the LC3-II/GAPDH ratio (n = 6). * p<0.05, repeated measurements ANOVA.


Figure 1Average piRNA precursor abundance relative to EV in EV and ints - 11 RNAi samples , normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs ( Chen et al . , 2013 ) . Error bars represent the standard error of the mean ( 5 replicates ) . Distributions of piRNA precursor length in ints - 11 RNAi - treated nematodes compared to empty vector - treated nematodes , obtained from deeply sequencing one pair of libraries ( inserts sequenced up to 75nt ) . Distributions of total sequences ( E ) and total read counts ( F ) are shown .

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Figure 1Average piRNA precursor abundance relative to EV in EV and ints-11 RNAi samples, normalized to the total number of short capped RNA reads mapping to annotated WormBase TSSs (Chen et al., 2013). Error bars represent the standard error of the mean (5 replicates).Distributions of piRNA precursor length in ints-11 RNAi-treated nematodes compared to empty vector-treated nematodes, obtained from deeply sequencing one pair of libraries (inserts sequenced up to 75nt). Distributions of total sequences (E) and total read counts (F) are shown.


Figure 2Co - localization between INTS - 6 : : eGFP and mCherry : : PRDE - 1 in germ cells . PRDE - 1 and INTS - 6 co - localize in early stages of meiosis ( 1 , 2 ) and up to mid - pachytene ( 3 ) , where an additional INTS - 6 focus appears ( 3 ) . When PRDE - 1 foci are about to disappear , co - localization is lost ( 4 ) , and the additional INTS - 6 focus remains present well after the loss of PRDE - 1 foci ( 5 ) .

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Figure 2Co-localization between INTS-6::eGFP and mCherry::PRDE-1 in germ cells. PRDE-1 and INTS-6 co-localize in early stages of meiosis (1,2) and up to mid-pachytene (3), where an additional INTS-6 focus appears (3). When PRDE-1 foci are about to disappear, co-localization is lost (4), and the additional INTS-6 focus remains present well after the loss of PRDE-1 foci (5).


Figure 3Length distributions of nucleoplasmic and chromatin - bound piRNA precursors in N2 empty vector , N2 ints - 11 RNAi , tfiis empty vector , and tfiis ints - 11 RNAi nematodes . Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows . The length distributions were calculated from two merged replicates ( see Appendix Figure S2 for the length distributions in each replicate ) .

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Figure 3Length distributions of nucleoplasmic and chromatin-bound piRNA precursors in N2 empty vector, N2 ints-11 RNAi, tfiis empty vector, and tfiis ints-11 RNAi nematodes. Length shifts in nascent RNA in tfiis mutants are indicated with dashed vertical lines and arrows. The length distributions were calculated from two merged replicates (see Appendix Figure S2 for the length distributions in each replicate).


Figure 4Signal of 5 ′ P small RNA 5 ′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs , after removal of reads corresponding to mature 21U - RNAs ( see Methods ) . The signal is normalized to counts per million of non - structural mapped reads . A peak of 5 ′ P ends centered at + 38 from piRNA TSSs is observed . Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength ( top panel ) . Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength ( bottom panel ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range . The data shown here corresponds to one of two N2 EV replicates , see Appendix Figure S6 for this analysis across replicates and genotypes .

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Figure 4Signal of 5′ P small RNA 5′ ends mapping to piRNA loci as a function of the distance to piRNA TSSs, after removal of reads corresponding to mature 21U-RNAs (see Methods). The signal is normalized to counts per million of non-structural mapped reads. A peak of 5′ P ends centered at +38 from piRNA TSSs is observed.Number of loci with detected cleavage fragments in N2 empty vector nematodes across percentile bins of increasing termination signal strength (top panel). Distributions of fragment read counts per locus for detected loci across percentile bins of increasing termination signal strength (bottom panel). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range. The data shown here corresponds to one of two N2 EV replicates, see Appendix Figure S6 for this analysis across replicates and genotypes.


Figure 5Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - dependent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile range . Average normalized CAGE signal ( transcripts per million ) 2 nt upstream of motif - dependent and motif - independent piRNAs ( excluding undetected loci ) . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile rangeDistributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif - independent piRNAs upon ints - 11 RNAi knockdown in an N2 and a tfiis mutant background . N2 empty vector was used as a baseline for fold change calculation . Boxplots show the interquartile range with a line at the median ; the whiskers extend to the greatest point no more than 1 . 5 times the interquartile

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

Figure 5Distributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-dependent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background calculated from two merged replicates. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range.Average normalized CAGE signal (transcripts per million) 2 nt upstream of motif-dependent and motif-independent piRNAs (excluding undetected loci). Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile rangeDistributions of log2 fold change in CAGE signal corresponding to 2 nt upstream motif-independent piRNAs upon ints-11 RNAi knockdown in an N2 and a tfiis mutant background. N2 empty vector was used as a baseline for fold change calculation. Boxplots show the interquartile range with a line at the median; the whiskers extend to the greatest point no more than 1.5 times the interquartile range


Figure 1B . Deconvolved fluorescence micrographs of Chm7 - GFP and truncations with Nup170 - mCherry NE marker and merge of green and red channels . Scale bar is 5 μm . C . Scatter plot of the mean fluorescence intensity with SD of individual NE foci ( horizontal line is mean ) . > 50 foci were measured for each GFP fusion ; p values from un - paired student ' s T - test . * * * is p ≤ 0 . 001 ; * * * * is p ≤ 0 . 0001 . D . Deconvolved fluorescence micrographs of Chm7 - GFP and Mps3 - mCherry with merge of green and red images ( right ) . Scale bar is 5 μm . E . 10 - fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid ( - ) or those expressing the indicated Chm7 constructs ( pRS416 - HA - CHM7 , pRS416 - HA - chm7 - NTD , pRS416 - HA - chm7 - CTD ) . See Appendix Figure S2A for protein levels .

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Figure 1B. Deconvolved fluorescence micrographs of Chm7-GFP and truncations with Nup170-mCherry NE marker and merge of green and red channels. Scale bar is 5 μm.C. Scatter plot of the mean fluorescence intensity with SD of individual NE foci (horizontal line is mean). >50 foci were measured for each GFP fusion; p values from un-paired student's T-test. *** is p ≤ 0.001; **** is p ≤ 0.0001.D. Deconvolved fluorescence micrographs of Chm7-GFP and Mps3-mCherry with merge of green and red images (right). Scale bar is 5 μm.E. 10-fold serial dilutions of chm7Δapq12Δ strains grown at the indicated temperatures with either an empty pRS416 plasmid (-) or those expressing the indicated Chm7 constructs (pRS416-HA-CHM7, pRS416-HA-chm7-NTD, pRS416-HA-chm7-CTD). See Appendix Figure S2A for protein levels.


Figure 2A . Deconvolved inverted fluorescence micrographs Chm7 - GFP and truncations in the indicated null strains . Scale bar is 5 μm . B . Plot of the percentage of cells with SD of Chm7 - GFP ( and truncations ) NE foci from A . Data are from 3 independent replicates where > 200 cells were counted for each strain . p values from un - paired student ' s T - test . * * is a p value ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . C . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in a heh1Δ strain containing either an empty pRS426 plasmid ( - ) or those expressing HEH1 ( pRS426 - HEH1 ) or HEH2 ( pRS426 - HEH2 ) . D . Plots of the percentage of cells from C with Chm7 - GFP NE - foci . Data are from 3 independent replicates where > 200 cells were counted for each strain . Error bars are SD . p values from T - test . * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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Figure 2A. Deconvolved inverted fluorescence micrographs Chm7-GFP and truncations in the indicated null strains. Scale bar is 5 μm.B. Plot of the percentage of cells with SD of Chm7-GFP (and truncations) NE foci from A. Data are from 3 independent replicates where >200 cells were counted for each strain. p values from un-paired student's T-test. ** is a p value ≤ 0.01; ****, p ≤ 0.0001.C. Deconvolved inverted fluorescence micrographs of Chm7-GFP in a heh1Δ strain containing either an empty pRS426 plasmid (-) or those expressing HEH1 (pRS426-HEH1) or HEH2 (pRS426-HEH2).D. Plots of the percentage of cells from C with Chm7-GFP NE-foci. Data are from 3 independent replicates where >200 cells were counted for each strain. Error bars are SD. p values from T-test. **, p ≤ 0.01; ****, p ≤ 0.0001.


Figure 3C . GST , GST - Snf7 and GST - snf7OPEN were immobilized on GT - resin and incubated with buffer ( - ) or recombinant heh2 ( 1 - 308 ) - His6 . Bound proteins separated by SDS - PAGE were visualized by coomassie stain and by Western blot with anti - His6 antibody and ECL detection ( WB ; top panel ) . Middle panel shows indicated cropped region of gel where contrast has been increased . Numbers on side of gel show position of molecular weight ( MW ) markers . D . In vitro transcription translation ( IVT ) reactions generating radiolabeled ( 35S ) truncations of Heh2 ( inputs ) were incubated with bead - bound GST , GST - snf7OPEN or GST - Snf7 before washing , elution and detection of bound proteins by autoradiography ( top panel ) or coomassie staining ( bottom ) . E . As in C except with GST - Chm7 constructs . Upper panel shows western blot ( WB ) with anti - His6 antibody . * chm7 - NTD is also expressed with a His6 tag . Bottom panel is coomassie stained . F . As in E except with MBP - Snf7 .

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

Figure 3C. GST, GST-Snf7 and GST-snf7OPEN were immobilized on GT-resin and incubated with buffer(-) or recombinant heh2(1-308)-His6. Bound proteins separated by SDS-PAGE were visualized by coomassie stain and by Western blot with anti-His6 antibody and ECL detection (WB; top panel). Middle panel shows indicated cropped region of gel where contrast has been increased. Numbers on side of gel show position of molecular weight (MW) markers.D. In vitro transcription translation (IVT) reactions generating radiolabeled (35S) truncations of Heh2 (inputs) were incubated with bead-bound GST, GST-snf7OPEN or GST-Snf7 before washing, elution and detection of bound proteins by autoradiography (top panel) or coomassie staining (bottom).E. As in C except with GST-Chm7 constructs. Upper panel shows western blot (WB) with anti-His6 antibody. *chm7-NTD is also expressed with a His6 tag. Bottom panel is coomassie stained.F. As in E except with MBP-Snf7.


Figure 4A . Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7 - VC with either Heh1 - VN or Heh2 - VN . Cell borders are outlined . Scale bar is 5 μm . B . Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1 - VN and Snf7 - VC with either Chm7 - mCherry or Mps3 - mCherry and merge of both channels . Scale bar is 5 μm . C . Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains . Cell borders outlined . Scale bar is 5 μm . D , E . Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds . Data are from three independent replicates where > 225 cells per strain per replicate were quantified . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 ; * * * * , p ≤ 0 . 0001 . F . Deconvolved inverted fluorescence micrographs of Heh1 - VN Snf7 - VC BiFC signal in a chm7Δ strain with either an empty plasmid ( " - " ; pRS416 ) or one expressing CHM7 ( pRS416 - HA - CHM7 ) . Cell borders outlined . Scale bar is 5 μm . G . Plot of the percentage of cells with BiFC signal from F . Error bars are SD from the mean of each replicate . p values from un - paired student ' s T - test where ns is p > 0 . 05 . * * * , p ≤ 0 . 001 .

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

Figure 4A. Deconvolved inverted fluorescent micrographs of BiFC signal of Chm7-VC with either Heh1-VN or Heh2-VN. Cell borders are outlined. Scale bar is 5 μm.B. Deconvolved inverted fluorescent micrographs of yeast strains expressing Heh1-VN and Snf7-VC with either Chm7-mCherry or Mps3-mCherry and merge of both channels. Scale bar is 5 μm.C. Deconvolved inverted fluorescent micrographs of BiFC signal of the indicated VN and VC fusions in the indicated strains. Cell borders outlined. Scale bar is 5 μm.D,E. Plots of the percentage of cells with BiFC signal of the indicated VN VC pairs in the indicated null backgrounds. Data are from three independent replicates where > 225 cells per strain per replicate were quantified. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.F. Deconvolved inverted fluorescence micrographs of Heh1-VN Snf7-VC BiFC signal in a chm7Δ strain with either an empty plasmid ("-"; pRS416) or one expressing CHM7 (pRS416-HA-CHM7). Cell borders outlined. Scale bar is 5 μm.G. Plot of the percentage of cells with BiFC signal from F. Error bars are SD from the mean of each replicate. p values from un-paired student's T-test where ns is p > 0.05. ***, p ≤ 0.001.


Figure 5A . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in the indicated strains . Scale bar is 5 μm . The percentage of cells ± SD with Chm7 - GFP foci is indicated below each panel . B . Deconvolved fluorescence images of SINC - containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7 - GFP and Nup170 - mCherry ( green , red and merged images are shown ) . Scale bar is 1 μm . C . Plot of the proportion of nup clusters with Chm7 - GFP enrichment in the indicated genetic backgrounds . Error bars are SD of the mean from 3 independent replicates . D . Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7 - GFP and clustered Nup170 - mCherry in a nup133Δ strain . Scale bar is 1 μm . E . Plot of the total fluorescence / integrated density ( in arbitrary units ( A . U . ) ) of Chm7 - GFP within ( and outside of ) the SINC . Error bars represent SD deviation of the mean from > 50 SINC or non - SINC accumulations pooled from 3 independent replicates . p values from un - paired student ' s T - tests with Welch ' s correction . * * * * is p ≤ 0 . 0001 . F . Correlation of the total fluorescence intensity / integrated density of SINC - enriched Chm7 - GFP and Nup170 - mCherry within individual SINCs . Linear regression calculated from 150 SINCs pooled from 3 independent replicates ; r is the linear correlation ( Pearson ' s ) coefficient .

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Figure 5A. Deconvolved inverted fluorescence micrographs of Chm7-GFP in the indicated strains. Scale bar is 5 μm. The percentage of cells ± SD with Chm7-GFP foci is indicated below each panel.B. Deconvolved fluorescence images of SINC-containing nuclei in vps4Δ and vps4Δpom152Δ cells expressing Chm7-GFP and Nup170-mCherry (green, red and merged images are shown). Scale bar is 1 μm.C. Plot of the proportion of nup clusters with Chm7-GFP enrichment in the indicated genetic backgrounds. Error bars are SD of the mean from 3 independent replicates.D. Deconvolved fluorescence micrograph showing the lack of colocalization of Chm7-GFP and clustered Nup170-mCherry in a nup133Δ strain. Scale bar is 1 μm.E. Plot of the total fluorescence/integrated density (in arbitrary units (A.U.)) of Chm7-GFP within (and outside of) the SINC. Error bars represent SD deviation of the mean from > 50 SINC or non-SINC accumulations pooled from 3 independent replicates. p values from un-paired student's T-tests with Welch's correction. **** is p ≤ 0.0001.F. Correlation of the total fluorescence intensity/integrated density of SINC-enriched Chm7-GFP and Nup170-mCherry within individual SINCs. Linear regression calculated from 150 SINCs pooled from 3 independent replicates; r is the linear correlation (Pearson's) coefficient.


Figure 6A . 10 - fold serial dilutions of the indicated yeast strains were grown on YPD at 23 ° C for 3 days before imaging . C . Deconvolved fluorescence micrographs of Sna3 - mCherry in the indicated genetic backgrounds . Scale bar is 5 μm . D . Deconvolved fluorescence micrographs ( maximum intensity projections of a z - series of images ) of GFP - Nup49 in the indicated yeast strains ( arrows indicate SINCs ) . E . Plot of proportion of cells in the indicated strains with SINCs . Error bars are the SD from the mean from 3 independent replicates quantifying > 300 cells for each strain . p values from un - paired student ' s T - tests where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * , p ≤ 0 . 001 .

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Figure 6A.10-fold serial dilutions of the indicated yeast strains were grown on YPD at 23°C for 3 days before imaging.C. Deconvolved fluorescence micrographs of Sna3-mCherry in the indicated genetic backgrounds. Scale bar is 5 μm.D. Deconvolved fluorescence micrographs (maximum intensity projections of a z-series of images) of GFP-Nup49 in the indicated yeast strains (arrows indicate SINCs).E. Plot of proportion of cells in the indicated strains with SINCs. Error bars are the SD from the mean from 3 independent replicates quantifying >300 cells for each strain. p values from un-paired student's T-tests where ns is p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.


Figure 7A . Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7 - GFP ( percentages ± SD of the proportion of cells with Chm7 - GFP foci is at bottom of each panel , see Figure EV5B ) . Scale bar is 5 μm . B . Plot of the percentage of cells with the indicated number of Chm7 - GFP foci . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns represents p > 0 . 05 ; * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . C . Deconvolved inverted fluorescencemicrographs of strains expressing Chm7 - GFP after 5 h at 23 ° C ( top ) or 37 ° C ( bottom ) . Scale bar is 5 μm . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . D . Plots of percentage of cells from C with the indicated number of Chm7 - GFP foci at 23 ° C ( left ) and 37 ° C ( right ) . Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain . p values from 2 - way ANOVA where ns is p > 0 . 05 ; * , p ≤ 0 . 05 ; * * * , p ≤ 0 . 001 .

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Figure 7A. Deconvolved inverted fluorescent micrographs of the indicated strains expressing Chm7-GFP (percentages ± SD of the proportion of cells with Chm7-GFP foci is at bottom of each panel, see Figure EV5B). Scale bar is 5 μm.B. Plot of the percentage of cells with the indicated number of Chm7-GFP foci. Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns represents p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.C. Deconvolved inverted fluorescencemicrographs of strains expressing Chm7-GFP after 5 h at 23°C (top) or 37°C (bottom). Scale bar is 5 μm. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).D. Plots of percentage of cells from C with the indicated number of Chm7-GFP foci at 23°C (left) and 37°C (right). Error bars are SD from the mean from 3 independent replicates of > 50 foci per strain. p values from 2-way ANOVA where ns is p > 0.05; *, p ≤ 0.05; ***, p ≤ 0.001.


Figure 8A . Plot of the percentage of nup116Δ cells incubated for 3 h at 37 ° C with nuclear rim - like accumulations of Chm7 - GFP . Error bars are SD from the mean from 3 independent replicates of > 100 cells . p values from unpaired student ' s T - test where * * * is p ≤ 0 . 001 . B . Deconvolved inverted fluorescence micrographs of Chm7 - GFP in nup116Δ cells at either 23 ° C or grown for 3 h at 37 ° C . Percentages ± SD of the proportion of cells with Chm7 - GFP NE foci are shown under each panel ( see Figure EV5F ) . C . Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37 ° C . N denotes nuclear interior . Scale bars are 100 nm . D . 10 - fold serial dilutions of indicated strains grown in medium permissive ( - URA ) or restrictive ( 5 - FOA ) of the retention of a URA3 / NUP116 plasmid . Images acquired after incubation for 3 days ( left ) or 6 days ( right ) at 23 ° C .

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

Figure 8A. Plot of the percentage of nup116Δ cells incubated for 3 h at 37°C with nuclear rim-like accumulations of Chm7-GFP. Error bars are SD from the mean from 3 independent replicates of >100 cells. p values from unpaired student's T-test where *** is p ≤ 0.001.B. Deconvolved inverted fluorescence micrographs of Chm7-GFP in nup116Δ cells at either 23°C or grown for 3 h at 37°C. Percentages ± SD of the proportion of cells with Chm7-GFP NE foci are shown under each panel (see Figure EV5F).C. Electron micrographs showing herniations of the NE in nup116Δ cells after incubation for 3 h at 37°C. N denotes nuclear interior. Scale bars are 100 nm.D. 10-fold serial dilutions of indicated strains grown in medium permissive (-URA) or restrictive (5-FOA) of the retention of a URA3/NUP116 plasmid. Images acquired after incubation for 3 days (left) or 6 days (right) at 23°C.


Figure 9A . Deconvolved fluorescence micrographs of NLS - GFP in the indicated yeast strains at either 30 ° C or after 2 h at 37 ° C . Scale bar is 5 μm . B . Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS - GFP from cells represented in A . Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain . p values from un - paired student ' s T - test where ns is p > 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 . Only chm7Δ and chm7Δapq12Δ cells have N : C ratios below 1 . 2 ( dotted red line ) . C . Re - plotting of data from B showing the percentage of cells with a N : C NLS - GFP fluorescence ratio < 1 . 2 . Error bars are the SD from the mean from 3 independent replicates of 150 cells / strain . p values from student ' s T - test where * , p ≤ 0 . 05 ; * * , p ≤ 0 . 01 ; * * * * , p ≤ 0 . 0001 .

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

Figure 9A. Deconvolved fluorescence micrographs of NLS-GFP in the indicated yeast strains at either 30°C or after 2 h at 37°C. Scale bar is 5 μm.B. Plot of the mean nuclear to cytosolic fluorescence intensity ratio of NLS-GFP from cells represented in A. Error bars are SD from the mean from 3 independent replicates of 150 cells from each strain. p values from un-paired student's T-test where ns is p > 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001. Only chm7Δ and chm7Δapq12Δ cells have N:C ratios below 1.2 (dotted red line).C. Re-plotting of data from B showing the percentage of cells with a N:C NLS-GFP fluorescence ratio <1.2. Error bars are the SD from the mean from 3 independent replicates of 150 cells/strain. p values from student's T-test where *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001.


Figure 1C . Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6 , 8 , 11 , or 15 . The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes . Ts , trisomy . D . CGH analysis of trisomic ES cell lines . The relative gene copy number is plotted using a log2 scale . E . CGH analysis of the genome stability of trisomic ES cells . F . CGH analysis of the genome integrity of trisomic ES cell subclones .

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Figure 1C. Karyograms of trisomic ES cells shows that these cell lines have extra copies of chromosomes 6, 8, 11, or 15. The Ts6 and Ts11 ES cells harbored isochromosomes of the two identical trisomic chromosomes. Ts, trisomy.D. CGH analysis of trisomic ES cell lines. The relative gene copy number is plotted using a log2 scale.E. CGH analysis of the genome stability of trisomic ES cells.F. CGH analysis of the genome integrity of trisomic ES cell subclones.


Figure 2A . Cumulative growth curves of trisomic and wild - type ES cells . The number of ES cells was counted at the indicated days in culture . The cell lines were tested in triplicates . Error bars , ± S . D . * P < 0 . 05 , * * P < 0 . 01 . B . Cell cycle distribution of trisomic and wild - type ES cell lines . The percentage of cells in G1 , S , and G2 / M phases was determined via flow cytometry . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 . C . The percentage of Annexin V + apoptotic cells was quantified . Error bars , ± S . D . n = 3 . D . Trisomic and wild - type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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Figure 2A. Cumulative growth curves of trisomic and wild-type ES cells. The number of ES cells was counted at the indicated days in culture. The cell lines were tested in triplicates. Error bars, ±S.D. * P < 0.05, ** P < 0.01.B. Cell cycle distribution of trisomic and wild-type ES cell lines. The percentage of cells in G1, S, and G2/M phases was determined via flow cytometry. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.C. The percentage of Annexin V+ apoptotic cells was quantified. Error bars, ±S.D. n=3.D. Trisomic and wild-type ES cells were treated with Cycloheximide or MG132 for 48 hours at the indicated doses. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


Figure 3A . The coloning - formation rates of the trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 . B . Alkaline phosphatase ( AP ) staining of trisomic and wild - type ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm . C . Evaluation of AP positive and negative colonies after LIF withdrawal . Black bars , undifferentiated colonies ; orange bars , partially differentiated colonies ; green bars , totally differentiated colonies . Error bars , ± S . D . n = 3 . * * , P < 0 . 01 . D . Boxplots of EB diameter at the indicated days of EB formation . Each boxplot was plotted for more than 20 EBs . * P < 0 . 05 , * * P < 0 . 01 . E . Time course analysis of marker genes expression during EB formation by RT - qPCR . Error bars , ± S . D . n = 3 . F . Quantification of Oct4 , Nanog and Rex1 expression in trisomic and wild - type ES cell derived EBs at day 8 of differentiation . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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Figure 3A. The coloning-formation rates of the trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.B. Alkaline phosphatase (AP) staining of trisomic and wild-type ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.C. Evaluation of AP positive and negative colonies after LIF withdrawal. Black bars, undifferentiated colonies; orange bars, partially differentiated colonies; green bars, totally differentiated colonies. Error bars, ±S.D. n=3. **, P < 0.01.D. Boxplots of EB diameter at the indicated days of EB formation. Each boxplot was plotted for more than 20 EBs. * P < 0.05, ** P < 0.01.E. Time course analysis of marker genes expression during EB formation by RT-qPCR. Error bars, ±S.D. n=3.F. Quantification of Oct4, Nanog and Rex1 expression in trisomic and wild-type ES cell derived EBs at day 8 of differentiation. Error bars, ±S.D. n=3. ** P < 0.01.


Figure 4A . Teratomagrowth curves of trisomic and wild - type ES cells after subcutaneous injection into SCIDmice . 1 × 106 cells were injected per site ( 6 sites / per group ) . Error bars , ± S . D . * P < 0 . 05 . B . Four - week teratomas derived from wild - type and trisomic ( Ts8 or Ts11 ) ES cell lines . 5 × 105 cells were injected per site ( 8 sites / per group ) . C . Representative images of undifferentiated regions in Ts8 ES cell derived teratomas ( middle , top and bottom ) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells ( right , top and bottom ) . The images of mature neuroectoderm from wild - type ES cell formed teratomas ( left , top and bottom ) were shown . The bottom panels were the enlarged views of the white dotted boxes of the top panels . Hematoxylin and eosin staining . Scale bars , 50 μm . D . Immunohistochemistry analysis of Oct4 expression in wild - type , Homo - 11 , Ts6 , Ts8 , Ts11 , or Ts15 ES cellteratomas produced via the injection of 5 × 105 ES cells . Scale bar , 100 μm . E . AP staining of the primary cultured teratomacells derived from trisomic and wild - type ES cells . Ts6 , Ts8 , or Ts11 ESC teratomacells grew ES - like colonies that were stained positive for AP . Scale bar , 200 μm . F . The expression of marker genes in cultured teratoma cells ( p3 ) . Error bars , ± S . D . n = 3 . * * P < 0 . 01 .

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Figure 4A. Teratomagrowth curves of trisomic and wild-type ES cells after subcutaneous injection into SCIDmice. 1×106 cells were injected per site (6 sites/per group). Error bars, ±S.D. * P < 0.05.B. Four-week teratomas derived from wild-type and trisomic (Ts8 or Ts11) ES cell lines. 5×105 cells were injected per site (8 sites/per group).C. Representative images of undifferentiated regions in Ts8 ES cell derived teratomas (middle, top and bottom) and the primitive neural rosettes in the teratomas derived from Ts15 ES cells (right, top and bottom). The images of mature neuroectoderm from wild-type ES cell formed teratomas (left, top and bottom) were shown. The bottom panels were the enlarged views of the white dotted boxes of the top panels. Hematoxylin and eosin staining. Scale bars, 50 μm.D. Immunohistochemistry analysis of Oct4 expression in wild-type, Homo-11, Ts6, Ts8, Ts11, or Ts15 ES cellteratomas produced via the injection of 5×105 ES cells. Scale bar, 100 μm.E. AP staining of the primary cultured teratomacells derived from trisomic and wild-type ES cells. Ts6, Ts8, or Ts11 ESC teratomacells grew ES-like colonies that were stained positive for AP. Scale bar, 200 μm.F. The expression of marker genes in cultured teratoma cells (p3). Error bars, ±S.D. n=3. ** P < 0.01.


Figure 5B . Karyograms of Di8 , Di11 and Di15 ES cell lines . Di8 was the corrected ES cell line from Ts8 , showing diploid karyotype and Y chromosome loss . Di11 and Di15 were from Ts11 and Ts15 ES cells respectively . C . AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal ( LIF 0 U / ml ) for 5 days . Scale bar , 500 μm . D . Teratomagrowth curves of trisomic and the corrected diploid ES cells . The injected cell number was 5 × 105 / site ( 8 sites / per group ) . Error bars , ± S . D . * * P < 0 . 01 . E . Scatterplot of four - week teratoma weight . The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells . * P < 0 . 05 .

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Figure 5B. Karyograms of Di8, Di11 and Di15 ES cell lines. Di8 was the corrected ES cell line from Ts8, showing diploid karyotype and Y chromosome loss. Di11 and Di15 were from Ts11 and Ts15 ES cells respectively.C. AP staining of trisomic and the corrected diploid ES cells after LIF withdrawal (LIF 0 U/ml) for 5 days. Scale bar, 500 μm.D. Teratomagrowth curves of trisomic and the corrected diploid ES cells. The injected cell number was 5×105/site (8 sites/per group). Error bars, ±S.D. ** P < 0.01.E. Scatterplot of four-week teratoma weight. The teratomas were formed by the corrected diploid ES cells and their paternal trisomic ES cells. * P < 0.05.


Figure 6F . RT - qPCR analysis of some gene expression in trisomic and wild - type ES cells . Error bars , ± S . D . n = 3 . * P < 0 . 05 , * * P < 0 . 01 .

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Figure 6F. RT-qPCR analysis of some gene expression in trisomic and wild-type ES cells. Error bars, ±S.D. n=3. * P < 0.05, ** P < 0.01.


Figure 7A . Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines . Hematoxylin and eosin staining . Scale bar , 100 μm . B . Quantification of Nanog expression in trisomic , wild - type , and mixed ES cell - derived EBs cultured for 8 days . Error bars , ± S . D . n = 3 . * * P < 0 . 01 . C . The morphology of EBs at day 10 of differentiation . M15 was the normal EB formation condition and was used as control . Black arrows indicate the cystic structures . D . Boxplots of EBs diameter at day 10 of EB formation under M15 , CM , and M15 supplemented with Bmp4 conditions . Each boxplot was plotted for more than 10 EBs . EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition . * P < 0 . 05 , * * P < 0 . 01 .

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Figure 7A. Morphology of EBs grew for 8 days derived from individual or mixed ES cell lines. Hematoxylin and eosin staining. Scale bar, 100 μm.B. Quantification of Nanog expression in trisomic, wild-type, and mixed ES cell-derived EBs cultured for 8 days. Error bars, ±S.D. n=3. ** P < 0.01.C. The morphology of EBs at day 10 of differentiation. M15 was the normal EB formation condition and was used as control. Black arrows indicate the cystic structures.D. Boxplots of EBs diameter at day 10 of EB formation under M15, CM, and M15 supplemented with Bmp4 conditions. Each boxplot was plotted for more than 10 EBs. EBs cultured under CM or Bmp4 condition were compared to EBs of the same cell line cultured under M15 condition. * P < 0.05, ** P < 0.01.


Figure 1C Confocal images of rNSC - derived astrocytes expressing GFP ( green ) and astrocyte - specific markers ( ACSBG1 , GFAP , S100β ; magenta ) at different timepoints ( P7 , P10 , P14 ) ; lower pictures depict single - channel images the astrocyte markers ( grey ) used above . Data information : ; scale bars ( C = 10 µm , D Representative pictures of recombined astrocytes ( GFP + / ACSBG1 + ; arrowheads ) during DG development ( P3 , P7 , P10 , P14 ) . E , Quantification of total numbers of recombined astrocytes ( GFP + / ACSBG1 + ) per area at postnatal timepoints in the total DG ( E ) Data information : All data are represented as mean ± SEM ; number of experimental animals throughout figure : n = 5 ( P3 ) and n = 6 ( P7 , P10 , P14 ) , ( E indicated by red dots dotted lines mark GZ borders ; scale bars ( D = 50 µm . , F Quantification of total numbers of recombined astrocytes ( GFP + / ACSBG1 + ) per area at postnatal timepoints in the distinct DG compartments ( F ) , i . e . hilus , GZ , and ML indicated by different colours . G Representative images of proliferating astrocytes ( ACSBG1 + , cyan ; arrowheads ) in combination with cell cycle markers KI67 or MCM2 ( magenta ; P3 , P7 : KI67 ; P10 , P14 : MCM2 ) . Data information : ; dotted lines mark GZ borders ; scale bars G ) = 50 µm . H Confocal image of proliferating astrocytes at P10 ( ACSBG1 + , cyan ; MCM2 + , magenta ) that did not express NESTIN ( white ) . I , J Quantification of total proliferating astrocytes ( ACSBG1 + / KI67 + for P3 / P7 ; ACSBG1 + / MCM2 + for P10 / P14 ) per area at distinct timepoints per total DG ( I ) or hilus , GZ and ML ( J ) . Data information : All data are represented as mean ± SEM ; number of experimental animals throughout figure : n = 5 ( P3 ) and n = 6 ( P7 , P10 , P14 ) , I ) indicated by red dots ; scale bars , H = 10 µm , K Confocal images of proliferating rNSC - derived astrocytes ( GFP + / ACSBG1 + / MCM2 + ; arrowheads ) at P10 . Data information : scale bars K ) = 10

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Figure 1C Confocal images of rNSC-derived astrocytes expressing GFP (green) and astrocyte-specific markers (ACSBG1, GFAP, S100β; magenta) at different timepoints (P7, P10, P14); lower pictures depict single-channel images the astrocyte markers (grey) used above. Data information: ; scale bars (C = 10 µm,D Representative pictures of recombined astrocytes (GFP+/ACSBG1+; arrowheads) during DG development (P3, P7, P10, P14). E, Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the total DG (E) Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), (E indicated by red dots dotted lines mark GZ borders; scale bars (D = 50 µm., F Quantification of total numbers of recombined astrocytes (GFP+/ACSBG1+) per area at postnatal timepoints in the distinct DG compartments (F), i.e. hilus, GZ, and ML indicated by different colours.G Representative images of proliferating astrocytes (ACSBG1+, cyan; arrowheads) in combination with cell cycle markers KI67 or MCM2 (magenta; P3, P7: KI67; P10, P14: MCM2). Data information: ; dotted lines mark GZ borders; scale bars G) = 50 µm.H Confocal image of proliferating astrocytes at P10 (ACSBG1+, cyan; MCM2+, magenta) that did not express NESTIN (white). I, J Quantification of total proliferating astrocytes (ACSBG1+/KI67+ for P3/P7; ACSBG1+/MCM2+ for P10/P14) per area at distinct timepoints per total DG (I) or hilus, GZ and ML (J). Data information: All data are represented as mean ± SEM; number of experimental animals throughout figure: n = 5 (P3) and n = 6 (P7, P10, P14), I) indicated by red dots; scale bars ,H = 10 µm,K Confocal images of proliferating rNSC-derived astrocytes (GFP+/ACSBG1+/MCM2+; arrowheads) at P10. Data information: scale bars K) = 10 µm


Figure 2B Representative pictures of P28 animals that received BrdU at P3 , P7 , P14 or P21 . For cell type identification of generated progeny , immunostainings against NESTIN ( white ) , GFAP ( green ) , and BrdU ( magenta ) were performed . rNSCs were identified as NESTIN + / GFAP + / BrdU + with a radial process ( as indicated with arrows ) , neurons as NESTIN - / GFAP - / BrdU + cells , and astrocytes as NESTIN - / GFAP + / BrdU + cells in the hilus , GZ , and ML ( arrowheads ) . Data information : dotted lines label GZ borders ; scale bars = 50 µm . C Quantification of total numbers of generated cell types ( rNSCs , neurons , astrocytes ) per area at distinct timepoints as indicated by different colours . Data information : Values represent mean ± SEM ; number of experimental animals : ( C n = 5 ( P3 , P21 ) , n = 4 ( P7 , P14 ) ; D Quantification of total numbers of newly generated astrocytes in the hilus , GZ , and ML at P3 , P7 , P14 , P21 . Data information : Values represent mean ± SEM ; number of experimental animals , D ) n = 5 ( P3 , P21 ) , n = 4 ( P7 , P14 ) G Representative pictures of immunostainings against cell type - specific proteins ( all depicted in white ) . Recombined cells were identified by GFP , rNSCs by their radial process labelled with NESTIN ( P3 , P7 ) or GFAP ( P10 , P14 ) , astrocytes by ACSBG1 , immature neurons expressed DCX , and proliferating cells the cell cycle markers KI67 ( P3 , P7 ) or MCM2 ( P10 , P14 ) . H Quantification of the percentages of GFP + recombined cell types out of all recombined cells at distinct timepoints . Intermediate precursor cells ( IPCs ) identified by TBR2 expression were added to DCX + neuroblasts . I Quantification of the proportions of proliferating cell types ( rNSCs , IPCs / neuroblasts , astrocytes ) out of all proliferating cells at P3 , P7 , P10 , P14 . Data information : Values represent mean ± SEM ; number of experimental animals : ( H , I ) n = 5 ( P3 ) , = 6 ( P7 , P10 , P14 ) ; dotted lines label GZ borders ; scale bars = 50 µm .

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Figure 2B Representative pictures of P28 animals that received BrdU at P3, P7, P14 or P21. For cell type identification of generated progeny, immunostainings against NESTIN (white), GFAP (green), and BrdU (magenta) were performed. rNSCs were identified as NESTIN+/GFAP+/BrdU+ with a radial process (as indicated with arrows), neurons as NESTIN-/GFAP-/BrdU+ cells, and astrocytes as NESTIN-/GFAP+/BrdU+ cells in the hilus, GZ, and ML (arrowheads). Data information: dotted lines label GZ borders; scale bars = 50 µm.C Quantification of total numbers of generated cell types (rNSCs, neurons, astrocytes) per area at distinct timepoints as indicated by different colours. Data information: Values represent mean ± SEM; number of experimental animals: (C n = 5 (P3, P21), n = 4 (P7, P14);D Quantification of total numbers of newly generated astrocytes in the hilus, GZ, and ML at P3, P7, P14, P21. Data information: Values represent mean ± SEM; number of experimental animals ,D) n = 5 (P3, P21), n = 4 (P7, P14)G Representative pictures of immunostainings against cell type-specific proteins (all depicted in white). Recombined cells were identified by GFP, rNSCs by their radial process labelled with NESTIN (P3, P7) or GFAP (P10, P14), astrocytes by ACSBG1, immature neurons expressed DCX, and proliferating cells the cell cycle markers KI67 (P3, P7) or MCM2 (P10, P14). H Quantification of the percentages of GFP+ recombined cell types out of all recombined cells at distinct timepoints. Intermediate precursor cells (IPCs) identified by TBR2 expression were added to DCX+ neuroblasts. I Quantification of the proportions of proliferating cell types (rNSCs, IPCs/neuroblasts, astrocytes) out of all proliferating cells at P3, P7, P10, P14. Data information: Values represent mean ± SEM; number of experimental animals: (H,I) n = 5 (P3), = 6 (P7, P10, P14); dotted lines label GZ borders; scale bars = 50 µm.