g , Survival curve of fasted neonates . Neonates obtained by Caesarean section resuscitated were fasted their survival monitored ( + / + : n = 13 ; G / + : n = 26 ; G / G : n = 10 ) . h , Survival curve of fasted neonates treated with rapamycin . Neonates obtained by Caesarean section resuscitated were fasted their survival monitored ( + / + : n = 13 ; G / + : n = 13 ; G / G : n = 6 ) . Numbers indicate median survival for each curve . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 005 .

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g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.


a , Glycaemia drop in RagA + / + , RagAGTP / + RagAGTP / GTP recovery in fasted RagA + / + RagAGTP / + but not in RagAGTP / GTP neonates ( + / + : n = 5 , 18 , 4 , 5 , 9 , 8 ; G / + : n = 10 , 26 , 10 , 13 , 26 , 21 ; G / G : n = 7 , 20 , 9 , 10 , 16 , 11 , for 0 , 1 , 2 , 3 , 6 10 h , respectively ; mean ± s . e . m . ) .

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a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.).


b , Rapamycin significantly increases glycaemia in RagAGTP / GTP fasted for 6 10 h ( mean ± s . e . m . ) . NS , not significant .

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b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant.


c , Extension of survival by glucose injections in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; glucose : n = 5 ) .

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c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5).


d , Normal expression of genes involved in glucose metabolism in neonatal liver ( + / + : n = 2 , G / + : n = 5 ; G / G : n = 4 ; mean ± s . e . m . ) .

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d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.).


e , Left : representative electron microscopy showing abundant glycogen content in RagA + / + RagAGTP / GTP hepatocytes before fasting ( 0 h , upper panels ) more pronounced glycogen depletion after 10 h of fasting ( lower panels ) in RagAGTP / GTP neonates . Right : of hepatic glycogen content ( + / + : n = 5 , 3 , 4 , 4 ; G / + : n = 11 , 7 , 14 , 15 ; G / G : n = 6 , 4 , 4 , 6 ; for 0 , 3 , 6 10 h , respectively ; mean ± s . e . m . ; AU , arbitrary units ) .

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e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units).


. f , Partial rescue of hepatic glycogen content in RagAGTP / GTP neonates fasted for 10 h treated with rapamycin ( rapa ) ( mean ± s . e . m . ) .

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. f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.).


g , of neonatal plasma amounts of branched - chain ( BCAA ) essential at birth ( left ) , after 10 h fasting ( middle ) after 10 h fasting with rapamycin treatment ( right ) ( n for + / + , G / + G / G , respectively : n = 4 , 5 4 for 0 h ; n = 4 , 4 3 for 10 h ; n = 2 , 5 3 for rapamycin ; mean ± s . d . ) . Values are expressed relative to RagA + / + amounts at each time point .

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g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point.


h , Extension of survival by injection of a combination of gluconeogenic ( a . a . ) in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; : n = 8 ) . * P 0 . 05 ; * * P 0 . 01 ; ns , P > 0 .

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h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0


a , Top : representative of autophagosomes autophagolysosomes in hepatocytes from RagA + / + neonates fasted for 1 h . Typical autophagosome with a double limiting ( arrows ) ; autophagosome several autolysosomes ( arrowheads ) . Scale bars , 5 µm . Bottom : frequency of two types of organelle ( early : autophagosomes ; late : autophagolysosomes ) detected in profiles of hepatocytes skeletal myocytes from RagA + / + RagAGTP / GTPneonates .

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a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates.


b , extracts from livers of neonates at Caesarean section ( 0 h ) fasted for 10 h were immunoblotted for autophagy markers LC3B p62 .

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b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62.


c , extracts from skeletal muscle heart from neonates at Caesarean section ( 0 h ) , fasted for 1 2 h , were immunoblotted for indicated .

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c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins.


d , Triggering of autophagy by - acid withdrawal in MEFs . MEFs were deprived of for indicated time points , whole - extracts were obtained mTORC1 activity autophagic activity determined by immunoblotting .

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d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting.


e , Recombinant LC3B - GFP was expressed in RagA + / + RagAGTP / GTPMEFs LC3B localization , in presence absence of , monitored by microscopy . LC3B - GFP ( green ) clustering , indicative of autophagy , was observed in acid - starved RagA + / + but not RagAGTP / GTPMEFs . Scale bar , 10 µm .

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e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm.


f , Localization of recombinant TFEB - GFP was determined in MEFs as in e . ( active ) TFEB was observed in RagA + / + MEFs upon - acid withdrawal .

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f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.


a ) AMPKa1 / a2 double knockout ( DKO ) wild - type ( WT ) MEFs were deprived of glucose for 1 h re - stimulated for 10 min . Wholecell extracts were immunoblotted for indicated .

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a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins.


b , Immortalized MEFs of indicated genotypes were deprived of growth factors , glucose , or glucose for 1 h re - stimulated with glucose / or for 10 min . Whole - lysates were immunoblotted for indicated .

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b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins.


c , RagA + / + RagAGTP / GTP immortalized MEFs were deprived of glucose or surviving quantified in triplicate after 48 h . number is indicated relative to number at start of treatment ; mean ± s . d . ; * * * P 0 .

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c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005


d , mTOR localization as detected by immunofluorescence . In HEK - 293T , glucose deprivation causes mTOR to localize to diffuse small throughout cytoplasm . Re - addition of glucose leads to mTOR shuttling to lysosomal surface , co - localizing with lysosomal Lamp2 . HEK - 293T - RagBGTP show mTOR localized at lysosomal surface , regardless of glucose concentrations . Scale bars , 10 μm .

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d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm.


e , Glucose affect binding of v - ATPase to Ragulator . HEK - 293T expressing Flag - p14 was deprived of glucose or for 90 min re - stimulated for 20 min . extracts immunoprecipitates were immunoblotted for indicated .

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e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins.


f , RagA + / + RagAGTP / GTP MEFs were cultured for 1 h in media with glucose - acid concentrations measured in neonates at birth ( 0 h ) or after fasting for 1 h ( 1 h ) whole - extracts were analysed by immunoblotting .

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f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.


( B ) Pol II ChIP - seq was performed in six different tissues shown in left panel ( scale bar - 100 µm ) . middle right panels show read normalized Pol II ChIP - seq tracks ( RPM ) from six tissues at genes . For each gene , grey red tracks indicate non - expressing tissues expressing tissue , respectively . middle panel shows Pol II profile at two non - paused genes , which have Pol II only in expressing tissues . expression is limited to specific tissues as shown in in situ from BDGP . right panel shows Pol II profile at a paused gene , which has Pol II in observed tissues , although expression is limited to specific tissues as shown in in situ from BDGP . Paused Pol II is generally highest in tissue with highest expression . We also found systematic differences between samples , thus some tissues have generally higher enrichments than others , presumably because they are easier to crosslink .

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(B) Pol II ChIP-seq was performed in six different tissues shown in the left panel (scale bar - 100 µm). The middle and the right panels show the read count normalized Pol II ChIP-seq tracks (RPM) from the six tissues at individual genes. For each gene, grey and red tracks indicate non-expressing tissues and expressing tissue, respectively. The middle panel shows the Pol II profile at two non-paused genes, which have Pol II only in the expressing tissues. The expression is limited to specific tissues as shown in the in situ images from BDGP. The right panel shows the Pol II profile at a paused gene, which has Pol II in all observed tissues, although the expression is limited to specific tissues as shown in the in situ images from BDGP. Paused Pol II is generally highest in the tissue with the highest expression. We also found systematic differences between samples, thus some tissues have generally higher enrichments than others, presumably because they are easier to crosslink.


( C , D ) Normalized gene expression ( read normalized for each , log2 ) in different tissues , from scRNA - seq experiment , for a TATA group gene , a highly paused group gene , are shown . ( C ) TATA gene , Ccp84Aa , shows noisy expression in epidermis , without detectable background expression in non - expressing tissues . ( D ) highly paused gene , Gip , shows very robust expression in crystal , but has high background expression in non - expressing tissues .

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(C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues.


C , D ) ATAC - seq accessibility was measured in different tissues isolated from 14 - 17 h using INTACT . ( C ) Read - normalized ATAC ( RPM ) signal at genes from TATA group ( Ccp84Aa ) highly paused gene group ( ect ) are shown . ( D ) Read - normalized ATAC signals ( RPM ) from different tissues were calculated for each gene from 150 bp upstream of TSS to TSS . Paused genes show higher average accessibility across tissues compared to TATA genes ( Wilcoxon two - sided , * P < 2 . 2 * 10 - 16 ) .

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C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).


B . MASTL was ablated in MDA - MB - 231 using isgMASTL system . 72 h after Dox , were starved of glucose in media in presence of 10 % dFBS for 1 h ( - ) then re - stimulated with 25 mM of glucose for 10 min before recovery ( + ) . Whole - lysates were blotted with indicated antibodies . * in indicates unspecific band . β - Actin was used as a loading control .

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B. MASTL was ablated in MDA-MB-231 cells using the isgMASTL system. 72 h after Dox, cells were starved of glucose in the media in presence of 10% dFBS for 1 h (-) and then re-stimulated with 25 mM of glucose for 10 min before recovery (+). Whole-cell lysates were blotted with the indicated antibodies. * in indicates unspecific band. β-Actin was used as a loading control.


C . MASTL was depleted in MDA - MB - 231 using shRNAs against MASTL ( + ) or scrambled shRNAs ( − ) as control . were starved from glucose for 2 h re - stimulated with 5 mM glucose for 15 min 1 h . Total extracts were recovered tested for indicated antibodies . β - Actin was used as a loading control . D . of phospho - AKT T308 in glucose deprivation or glucose stimulation ( 15 min ) conditions in MDA - MB - 231 control MASTL - depleted either using shRNA or inducible sgRNA . Bars displays mean data + from three independent experiments . Significance determined by Student ' s t - comparing feedback activation in MASTL - depleted versus control ( ns , not significant ; * P < 0 . 05 ) .

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C. MASTL was depleted in MDA-MB-231 cells using shRNAs against MASTL (+) or scrambled shRNAs (−) as control. Cells were starved from glucose for 2 h and re-stimulated with 5 mM glucose for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β-Actin was used as a loading control. D. Quantification of phospho-AKT T308 levels in glucose deprivation or glucose stimulation (15 min) conditions in MDA-MB-231 control cells and MASTL-depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).


E . Immunoblot with indicated antibodies in BT - 549 upon glucose deprivation 15 min glucose stimulation . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . β - Actin was used as a loading control . Bar chart displays mean data + from 3 experiments . Significance determined by Student ' s t - comparing feedback activation in MASTL - depleted versus control ( ns , not significant ) . F . Immunoblot with indicated antibodies in MCF - 7 upon glucose deprivation glucose stimulation for 15 min . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . Bar chart displays mean data + from 3 experiments . Significance determined by Student ' s t - comparing feedback activation in MASTL - depleted versus control ( ns , not significant ; * P < 0 . 05 ) .

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E. Immunoblot analysis with the indicated antibodies in BT-549 cells upon glucose deprivation and 15 min glucose stimulation. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. β-Actin was used as a loading control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant). F. Immunoblot analysis with the indicated antibodies in MCF-7 cells upon glucose deprivation and glucose stimulation for 15 min. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).


A . Time course experiment at indicated time points after 100 nM insulin stimulation in 6 h serum - starved control MASTL knockout MDA - MB - 231 using isgMASTL system . plot shows of phospho - AKT T308 after 30 min of insulin stimulation . Plots displays mean data + from 5 independent experiments . * P < 0 . 05 , Student ' s t - .

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A. Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum-starved control and MASTL knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.


B . TSC2 was knocked down in control or MASTL - null MDA - MB - 231 using specific ( + ) or Scrambled ( − ) shRNAs . were starved for glucose for 1 h . plot shows of phospho - AKT T308 . Data are mean + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

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B. TSC2 was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose for 1 h. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.


C . MDA - MB - 231 control ( − ) or MASTL null ( + ) were treated with 100 nM rapamycin or vehicle as control . were starved for glucose for 1 h re - stimulated with glucose for 15 min ; rapamycin was added 15 min before glucose stimulation . plot shows of phospho - AKT T308 . Data are mean + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

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C. MDA-MB-231 control (−) or MASTL null (+) cells were treated with 100 nM rapamycin or vehicle as control. Cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min; rapamycin was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.


A . Immunofluorescence for GLUT4 ( magenta ) in MDA - MB - 231 in conditions of serum starvation insulin stimulation . DAPI ( ) is in cyan a mask is in yellow . Arrowheads indicate positive for GLUT4 enrichment at plasma ( PM ) . Representative magnifications are also shown . Scale bars , 10 μm plot shows of of positive for GLUT4 translocation at PM . Data are mean fold increase vs serum deprivation in scramble condition + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA . Control inmunoblotting in conditions of insulin stimulation is also shown for indicated antibodies .

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A. Immunofluorescence for GLUT4 (magenta) in MDA-MB-231 cells in conditions of serum starvation and insulin stimulation. DAPI (DNA) is in cyan and a cell mask is in yellow. Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM. Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies.


E . GLUT4 ( magenta ) in skeletal muscle sections . Vinculin ( ) DAPI ( ) is shown in yellow cyan , respectively . Scale bars , 50 µm . Mice were fasted overnight for 16 h , re - fed for 2 h before sample collection . of mean [ relative units x103 ; n = 3 Mastl ( + / + ) 3 Mastl ( ∆ / ∆ ) ] is shown . Data points reflect mean + ; * P < 0 . 05 ; * * * P < 0 . 001 ; unpaired Student ' s t - .

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E. GLUT4 staining (magenta) in skeletal muscle sections. Vinculin (membrane) and DAPI (DNA) staining is shown in yellow and cyan, respectively. Scale bars, 50 µm. Mice were fasted overnight for 16 h, and re-fed for 2 h before sample collection. Quantification of the mean intensity [relative units x103; n=3 Mastl(+/+) and 3 Mastl(∆/∆)] is shown. Data points reflect the mean + SEM; *P<0.05; ***P <0.001; unpaired Student's t-test.


G . Immunoblot with indicated antibodies in liver tissues from Mastl ( + / + ) ( n = 6 ) Mastl ( Δ / Δ ) ( n = 7 ) mice . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg ) , sacrificed 30 min later for sample collection . of relative fold change signal of phospho - AKT T308 ( normalized to total AKT level ) . n = 6 Mastl ( + / + ) 7 Mastl ( Δ / Δ ) mice . Charts reflect mean + ; * P < 0 . 05 ; unpaired Student ' s t - .

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G. Immunoblot with the indicated antibodies in liver tissues from Mastl(+/+) (n=6) and Mastl(Δ/Δ) (n=7) mice. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of phospho-AKT T308 (normalized to total AKT level). n=6 Mastl(+/+) and 7 Mastl(Δ/Δ) mice. Charts reflect the mean + SEM; *P<0.05; unpaired Student's t-test.


A . Co - depletion of ENSA ARPP19 in MDA - MB - 231 using specific siRNAs or control siRNAs against luciferase ( Luc ) . 72 h after transfection were starved for glucose for 1 h re - stimulated with glucose for 15 min . Total extracts were collected analyzed for indicated antibodies .

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A. Co-depletion of ENSA and ARPP19 proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase (Luc). 72 h after transfection cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.


B . isgMASTL were previously infected with lentiviral supernatants expressing FLAG - HA - tagged versions of ENSA ARPP19 phospho - mimetic mutants ( S67D S62D ) or GFP as a control . were treated with Dox to induce MASTL CRISPR / Cas9 - dependent knock - out , 72 h later were treated as specified in A . plot displays mean + from 3 independent experiments . Significance determined by Student ' s t - comparing ENSA / ARPP19 overexpression versus control ( ns , not significant ; * P < 0 . 05 ) .

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B. isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG- and HA-tagged versions of ENSA and ARPP19 phospho-mimetic mutants (S67D and S62D) or GFP as a control. Cells were treated with Dox to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19 overexpression versus control cells (ns, not significant; *P<0.05).


C . Control ( Dox − ) MASTL knockout ( Dox + ) were treated with 5 µM fostriecin , 50 nM okadaic acid ( OA ) or vehicle ( Veh ) 10 min before insulin stimulation for 30 min . Total lysates were analyzed for indicated antibodies P - T308 - AKT signal was quantified . β - Actin was used as a loading control . plot represents mean + from 3 independent experiments , except for fostriecin treatment in which only data from two experiments were obtained no error bars are shown . * P < 0 . 05 ; Student ' s t - .

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 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 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

C. Control (Dox −) and MASTL knockout (Dox +) cells were treated with 5 µM fostriecin, 50 nM okadaic acid (OA) or vehicle (Veh) 10 min before insulin stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.


A . MDA - MB - 231 were co - transduced with specific siRNAs against PPP2R2A ( B55α ) PPP2R2D ( B55δ ) transcripts ( B55αδ ) or against luciferase ( Luc ) as a control . Asynchronous were collected blotted for indicated antibodies . histograms represent quantifications of specified phospho - residues . Plots show mean + from 3 independent experiments . * P < 0 . 05 , Student ' s t - .

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A. MDA-MB-231 cells were co-transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phospho-residues. Plots show mean + SEM from 3 independent experiments. *P<0.05, Student's t-test.


B . Immunodetection of indicated antigens in control ( − ) MASTL knockout ( + ) starved from glucose re - stimulated with glucose for 15 min .

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B. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re-stimulated with glucose for 15 min.


C . siRNA - mediated genetic depletion of B55alpha delta subunits ( B55αδ ) in control ( Dox − ) MASTL - null ( Dox + ) . phosphorylation status of different was scored in conditions of glucose re - addition . n = 3 independent experiments . plot shows mean + from 3 independent experiments . * P < 0 . 05 , 1 - way ANOVA .

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C. siRNA-mediated genetic depletion of B55alpha and delta subunits (B55αδ) in control (Dox −) and MASTL-null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.


D . Immunoblot with indicated antibodies in skeletal muscle ( gastrocnemius ) extracts . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg ) , sacrificed 30 min later for sample collection . of relative fold change signal of total IRS1 , phospho - S6K1 T389 ( normalized to total S6K1 level ; n = 6 Mastl ( + / + ) 7 Mastl ( ∆ / ∆ ) ) . vinculin were used as a loading control . Data are mean + ; * P < 0 . 05 , unpaired Student ' s t - .

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D. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1 T389 (normalized to total S6K1 level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). vinculin were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.


A . MDA - MB - 231 were starved for 1 h re - stimulated with glucose for 15 min T194 phosphorylation was tested with a specific antibody . plot shows of phospho - MASTL T194 ( n = 2 independent experiments ) . Data are mean + from 3 independent experiments . ns , not significant ; Student ' s t - .

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A. MDA-MB-231 cells were starved for 1 h and re-stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody. The plot shows the quantification of phospho-MASTL T194 (n=2 independent experiments). Data are mean + SEM from 3 independent experiments. ns, not significant; Student's t-test.


B . Cas9 - expressing control ( − ) or with CRIPSR / Cas9 - mediated TSC2 knockout ( sgTSC2 ) were treated with rapamycin ( Rap ) , Torin1 ( Tor ) , or vehicle as control ( V ) . MASTL - knockdown ( shMASTL ) were used as an additional control of specificity of antibody . Inhibitors were added 15 min before glucose re - stimulation for additional 15 min total lysates were harvested for immunoblot . plot shows of phospho - MASTL T194 as mean from 2 independent 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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

B. Cas9-expressing control (−) cells or cells with CRIPSR/Cas9-mediated TSC2 knockout (sgTSC2) were treated with rapamycin (Rap), Torin1 (Tor), or vehicle as control (V). MASTL-knockdown cells (shMASTL) were used as an additional control of the specificity of the antibody. Inhibitors were added 15 min before glucose re-stimulation for additional 15 min and total cell lysates were harvested for immunoblot. The plot shows the quantification of phospho-MASTL T194 as mean from 2 independent experiments.


C . In vitro kinase using purified GST - human MASTL as a substrate , recombinant mTORC1 S6K1 kinases . Red arrowheads show phosphorylation of MASTL in presence of mTORC1 or S6K1 . Note appearance of other phosporylation signals in mTORC1 S6K1 reactions , independently of presence of MASTL , which correspond to autophosphorylation of these kinases . Imperial shows amount of GST - MASTL used as a substrate in each reaction .

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C. In vitro kinase assay using purified GST-human MASTL as a substrate, and recombinant mTORC1 and S6K1 kinases. Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1. Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions, independently of the presence of MASTL, which correspond to autophosphorylation of these kinases. Imperial staining shows the amount of GST-MASTL protein used as a substrate in each reaction.


F . In vitro kinase activity of Mastl mutant isoforms . HEK - 293T were transiently transfected with indicated GFP fusions of mouse Mastl mutants . Anti - GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 ( circles ) or ENSA ( filled circles ) as a substrate . graph shows relative catalytic activity of indicated mutants . Kinase activity of GFP - mMastl wild - type was set as 1 ( n = 3 independent experiments ) . Data are mean + from 3 independent experiments . * * * P < 0 . 001 ; 1 - way ANOVA .

F. In vitro kinase activity of Mastl mutant isoforms. HEK-293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants. Anti-GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 (white circles) or ENSA (filled circles) as a substrate. The graph shows the relative catalytic activity of the indicated mutants. Kinase activity of GFP-mMastl wild-type was set as 1 (n=3 independent experiments). Data are mean + SEM from 3 independent experiments. ***P <0.001; 1-way ANOVA.


G . isgMASTL MDA - MB - 231 were transduced with indicated Mastl mutants or GFP alone as a control , treated with doxycycline ( Dox ) to induce Mastl deletion . were collected after glucose stimulation , total lysates blotted for indicated antibodies .

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G. isgMASTL MDA-MB-231 cells were transduced with the indicated Mastl mutants or GFP alone as a control, and treated with doxycycline (Dox) to induce Mastl deletion. Cells were collected after glucose stimulation, and total lysates blotted for the indicated antibodies.


B sgRNA frequencies ( log2 of fold change ) in PD - L1low unstimulated ( left ) or IFN - γ stimulated ( right ) . Enriched sgRNAs ( dashed ) are highlighted ; hit genes ( black , red ) controls ( green , yellow or purple ) are shown labeled with target gene names .

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B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names.


C sgRNA frequencies ( log2 of fold change ) in viability screen . sgRNAs targeting controls are highlighted ; essential ( orange ) , non - essential ( blue ) . Genes attributed to PD - L1 modulation are shown in different colors ( red , yellow purple ) .

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C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).


A PD - L1 surface presentation ( ± SD ) in RKO mock control ( black ) or monoclonal MLLT6 knockout ( grey ) ( Student ' s t - ; * * p < 0 . 01 ; n = 3 biological replicates ; degrees of freedom ( df ) = 2 ) .

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A PD-L1 cell surface presentation (percentage ± SD) in RKO mock control (black) or monoclonal MLLT6 knockout cells (grey) (Student's t-test; **p < 0.01; n=3 biological replicates; degrees of freedom (df)=2).


B MLLT6 BAC mediated rescue in U2OS ( left ) showing ± SD of PD - L1 surface presentation in mock control ( black ) , MLLT6 polyclonal knockout ( grey ) MLLT6 knockout + BAC ( grey ) ( Student ' s t - ; * p < 0 . 05 ; n = 3 biological replicates ; df = 2 ) . Representative flow cytometry plots ( right ) of PD - L1 surface presentation after with anti - PD - L1 antibody ( top right , bottom left , bottom right ) isotype control ( top left ) . Percentages of in PD - L1 + gate as indicated .

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B MLLT6 BAC mediated rescue in U2OS cells (left) showing percentage ± SD of PD-L1 cell surface presentation in mock control (black), MLLT6 polyclonal knockout (light grey) and MLLT6 knockout + BAC cells (dark grey) (Student's t-test; *p < 0.05; n=3 biological replicates; df=2). Representative flow cytometry plots (right) of PD-L1 cell surface presentation after staining with anti-PD-L1 antibody (top right, bottom left, bottom right) and isotype control (top left). Percentages of cells in the PD-L1+ gate as indicated.


C Total of PD - L1 ( top ) GAPDH ( bottom ) in monoclonal RKO MLLT6 knockout , mock control or MLLT6 knockout + BAC visualized by immunoblotting . Numbers indicate relative band intensities of PD - L1 normalized to GAPDH .

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C Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in monoclonal RKO MLLT6 knockout, mock control or MLLT6 knockout + BAC cells visualized by immunoblotting. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


D PD - L1 transcript in U2OS mock ( black ) polyclonal MLLT6 knockout lines ( grey ) treated with without IFN - γ normalized to expression in mock U2OS without IFN - γ treatment ( ANOVA ; * * p < 0 . 01 ; n = 4 biological replicates ; df = 12 ) .

D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).


E Representative flow cytometry plots of PD - L1 surface presentation in presence absence of IFN - γ in mock , polyclonal MLLT6 knockout PD - L1 knockout . Percentages of in PD - L1 + gate after with anti - PD - L1 antibody or isotype control as indicated .

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E Representative flow cytometry plots of PD-L1 cell surface presentation in the presence and absence of IFN-γ in mock, polyclonal MLLT6 knockout and PD-L1 knockout cells. Percentages of cells in the PD-L1+ gate after staining with anti-PD-L1 antibody or isotype control as indicated.


F Total of PD - L1 ( top ) GAPDH ( bottom ) in U2OS polyclonal MLLT6 knockout or mock control with without IFN - γ stimulation . Numbers indicate relative band intensities of PD - L1 normalized to GAPDH .

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F Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN-γ stimulation. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


B Relative numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( black ) or polyclonal MLLT6 knockout ( grey ) treated with varying amounts of EpCAM - CD3 BiTE CTLs ( Student ' s t - ; * * p < 0 . 01 ; degrees of freedom ( df ) = 4 ) .

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B Relative cell numbers (± SD; n=3; biological replicates) of U2OS mock (black) or polyclonal MLLT6 knockout (grey) cells treated with varying amounts of EpCAM-CD3 BiTE and CTLs (Student's t-test; **p < 0.01; degrees of freedom (df)=4).


C Kaplan Meier survival plot ( left ) showing changes in survival ( ) of mock ( grey ) MLLT6 knockout ( red ) treated with CTLs EpCAM - CD3 BiTE over a course of 9 . 5 hours . Representative ( right ) of time - lapse microscopy following U2OS mock ( eGFP tagged , green ) MLLT6 knockout ( mCherry tagged , red ) immediately ( top left bottom left ) after adding CTLs EpCAM - CD3 BiTE 9 . 5 hours later ( top right bottom right ) ( scale bars = 100 μm ) .

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C Kaplan Meier survival plot (left) showing changes in survival (percent) of mock (grey) and MLLT6 knockout (red) cells treated with CTLs and EpCAM-CD3 BiTE over a course of 9.5 hours. Representative images (right) of time-lapse fluorescence microscopy following U2OS mock (eGFP tagged, green) and MLLT6 knockout (mCherry tagged, red) cells immediately (top left and bottom left) after adding CTLs and EpCAM-CD3 BiTE and 9.5 hours later (top right and bottom right) (scale bars = 100 μm).


D Relative numbers ( left ) ( ± SD ; n = 3 ; biological replicates ) of mock ( black ) , PD - L1 ( grey ) or MLLT6 ( grey ) knockout treated with BiTE or bi - specific antibodies CTLs as indicated ( Student ' s t - ; * * p < 0 . 01 ; df = 4 ) . Representative ( right ) from bright field microscopy of polyclonal U2OS mock ( top ) , PD - L1 ( middle ) or MLLT6 ( bottom ) knockout treated without or with EpCAM - CD3 BiTE or bispecific antibodies .

D Relative cell numbers (left) (± SD; n=3; biological replicates) of mock (black), PD-L1 (dark grey) or MLLT6 (light grey) knockout cells treated with BiTE or bi-specific antibodies and CTLs as indicated (Student's t-test; **p < 0.01; df=4). Representative images (right) from bright field microscopy of polyclonal U2OS mock (top), PD-L1 (middle) or MLLT6 (bottom) knockout cells treated without or with EpCAM-CD3 BiTE or bispecific antibodies.


E Relative normalized numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( left ) , MLLT6 ( middle ) or PD - L1 ( right ) knockout treated with EpCAM - CD3 BiTE CTLs in presence ( grey ) or absence ( black ) of IFN - γ ( Student ' s t - ; * * p < 0 . 01 ; df = 4 ) .

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E Relative normalized cell numbers (± SD; n=3; biological replicates) of U2OS mock (left), MLLT6 (middle) or PD-L1 (right) knockout cells treated with EpCAM-CD3 BiTE and CTLs in the presence (grey) or absence (black) of IFN-γ (Student's t-test; **p < 0.01; df=4 ).


A Scatter plots of pairwise comparisons of transcript expression in U2OS . Plots describe log2 fold change in transcript expression in mock polyclonal MLLT6 knockout treated with without IFN - γ as indicated . Horizontal axis shows log2 fold change in transcript expression vertical axis represents statistical significance ( log10 of p - value , n = 3 ) with candidates nominated for further validation ( red ) .

A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red).


B Correlation plots describing log2 fold change of transcripts in sequencing ( x axis ) Nanostring ( y axis ) measurements in mock + / - IFN - γ ( left ) MLLT6 knockout with IFN - γ ( right ) . Transcripts previously described to have a role in immunity ( red ) PD - L1 ( blue ) are represented on plots as indicated .

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B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated.


C Changes ( log2 fold ) in transcript expression ( left ) of select immune - related genes in U2OS mock or MLLT6 knockout treated with or without IFN - γ total measured by immunoblotting ( right ) . Numbers indicate band intensities normalized to GAPDH .

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C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.


A Immunoblot expression of implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout treated with or without IFN - γ . Numbers indicate band intensities normalized to GAPDH .

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A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.


B Immunoblot showing total of STAT1 isoforms ( α β ) , IDO1 , GBP5 CD74 in wild type U2OS transfected with esiRNAs targeting renilla luciferase ( Rluc , negative control ) , total STAT1 , STAT1α STAT1β treated with without IFN - γ .

B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ.


C . Representative of calyx synapses from littermate control Nrxn123 TKO mice . Brainstem sections were labeled with antibodies to vGluT1 ( red ) Syt2 ( green ) . Scale bar , 10 μm . D . Summary graphs of synaptic vGluT1 Syt2 immunostaining ( normalized to control ) .

C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm. D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).


E - H . Representative traces of spontaneous EPSCs ( sEPSCs ) ( E ) , summary graphs of sEPSC frequency ( F ) , amplitude ( G ) , kinetics ( H ) recorded from littermate control Nrxn123 TKO mice .

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E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.


A . Representative traces of EPSCs evoked by paired stimuli separated by 10 ms repeated every 20 s , recorded in a standard bath solution containing 2 mM Ca2 + . B . Pan - neurexin deletion suppresses synaptic transmission . Summary graphs show amplitude ( left ) charge transfer ( right ) of first EPSC recorded in response to paired stimuli . C . Pan - neurexin deletion more than doubles paired - ( PPR ) , suggesting a large decrease in release probability . D . Pan - neurexin deletion decelerates EPSC time course . Summary graphs show rise time ( left ) decay time constants ( right ) of first EPSC in response to paired stimuli .

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A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+. B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli. C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability. D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.


E - F . Pan - neurexin deletion does not significantly alter cumulative EPSC amplitude during a high - frequency stimulus train ( 100 Hz for 0 . 5 sec ) . Left , representative ESPC traces ; right , cumulative summary plot of EPSC amplitudes during train ( dotted lines show linear regression fits for estimating cumulative EPSC amplitude by back - extrapolation to zero time , which is used to correct for vesicle replenishment during train ) .

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E-F. Pan-neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high-frequency stimulus train (100 Hz for 0.5 sec). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back-extrapolation to zero time, which is used to correct for vesicle replenishment during the train).


G . Pan - neurexin deletion does not decrease readily - releasable pool of vesicles , but lowers initial release probability during a high - frequency stimulus train . Summary graphs show cumulative EPSC amplitudes extrapolated to time zero as an estimate of readily - releasable pool ( left ) , of first EPSC amplitude divided by cumulative EPSC amplitudes extrapolated to time zero as an estimate of initial release probability ( right ) .

G. Pan-neurexin deletion does not decrease the readily-releasable pool of vesicles, but lowers the initial release probability during a high-frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily-releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).


A - D . Same as Fig 2A - D , except that ACSF contained only 1 mM Ca2 + , which is close to estimated physiological of extracellular Ca2 + ( Forsberg et al . , 2019 ) .

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A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019).


E - H . Same as Fig 2A - D , except that slice were incubated with 0 . 2 mM EGTA - AM for 30 mins before recording , which introduces EGTA into presynaptic terminals .

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E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.


A . Example traces of presynaptic Ca2 + - currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV . B . Summary graphs of I - V relationship for peak Ca2 + - current amplitude ( left ) of peak Ca2 + - current density ( right ) .

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A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).


C . Example traces ( left ) summary graphs ( right ) of Ca2 + - current activation deactivation kinetics . Currents were induced by a 50 ms step depolarization from - 80 mV to + 10 mV .

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C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.


D . Example traces of Ca2 + - currents evoked by an action potential - equivalent depolarization ( APe , from - 80 mV to + 17 mV for 1 ms ; left ) , summary graph of APe - evoked Ca2 + - current peak amplitude charge ( right ) .

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D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).


E . Example traces of presynaptic Ca2 + - currents induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV before after perfusion of 200 nM agatoxin ( Agtx ) , a selective P / Q - type Ca2 + channel blocker .

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E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.


F . Summary graph of relative contribution of P / Q - type Ca2 + channels to total presynaptic Ca2 + currents , as quantified by relative reduction in Ca2 + currents evoked by step depolarization to + 10 mV , in control Nrxn123 TKO synapses .

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F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.


B . Example traces of presynaptic capacitance Ca2 + - currents at calyx terminals induced by a 20 ms step depolarization from − 80 mV to + 10 mV with an extracellular solution containing 2 mM Ca2 + , an internal pipette solution containing a standard 0 . 05 mM BAPTA . C . Summary graphs of depolarization - induced capacitance jump that monitors synaptic vesicle exocytosis ( left ) , peak Ca2 + - current density ( right ) . D , E . Same as ( B , C ) , except that 0 . 5 mM EGTA was added in addition to 0 . 05 mM BAPTA to patch pipette internal solution . F , G . Same as ( B , C ) , except that 1 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to patch pipette internal solution . H , I . Same as ( B , C ) , except that 10 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to patch pipette internal solution .

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B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.


J , K . Summary plots of relationship between total capacitance jump ( J ) or between Ca2 + - current density ( K ) of EGTA in patch pipette .

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J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.


A . Representative traces of Ca2 + - currents 4 - AP insensitive K + - currents evoked by step depolarizations ( from − 50 mV to + 10 mV in 10 mV increments ) , recorded from calyx terminals in acute slices from littermate control Nrxn123 TKO mice at P12 - P14 . B . Same as ( A ) but after addition of 200 nM iberiotoxin ( IbTx ) by perfusion . C . Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in ( B ) from currents shown in ( A ) . D . Summary graphs of capacitance , Ca2 + - current density , BK - current density .

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A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.


A . Representative of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 ( green ) , CaV2 . 1 - type Ca2 + - channels ( red ) , active zone Bassoon ( purple ) . Cryosections were obtained from littermate control Nrxn123 TKO mice at P12 - 14 . Scale bar , 10 μm .

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A. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), CaV2.1-type Ca2+-channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12-14. Scale bar, 10 μm.


B . Summary graphs of vGluT1 , CaV2 . 1 , Bassoon immunostaining ( normalized to control ; CaV2 . 1 Bassoon signals were quantified over vGluT1 - positive to measure active zone - localized ) .

B. Summary graphs of the vGluT1, CaV2.1, and Bassoon immunostaining intensity (normalized to control; CaV2.1 and Bassoon signals were quantified over the vGluT1-positive area to measure active zone-localized proteins).


C . Summary graphs of diameter total of vGluT1 - positive presynaptic of calyx of Held .

C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect total number of Ki - 67 + ( A ) ( S - phase ) in hair matrix . Unpaired t - performed using N of 9 - 12 HFs from 3 .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect total number of EdU + ( B ) ( S - phase ) in hair matrix . Unpaired t - performed using N of 9 - 12 HFs from 3 .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


C ) 100 nM paclitaxel treatment ( 24h ) significantly ( p = < 0 . 0001 [ * * * * ] ) increased number of mitotic phospho - histone H3 ( pH3 ) + in hair matrix . Welch ' s t - performed using N of 9 HFs from 3 .

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C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.


D ) 100 nM docetaxel treatment ( 24h ) significantly ( p = 0 . 0004 [ * * * ] ) increases number of pH3 + in hair matrix . Unpaired t - performed using N of 8 - 9 HFs from 3 .

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D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.


E ) Representative immunofluorescence highlight effects of 24h 100 nM taxane treatment on ( i ) Ki - 67 expression [ paclitaxel ] ; ( ii ) EdU incorporation pH3 immunoreactivity [ paclitaxel ] ; ( iii ) pH3 immunoreactivity [ docetaxel ] . 20 µm scale . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


presence of micronucleated in hair matrix in paclitaxel ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U performed using N of 12 - 13 HFs ( paclitaxel ) from 3 .

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The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.


presence of micronucleated in hair matrix in docetaxel treated ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U performed using N of 8 HFs ( docetaxel ) from 3 .

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The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.


C ) Hoechst 33342 of healthy nuclei comprising hair matrix ( lined ) dermal papilla in untreated ( vehicle ) human HFs . 20 µm scale .

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C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.


D ) Paclitaxel treatment ( 100 nM , 24h ) induces formation of micronucleated bodies , as visualised by Hoechst 33342 ( arrows ) , localising to proliferative region of hair matrix . i - 20 µm scale ; ii - 10 µm scale .

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D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.


E ) 100 nM docetaxel treatment was also seen to promote formation of micronucleated bodies ( arrows ) . 10 µm scale . Error bars are standard error .

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E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.


A ) Nascent synthesis , as detected by ethynyl uridine ( EU ) incorporation , is blocked within clusters of nuclei in hair matrix ( arrows ) following paclitaxel treatment ( 100 nM , 24h ) . 20 µm scale .

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A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.


B ) Quantitative highlights a significant ( p = < 0 . 0001 [ * * * * ] ) decrease in number of EU + nuclei following 24h paclitaxel treatment . Welch ' s t - performed using N of 11 - 12 hair follicles ( HFs ) from 3 .

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B) Quantitative analysis highlights a significant (p =<0.0001 [****]) decrease in the number of EU+ nuclei following 24h paclitaxel treatment. Welch's t-test performed using N of 11-12 hair follicles (HFs) from 3 patients.


C ) Representative dual stain highlights how EU incorporation in hair matrix is blocked within pH3 + population that accumulates in response to paclitaxel treatment 10 µm scale .

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C) Representative dual fluorescence stain highlights how EU incorporation in the hair matrix is blocked within the pH3+ cell population that accumulates in response to paclitaxel treatment 10 µm scale.


D ) caspase 3 expression in hair matrix following 24h paclitaxel treatment . 20 µm scale .

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D) Cleaved caspase 3 expression in the hair matrix following 24h paclitaxel treatment. 20 µm scale.


E ) 100 nM paclitaxel treatment significantly ( p = 0 . 0016 [ exact ] [ * * ] ) increases number of caspase 3 + in hair matrix after 24h . Mann Whitney U performed using N of 16 - 18 HFs from 5 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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E) 100 nM paclitaxel treatment significantly (p = 0.0016 [exact] [**]) increases the number of cleaved caspase 3+ cells in the hair matrix after 24h. Mann Whitney U test performed using N of 16-18 HFs from 5 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


Ai ) Paclitaxel treatment promotes mitotic arrest ( orange arrows / pH3 + ) apoptosis ( arrows / caspase 3 + ) , within K15 + bulge K15 + proximal bulb outer root sheath ( pbORS ) / progenitor compartments of human hair follicle ( HF ) . 20 µm scale . Aii ) High magnification montage demonstrating mitotic arrest ( orange arrow ) apoptosis / caspase 3 positivity ( arrow ) within K15 + bulge . 10 µm scale . pbORS - proximal bulb outer root sheath .

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Ai) Paclitaxel treatment promotes mitotic arrest (orange arrows/pH3+ cells) and apoptosis (white arrows/caspase 3+ cells), within the K15+ bulge and K15+ proximal bulb outer root sheath (pbORS) stem/progenitor cell compartments of the human hair follicle (HF). 20 µm scale. Aii) High magnification montage demonstrating mitotic arrest (orange arrow) and apoptosis/caspase 3 positivity (white arrow) within the K15+ bulge. 10 µm scale. pbORS - proximal bulb outer root sheath.


B ) Representative immunofluorescence of heightened caspase 3 immunoreactivity within K15 + bulge following extended paclitaxel organ culture experiments 50 µm scale . EC - extended cultures .

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B) Representative immunofluorescence images of heightened cleaved caspase 3 immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments 50 µm scale. EC - extended cultures.


C ) Graph showing significantly ( p = 0 . 029 [ * * ] ) increased K15 / caspase 3 double + within bulge following extended paclitaxel HF organ cultures . Welch ' s t - performed using N of 8 - 9 HFs from 3 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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C) Graph showing significantly (p = 0.029 [**]) increased K15/caspase 3 double+ cells within the bulge following extended paclitaxel HF organ cultures. Welch's t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) K15 + of human HF bulge express Ki - 67 during extended organ culture experiments . Paclitaxel treatment did not significantly affect number of bulge K15 / Ki - 67 double + . Unpaired t - performed using N of 8 - 9 HFs from 3 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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D) K15+ cells of the human HF bulge express Ki-67 during extended organ culture experiments. Paclitaxel treatment did not significantly affect the number of bulge K15/Ki-67 double+ cells. Unpaired t-test performed using N of 8-9 HFs from 3 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Representative double immunofluorescence of elevated γH2A . X immunoreactivity within K15 + bulge following extended paclitaxel organ culture experiments ( see methods ) . 50 µm scale . EC - extended cultures .

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E) Representative double immunofluorescence images of elevated γH2A.X immunoreactivity within the K15+ bulge following extended paclitaxel organ culture experiments (see methods). 50 µm scale. EC - extended cultures.


F ) γH2A . X showing a significant ( p = 0 . 065 [ * * ] ) increase in number of with double - strand breaks in K15 + bulge following extended organ culture experiments . Mann Whitney U performed using N of 5 - 6 HFs from 2 . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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F) γH2A.X analysis showing a significant (p = 0.065 [**]) increase in the number of cells with DNA double-strand breaks in the K15+ bulge following extended organ culture experiments. Mann Whitney U test performed using N of 5-6 HFs from 2 patients. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


B ) Palbociclib - only palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki - 67 expression in hair matrix beyond that seen with just 24h treatment Data also confirms that Ki - 67 expression is unaffected by paclitaxel treatment performed using N of 6 - 9 HFs per condition from 3 . Ordinary one - way ANOVA with multiple comparisons performed . Adjusted p values = 0 . 0001 [ * * * ] 0 . 0001 [ * * * * ] respectively . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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B) Palbociclib-only and palbociclib plus paclitaxel dual treated HFs show marked reductions in Ki-67 expression in the hair matrix beyond that seen with just 24h treatment Data also confirms that Ki-67 expression is unaffected by paclitaxel treatment Analysis performed using N of 6-9 HFs per condition from 3 patients. Ordinary one-way ANOVA with multiple comparisons performed. Adjusted p values = 0.0001[***] and 0.0001 [****] respectively. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


C ) Data confirming that paclitaxel treatment significantly increases ( adjusted p value = 0 . 0001 [ * * * ] ) number of pH3 + in hair matrix This effect was not observed when paclitaxel treated hair follicles were pre - co - incubated with palbociclib . performed using N of 6 - 9 HFs per condition from 3 Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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C) Data confirming that paclitaxel treatment significantly increases (adjusted p value = 0.0001[***]) the number of pH3+ cells in the hair matrix This effect was not observed when paclitaxel treated hair follicles were pre- and co-incubated with palbociclib. Analysis performed using N of 6-9 HFs per condition from 3 patients Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


D ) caspase 3 in hair follicles from a donor that showed apoptotic sensitivity to paclitaxel treatment ( adjusted p value = 0 . 0056 [ * * ] ) . Paclitaxel treated hair follicles pre - co - incubated with palbociclib do not show enhanced caspase 3 expression , contrasting with paclitaxel - only treatment . Ordinary one - way ANOVA with multiple comparisons performed using N of 4 - 5 HFs per condition . Values plotted represent mean number of positive counted per HF analysed . Error bars are standard error .

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

D) Cleaved caspase 3 analysis in hair follicles from a patient donor that showed apoptotic sensitivity to paclitaxel treatment (adjusted p value = 0.0056 [**]). Paclitaxel treated hair follicles pre- and co-incubated with palbociclib do not show enhanced cleaved caspase 3 expression, contrasting with paclitaxel-only treatment. Ordinary one-way ANOVA with multiple comparisons performed using N of 4-5 HFs per condition. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


E ) Immunofluorescence data representing how palbociclib / or paclitaxel affect ( i ) Ki - 67 expression , ( ii ) pH3 immunoreactivity ( iii ) caspase 3 expression . 20 µm scale .

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E) Immunofluorescence data representing how palbociclib and/or paclitaxel affect (i) Ki-67 expression, (ii) pH3 immunoreactivity and (iii) cleaved caspase 3 expression. 20 µm scale.


B ) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle . synthesis in hair matrix resumed in palbociclib - only palbociclib plus paclitaxel dual treated conditions following washout Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 13 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

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 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

B) No significant differences in EdU incorporation were found in any experimental conditions compared to vehicle. DNA synthesis in the hair matrix resumed in the palbociclib-only and palbociclib plus paclitaxel dual treated conditions following washout Ordinary one-way ANOVA with multiple comparisons performed using N of 8-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


C ) A trending increase in number of caspase 3 + was seen in hair matrix of HFs dual treated with paclitaxel palbociclib following drug washout , but this was not significant , whereas paclitaxel - only treatment significantly increased number of caspase 3 + in hair matrix ( adjusted p value = 0 . 0003 [ * * * ] ) . Palbociclib - only treatment did not increase number of caspase 3 + in hair matrix following drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

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 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 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

C) A trending increase in the number of cleaved caspase 3+ cells was seen in the hair matrix of HFs dual treated with paclitaxel and palbociclib following drug washout, but this was not significant, whereas paclitaxel-only treatment significantly increased the number of cleaved caspase 3+ cells in the hair matrix (adjusted p value = 0.0003 [***]). Palbociclib-only treatment did not increase the number of cleaved caspase 3+ cells in the hair matrix following drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


D ) HFs treated with paclitaxel alone showed a significant ( adjusted p value = 0 . 0316 [ * ] ) increase in number of pH3 + in matrix after washout . A significant ( adjusted p value = 0 . 0278 [ * ] ) increase in number pH3 + was seen in HFs dual treated with both paclitaxel palbociclib following drug washout . Palbociclib treatment alone showed no significant change in number of pH3 + hair matrix keratinocytes after drug washout . Ordinary one - way ANOVA with multiple comparisons performed using N of 8 - 12 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard error .

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 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

D) HFs treated with paclitaxel alone showed a significant (adjusted p value = 0.0316 [*]) increase in the number of pH3+ cells in the matrix after washout. A significant (adjusted p value = 0.0278 [*]) increase in the number pH3+ cells was seen in HFs dual treated with both paclitaxel and palbociclib following drug washout. Palbociclib treatment alone showed no significant change in the number of pH3+ hair matrix keratinocytes after drug washout. Ordinary one-way ANOVA with multiple comparisons performed using N of 8-12 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error.


E ) Representative of EdU pH3 / caspase 3 in paclitaxel - only palbociclib plus paclitaxel dual treated HFs following drug washout . Where synthesis has resumed following reversal of G1 arrest , paclitaxel cytotoxicity is emergent . HS - hair shaft ; " wo " - washout . 20 µm scale .

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E) Representative fluorescence images of EdU and pH3/cleaved caspase 3 staining in paclitaxel-only and palbociclib plus paclitaxel dual treated HFs following drug washout. Where DNA synthesis has resumed following reversal of the G1 arrest, paclitaxel cytotoxicity is emergent. HS - hair shaft; "wo" - washout. 20 µm scale.


B ) Paclitaxel treatment did not affect number of EdU + within HF outer root sheath ( ORS ) following washout . In contrast , palbociclib treated HFs lacked EdU incorporation following drug washout ( adjusted p value = 0 . 0001 [ * * * ] ) . number of EdU + was significantly decreased in outer root sheath ( ORS ) of HFs dual treated with both paclitaxel palbociclib ( adjusted p value = 0 . 0021 [ * * ] ) . Ordinary one - way ANOVA with multiple comparisons performed using N of 12 - 13 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

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 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 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 O O O O O O O O O

B) Paclitaxel treatment did not affect the number of EdU+ cells within the HF outer root sheath (ORS) following washout. In contrast, palbociclib treated HFs lacked EdU incorporation following drug washout (adjusted p value = 0.0001 [***]). The number of EdU+ cells was significantly decreased in the outer root sheath (ORS) of HFs dual treated with both paclitaxel and palbociclib (adjusted p value = 0.0021 [**]). Ordinary one-way ANOVA with multiple comparisons performed using N of 12-13 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


C ) HFs treated with paclitaxel showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in number of caspase 3 + in ORS following drug washout . Palbociclib - only palbociclib plus paclitaxel dual treated HFs showed no change in number of caspase 3 + ORS . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

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 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

C) HFs treated with paclitaxel showed a significant (adjusted p value = 0.0001 [****]) increase in the number of cleaved caspase 3+ cells in the ORS following drug washout. Palbociclib-only and palbociclib plus paclitaxel dual treated HFs showed no change in the number of cleaved caspase 3+ ORS cells. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


D ) Paclitaxel - only treated HFs showed a significant ( adjusted p value = 0 . 0001 [ * * * * ] ) increase in number of pH3 + ORS following drug washout . Palbociclib treatment alone had no significant effect on number of pH3 + ORS . There was a trending , yet non - significant , increase in number of pH3 + ORS in HFs dual treated with palbociclib plus paclitaxel . Ordinary one - way ANOVA with multiple comparisons performed using N of 9 - 10 HFs from 3 . Values plotted represent mean total number of counted per HF analysed . Error bars are standard

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

D) Paclitaxel-only treated HFs showed a significant (adjusted p value = 0.0001 [****]) increase in the number of pH3+ ORS cells following drug washout. Palbociclib treatment alone had no significant effect on the number of pH3+ ORS cells. There was a trending, yet non-significant, increase in the number of pH3+ ORS cells in HFs dual treated with palbociclib plus paclitaxel. Ordinary one-way ANOVA with multiple comparisons performed using N of 9-10 HFs from 3 patients. Values plotted represent the mean total number of cells counted per HF analysed. Error bars are standard error


E ) Representative image of ORS EdU incorporation in palbociclib plus paclitaxel treated HFs following drug washout , whereby synthesis has not resumed . ; wo - washout . 50 µm scale ;

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E) Representative image of ORS EdU incorporation staining in palbociclib plus paclitaxel treated HFs following drug washout, whereby DNA synthesis has not resumed. ; wo - washout. 50 µm scale;


F ) Representative of pH3 / caspase 3 within ORS of paclitaxel - only palbociclib plus paclitaxel treated HFs following drug washout . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 50 µm

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F) Representative images of pH3/caspase 3 staining within the ORS of paclitaxel-only and palbociclib plus paclitaxel treated HFs following drug washout. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 50 µm scale


Immunofluorescence illustrating how palbociclib blocks paclitaxel - induced accumulation of caspase 3 + ( G ) in K15 + bulge proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

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Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of cleaved caspase 3+ (G) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


Immunofluorescence illustrating how palbociclib blocks paclitaxel - induced accumulation of pH3 + ( H ) in K15 + bulge proximal bulb ORS ( pbORS ) compartments . CTS - connective tissue sheath ; HS - hair shaft ; pbORS - proximal bulb outer root sheath ; wo - washout . 20 µm scale .

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Immunofluorescence images illustrating how palbociclib blocks the paclitaxel-induced accumulation of pH3+ (H) cells in the K15+ bulge and proximal bulb ORS (pbORS) compartments. CTS - connective tissue sheath; HS - hair shaft; pbORS - proximal bulb outer root sheath; wo - washout. 20 µm scale.


( A - C ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS ( Earle ' s Balanced Salt Solution ) for 30 min . Total membranes ( mem ) from lysed were collected incubated in a lipidation reaction with cytosols prepared from starved HEK293T . Reactions contained indicated concentrations of PI3K inhibitor ( PI3KI ) 3 - methyladenine ( 3 - MA ) ( B ) or FYVE ( C ) . A diagram of experimental scheme is shown in ( A ) . RPN1 , Ribophorin

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

(A-C) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS (Earle's Balanced Salt Solution) for 30 min. Total membranes (mem) from lysed cells were collected and incubated in a lipidation reaction with cytosols prepared from starved HEK293T cells. Reactions contained the indicated concentrations of PI3K inhibitor (PI3KI) 3-methyladenine (3-MA) (B) or FYVE protein (C). A diagram of the experimental scheme is shown in (A). RPN1, Ribophorin 1


( D , E ) Atg5 KO MEFs were either untreated ( NT ) or starved ( ST ) with EBSS in absence or presence of 20 nM wortmannin ( Wtm ) or 10 mM 3 - methyladenine ( 3 - MA ) for 30 min . Membranes from each treated sample were collected subjected to a differential centrifugation to separate 3K × g , 25K × g 100K × g pellet fractions followed by a lipidation as above ( E ) . A diagram is shown in ( D ) .

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(D, E) Atg5 KO MEFs were either untreated (NT) or starved (ST) with EBSS in the absence or presence of 20 nM wortmannin (Wtm) or 10 mM 3-methyladenine (3-MA) for 30 min. Membranes from each treated cell sample were collected and subjected to a differential centrifugation to separate the 3K ×g, 25K ×g and 100K ×g pellet fractions followed by a lipidation assay as above (E). A diagram is shown in (D).


( F , G ) Atg5 KO MEFs were starved for 30 min . Membranes in 25K × g 100K × g pellets from a differential centrifugation were collected as described above . A similar lipidation was performed in presence of indicated concentrations ( Conc in G ) of 3 - MA , wortmannin ( F ) FYVE as well as a PI3P binding - deficient FYVE mutant ( C / S ) ( G ) . of lipidation activity is shown as of LC3 - II to LC3 - I ( II / I ) .

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(F, G) Atg5 KO MEFs were starved for 30 min. Membranes in the 25K ×g and 100K ×g pellets from a differential centrifugation were collected as described above. A similar lipidation assay was performed in the presence of indicated concentrations (Conc in G) of 3-MA, wortmannin (F) and FYVE protein as well as a PI3P binding-deficient FYVE mutant protein (C/S) (G). Quantification of lipidation activity is shown as the ratio of LC3-II to LC3-I (II/I).