Figure 6A Representative images of control housing ( CH ) and environmental enrichment ( EE ) cages . B Schematic diagram of the experimental design . C - D Representative images of DCX + cells in the DG of CH and EE animals ( C ) , and quantification of the number of DCX + cells of WT and Tau - / - animals in CH or EE conditions ( D ) . E Representative images of 8 - week - old newborn granule neurons labeled with IdU ( red ) belonging to WT CH , WT EE , Tau - / - CH and Tau - / - EE mice . F - G Quantification of the number of 8 - week - old IdU + cells ( F ) and apoptotic fractin + cells ( G ) .

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Figure 6A Representative images of control housing (CH) and environmental enrichment (EE) cages.B Schematic diagram of the experimental design.C-D Representative images of DCX+ cells in the DG of CH and EE animals (C), and quantification of the number of DCX+ cells of WT and Tau-/- animals in CH or EE conditions (D).E Representative images of 8-week-old newborn granule neurons labeled with IdU (red) belonging to WT CH, WT EE, Tau-/- CH and Tau-/- EE mice.F-G Quantification of the number of 8-week-old IdU+ cells (F) and apoptotic fractin+ cells (G).


Figure 7A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD - 65 antibody ( red ) , and high - power magnifications of the DG molecular layer showing GAD - 65 + terminals in WT CH , Tau - / - CH , WT EE and Tau - / - EE mice . Cell nuclei were labeled with DAPI ( blue ) . White scale bar 300 µm . Red scale bar 50 µm . Green scale bar 5 µm . B - C Quantification of the density ( B ) and area ( C ) of GAD - 65 + terminals in the molecular layer of mice in each experimental condition ( n = 10 mice WT CH , n = 8 miceTau - / - CH , n = 6 mice WT EE , n = 10 miceTau - / - EE ) .

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Figure 7A Representative images of the whole hippocampus showing GABAergic innervation labeled with GAD-65 antibody (red), and high-power magnifications of the DG molecular layer showing GAD-65+ terminals in WT CH, Tau-/- CH, WT EE and Tau-/- EE mice. Cell nuclei were labeled with DAPI (blue). White scale bar 300 µm. Red scale bar 50 µm. Green scale bar 5 µm.B-C Quantification of the density (B) and area (C) of GAD-65+ terminals in the molecular layer of mice in each experimental condition (n= 10 mice WT CH, n= 8 miceTau-/- CH, n= 6 mice WT EE, n= 10 miceTau-/- EE).


Figure 8B Representative images of 8 - week - old newborn granule neurons of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CH image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) . E - F Representative images of an 8 - week - old newborn granule neuron in which proximal and distal dendrites are highlighted ( E ) and of PSDs ( green ) in distal and proximal dendrites of WT and Tau - / - mice housed under either CH or EE conditions ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) . I Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) . J - K Representative images ( J ) and quantification of the area ( K ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CH , WT EE , Tau - / - CH , and Tau - / - EE mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

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Figure 8B Representative images of 8-week-old newborn granule neurons of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice infected with a PSD95-GFP-expressing retrovirus. In the WT CH image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).E-F Representative images of an 8-week-old newborn granule neuron in which proximal and distal dendrites are highlighted (E) and of PSDs (green) in distal and proximal dendrites of WT and Tau-/- mice housed under either CH or EE conditions (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).I Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).J-K Representative images (J) and quantification of the area (K) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CH, WT EE, Tau-/- CH, and Tau-/- EE mice (n= 3 mice per experimental condition). Arrows indicate MFTs.


Figure 1ChEP analysis using EGFP ( control ) - or Piwi - KD OSCs . Signals with an adjusted p - value ( Bonferroni test ) of less than 0 . 05 are highlighted in red . The signals for Lam , LamC , and H1 are labelled within the plots . Lam - or LamC - KD followed by qRT - PCR of RNA levels for piRNA target ( mdg1 and gypsy ) or non - target ( roo ) TEs . Error bars indicate SD ( n = 3 ) . * = p - value < 0 . 05 ( t - test ) . Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells .

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Figure 1ChEP analysis using EGFP (control)- or Piwi-KD OSCs. Signals with an adjusted p-value (Bonferroni test) of less than 0.05 are highlighted in red. The signals for Lam, LamC, and H1 are labelled within the plots.Lam- or LamC-KD followed by qRT-PCR of RNA levels for piRNA target (mdg1 and gypsy) or non-target (roo) TEs. Error bars indicate SD (n=3). * = p-value<0.05 (t-test). Note that the complete depletion of Lam and LamC is impossible since their KD causes severe damage to cells.


Figure 2Boxplots showing fold changes in the expression of piRNA target or non - target TEs based on RNA - seq upon Piwi - KD ( left ) , and the frequency of piRNAs , which target piRNA target TEs or non - piRNA target TEs ( right ) . Definition of piRNA target and non - target TEs is in Fig EV1E . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test . Boxplot showing DamID - seq signals of piRNA target or non - target TEs upon EGFP ( control ) - or Piwi - KD . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated using the Wilcoxon rank - sum test . Oligo - FISH images for the representative piRNA target region described in Fig 4A , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin immunofluorescence staining . Scale bar , 5μm . Ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Figure 2Boxplots showing fold changes in the expression of piRNA target or non-target TEs based on RNA-seq upon Piwi-KD (left), and the frequency of piRNAs, which target piRNA target TEs or non-piRNA target TEs (right). Definition of piRNA target and non-target TEs is in Fig EV1E. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.Boxplot showing DamID-seq signals of piRNA target or non-target TEs upon EGFP (control)- or Piwi-KD. Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated using the Wilcoxon rank-sum test.Oligo-FISH images for the representative piRNA target region described in Fig 4A, upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin immunofluorescence staining. Scale bar, 5μm. Ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Figure 3Oligo - FISH images for changes in the intra - TAD interaction at the TAD described in ( C ) , upon EGFP ( control ) - or Piwi - KD . Gray indicates DAPI staining for DNA , green and magenta indicates FISH signal at the different positions of TADs ( TAD - 1 and TAD - 2 ) . Details of TAD probe design is described in Fig EV2C . Scale bar , 5μm . Distance between two FISH signals is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Figure 3Oligo-FISH images for changes in the intra-TAD interaction at the TAD described in (C), upon EGFP (control)- or Piwi-KD. Gray indicates DAPI staining for DNA, green and magenta indicates FISH signal at the different positions of TADs (TAD-1 and TAD-2). Details of TAD probe design is described in Fig EV2C. Scale bar, 5μm. Distance between two FISH signals is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Figure 4Boxplots showing fold changes in the histone mark association of piRNA target or non - target TEs based on ChIP - seq upon Piwi - KD ( Definition of piRNA target and non - target TEs are described in Fig EV1E ) . Boxplot whiskers , box , and central band show maximum , third quartile , median , first quartile , and minimum , respectively ( n = 10 for piRNA target TEs , n = 80 for non - piRNA target TEs ) . P - values were calculated by Wilcoxon rank - sum test . ChIP ‐ qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN - Nxf2 expression vector or control EGFP expression vector . Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid . Error bars indicate SD ( n = 3 ) , * = p - value < 0 . 01 ( t - test ) . .

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Figure 4Boxplots showing fold changes in the histone mark association of piRNA target or non-target TEs based on ChIP-seq upon Piwi-KD (Definition of piRNA target and non-target TEs are described in Fig EV1E). Boxplot whiskers, box, and central band show maximum, third quartile, median, first quartile, and minimum, respectively (n=10 for piRNA target TEs, n= 80 for non-piRNA target TEs). P-values were calculated by Wilcoxon rank-sum test.ChIP‐qPCR analysis of H3K27Ac and H3K4me3 occupancy on the reporter gene upon transfection of λN-Nxf2 expression vector or control EGFP expression vector. Bar graph shows the occupancy relative to that of the sample transfected with a control plasmid. Error bars indicate SD (n = 3), * = p-value<0.01 (t-test)..


Figure 5Immunoprecipitation ( IP ) from OSC lysate using anti - Lam , or myc antibody , followed by WB using indicated antibodies . Mouse immunoglobulin G ( n . i . ) was used for control IP . Note that OSCs used at the right panel is a line which stably expresses myc - Nxf2 . Density plots for DamID - seq signals over consensus sequences from piRNA target ( gypsy and mdg1 , orange ) and non - target TEs ( roo , blue ) in EGFP ( control ) - or Nxf2 - KD cells . Oligo - FISH images for the reporter of the tethering system , at 0h ( control ) , 48h hours , 96 hours after transfection of λN - Nxf2 expression vector . Gray indicates DAPI staining for DNA , magenta indicates FISH signal , and green indicates Lamin staining . Scale bar , 5μm . The ratio of distance from FISH signal to the nuclear periphery ( DAPI surface ) is quantified at right , dot in the box plot indicates mean value ; Boxplot whiskers , box , and central band show minimum , first quartile , median , third quartile , and maximum , respectively . P - values calculated with Mann - Whitney U test .

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Figure 5Immunoprecipitation (IP) from OSC lysate using anti-Lam, or myc antibody, followed by WB using indicated antibodies. Mouse immunoglobulin G (n.i.) was used for control IP. Note that OSCs used at the right panel is a line which stably expresses myc-Nxf2.Density plots for DamID-seq signals over consensus sequences from piRNA target (gypsy and mdg1, orange) and non-target TEs (roo, blue) in EGFP (control)- or Nxf2-KD cells.Oligo-FISH images for the reporter of the tethering system, at 0h (control), 48h hours, 96 hours after transfection of λN-Nxf2 expression vector. Gray indicates DAPI staining for DNA, magenta indicates FISH signal, and green indicates Lamin staining. Scale bar, 5μm. The ratio of distance from FISH signal to the nuclear periphery (DAPI surface) is quantified at right, dot in the box plot indicates mean value; Boxplot whiskers, box, and central band show minimum, first quartile, median, third quartile, and maximum, respectively. P-values calculated with Mann-Whitney U test.


Figure 6Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in intra - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 ) Dot plot showing the correlation between the normalized density of indicated TEs per TAD ( x - axis ) and changes in inter - TAD interactions upon H1 ( left ) - , Egg ( middle ) - , or HP1a ( right ) - KD ( y - axis ) . Trend lines are in red . Spearman ' s rank correlation rho is indicated along with the p - value ( algorithm AS 89 ) . n . s . ; not significant ( p > 0 . 05 )

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Figure 6Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in intra-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05) Dot plot showing the correlation between the normalized density of indicated TEs per TAD (x-axis) and changes in inter-TAD interactions upon H1(left)-, Egg(middle)-, or HP1a(right)-KD (y-axis). Trend lines are in red. Spearman's rank correlation rho is indicated along with the p-value (algorithm AS 89). n.s.; not significant (p > 0.05)


Figure 1A , Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA , and β - actin was used as a loading control . B , ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines . For NPECs , n = 3 ; for NPC , n = 7 . C , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in training cohort . E , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 1 ( GZ ) . F , The concentration of plasma INSL5 in healthy controls ( normal EBV ( - ) ) , non - tumor individuals with VCA - IgA positive ( normal EBV ( + ) ) and NPC patients , in validation cohort 2 ( ZS ) .

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Figure 1A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ). F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).


Figure 2A - C , Kaplan - Meier curves showing the overall survival ( OS ) curves of NPC patients with low or high INSL5 concentration ( A ) , low ( ≤ 4000 copy / ml ) or high ( > 4000 copy / ml ) EBV DNA copy number ( B ) , and the combination of INSL5 concentration with EBV DNA copy number ( C ) . D - F , Kaplan - Meier curves showing the disease - free survival ( DFS ) curves of NPC patients with low or high INSL5 concentration ( D ) , low or high EBV DNA copy number ( E ) , and the combination of INSL5 concentration with EBV DNA copy number ( F ) .

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Figure 2A-C, Kaplan-Meier curves showing the overall survival (OS) curves of NPC patients with low or high INSL5 concentration (A), low (≤4000 copy/ml) or high (>4000 copy/ml) EBV DNA copy number (B), and the combination of INSL5 concentration with EBV DNA copy number (C).D-F, Kaplan-Meier curves showing the disease-free survival (DFS) curves of NPC patients with low or high INSL5 concentration (D), low or high EBV DNA copy number (E), and the combination of INSL5 concentration with EBV DNA copy number (F).


Figure 3A . Exogenous expression of INSL5 in NPC cells . Representative immunoblotting ( upper panel ) and immunofluorescent staining ( lower panel ) showed stable exogenous expression of INSL5 in both CNE1 , CNE2 and HK1 NPC cell lines . Scale bars represent 20μm . BMTT assay of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines ( upper panel ) either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) ( lower panel ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for HK1 . colony formation ( C and D ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( C ) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments . Brdu incorporation ( E and F ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( E ) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments . Scale bars represent 20μm in ( E ) migration assays ( G and H ) of vector control or INSL5 overexpressing CNE1 , CNE2 and HK1 NPC cell lines either transfected with control siRNA ( NC ) or GPCR142 siRNA ( # 1 and # 2 ) . Representative images are shown in ( G ) for migration assays migrated cells per field of view were plotted in H , respectively . The results are from three different experiments . Scale bars represent 100μm in ( G ) I , J , Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor size ( I ) n = 11 mice per group . Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice . . Tumor weight of two groups . n = 11 mice per group .

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Figure 3A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.BMTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.colony formation (C and D) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C) for colony formation Number of colonies per field of view were plotted in D The results are from three different experiments.Brdu incorporation (E and F) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (E) Brdu incorporation the percentage of Brdu positive cells were plotted in The results are from three different experiments. Scale bars represent 20μm in (E)migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (G) for migration assays migrated cells per field of view were plotted in H, respectively. The results are from three different experiments. Scale bars represent 100μm in (G)I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) n=11 mice per group.Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor weight of two groups. n=11 mice per group.


Figure 4A , Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control . The genes are shaded with green , black or red in the heat map to indicate low , intermediate or high expression , respectively . C , The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis . D , Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock - down cell lines . E , Metabolites were purified from five ( CNE1 cell line and HK1 cell line ) or seven ( CNE2 cell line ) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography - mass spectrometry ( LC - MS ) . Red : glycolytic intermediate metabolites . Green : tricarboxylic acid cycle ( TCA cycle ) intermediate metabolites . PPP : pentose phosphate pathway . The color scale bar represents Z - score of the metabolites ' level . F , The extracellular acidification rate ( ECAR ) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer . Glu , glucose ; Oligo , oligomycin . G , Statistical analysis of the effects of INSL5 overexpression on glycolytic activity . H , CNE1 - vector and CNE1 - INSL5 cells were grown in the presence of the fluorescent glucose analog 2 - NBDG ( Invitrogen ) for 15min , and glucose uptake was then quantified using flow cytometry ( FACS ) ( left panel ) . Statistical analysis of the glucose uptake was showed in the right panel . glucose consumption and lactate production ( I ) in CNE1 - vector and CNE1 - INSL5 cells . basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2 - DG for 24h ( J ) in CNE1 - vector and CNE1 - INSL5 cells . HK2 enzyme activity ( K ) in CNE1 - vector and CNE1 - INSL5 cells . L , ATP concentration , HK2 enzyme activity and lactate production in INSL5 wide - type or knock down HNE1 - EBV cells .

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Figure 4A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.glucose consumption and lactate production (I) in CNE1-vector and CNE1-INSL5 cells.basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J) in CNE1-vector and CNE1-INSL5 cells.HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.


Figure 5B , phosphorylation of STAT5 under INSL5 stimulation . CNE1 cells were starved in FBS - free medium for 24h and then treated with INSL5 peptide ( 50ng / mL ) in the presence of an anti - INSL5 antibody at 100ug / ml , or an isotype - matched IgG control ( IgG ) for 30min . C , Localization of STAT5 . A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5 . Scale bars represent 20μmD , Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated . The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions . E , INSL5 overexpression activated AKT , ERK , JAK1 and STAT5 phosphorylation , not STAT3 in HK1 cells . INSL5 overexpressing HK1 cells were treated with MK2206 ( 50uM ) , U0126 ( 20uM ) , MK2206 + U0126 , or Ruxolitinib ( 5uM ) for 24h . Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level . F , ChIP - qPCR analyzed the occupancy of potential E - boxes by STAT5 in the genes of HK2 , Glut3 and PFK1 in INSL5 - overexpressing CNE1 cells using IgG or anti - STAT5 antibodies . G , Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector . H , Glucose uptake assessed by 2 - NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down . I , Glucose uptake was assessed by 2 - BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor ( Ruxolitinib ) . J , MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor .

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Figure 5B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μmD, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.


Figure 6A , Brdu assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 20μm . B , similar to ( A ) , migration and invasion assay for CNE1 cells stimulated with 100ng / ml INSL5 peptide in the presence of an anti - INSL5 antibody at 50 or 100ug / ml , or an isotype - matched IgG control ( IgG ) for 24h . Scale bars represent 100μm . C - E Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of glycolysis inhibitor , 2 - DG . CNE1 cells ( C ) , CNE2 cells ( D ) and HK1 cells ( E ) . n = 4 biological replicates for CNE1 and CNE2 cell line , n = 6 biological replicates for HK1F , Growth curves of cell lines stably expressing INSL5 or vector 48 h post - treatment with the indicated dose of cis - platinum ( DDP ) and 2 - DG . n = 4 biological replicates per group . Tumor size ( G ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group . tumor weight ( H ) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti - INSL5 , anti - GPCR142 or isotype - matched IgG control ( IgG ) every two days . n = 6 mice per group .

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Figure 6A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.Tumor size (G) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.


Figure 1Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane . The apical MTs were associated with both LUZP1 and TJs . Scale bar , 10 μm ( low magnification ) and 1 μm ( high magnification ) . Representative immunoelectron micrographs of Eph4 cells . Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin . MV , microvilli ; AJ , adherens junction , DS , desmosome . Scale bars , 200 nm . Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ZO - 1 / - 2 DKO cells were marked by asterisks ( * ) . The LUZP1 junctional localization was apparently disrupted in TJ - deficient ZO - 1 / - 2 DKO cells . Scale bar , 10 μm . Representative confocal micrographs of transient E - cadherin knockout ( KO ) in WT Eph4 cells . E - cadherin KO cells were marked by asterisks ( * ) . The LUZP1 junctional localization seemed not to change between AJ - deficient E - cadherin KO and WT cells . Scale bar , 10 μm .

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Figure 1Representative confocal micrographs of immunostained Eph4 epithelial cell sheets in the apical plane. The apical MTs were associated with both LUZP1 and TJs. Scale bar, 10 μm (low magnification) and 1 μm (high magnification).   Representative immunoelectron micrographs of Eph4 cells. Immunogold particles for LUZP1 preferentially accumulated at the level of TJs that are positive for cingulin. MV, microvilli; AJ, adherens junction, DS, desmosome. Scale bars, 200 nm.   Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ZO-1/-2 DKO cells were marked by asterisks (*). The LUZP1 junctional localization was apparently disrupted in TJ-deficient ZO-1/-2 DKO cells. Scale bar, 10 μm.   Representative confocal micrographs of transient E-cadherin knockout (KO) in WT Eph4 cells. E-cadherin KO cells were marked by asterisks (*). The LUZP1 junctional localization seemed not to change between AJ-deficient E-cadherin KO and WT cells. Scale bar, 10 μm.


Figure 2Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [ 19 . 19 ± 10 . 02 arbitrary units ( a . u . ) ( WT ) vs . 2 . 92 ± 1 . 81 a . u . ( LUZP1 KO ) ] . n = 3 . * * p < 0 . 01 ( Mann - Whitney U test ) . Bars and error bars represent the mean ± SD . Representative confocal micrographs of co - cultures of wild - type ( WT ) and ZO - 1 / - 2 double knockout ( DKO ) Eph4 cells . ppMLC levels within CRs were clearly reduced in ZO - 1 / - 2 DKO cells compared to those in WT cells . Scale bar , 10 μm . Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO - 1 / - 2 DKO cells [ 119 . 62 ± 31 . 62 μm2 ( WT ) vs . 199 . 96 ± 94 . 22 μm2 ( LUZP1 KO ) ] ( See also Fig 1I ) . n = 3 . * * p < 0 . 01 ( unpaired t test ) . Bars and error bars represent the mean ± SD .

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Figure 2Bar plots with dot density plots showing that ppMLC levels within CRs were significantly reduced in LUZP1 KO cells compared to those in WT cells [19.19 ± 10.02 arbitrary units (a.u.) (WT) vs. 2.92 ± 1.81 a.u. (LUZP1 KO)]. n = 3. **p < 0.01 (Mann-Whitney U test). Bars and error bars represent the mean ± SD.Representative confocal micrographs of co-cultures of wild-type (WT) and ZO-1/-2 double knockout (DKO) Eph4 cells. ppMLC levels within CRs were clearly reduced in ZO-1/-2 DKO cells compared to those in WT cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that apical areas of WT cells were significantly smaller than those of ZO-1/-2 DKO cells [119.62 ± 31.62 μm2 (WT) vs. 199.96 ± 94.22 μm2 (LUZP1 KO)] (See also Fig 1I). n = 3. **p < 0.01 (unpaired t test). Bars and error bars represent the mean ± SD.


Figure 3In vitro direct binding assay between FLAG - LUZP1 and GST - MLC . Representative immunoblots are shown . S , supernatant ; P , pellet . A representative plot in which the amount of bound GST - WT - , AA - , or DD - MLC / FLAG - LUZP1 was plotted with the Michaelis - Menten fitted curve . Bar plots with dot density plots showing that the dissociation constant ( Kd ) of LUZP1 from DD - MLC was significantly lower than that from WT - MLC and AA - MLC [ 4 . 04 ± 1 . 05 μM ( WT - MLC ) vs . 4 . 88 ± 1 . 79 μM ( AA - MLC ) vs . 0 . 85 ± 0 . 39 μM ( DD - MLC ) ] . n = 5 ( WT - MLC and AA - MLC ) and n = 4 ( DD - MLC ) . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel - Dwass test ] . Bars and error bars represent the mean ± SD .

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Figure 3In vitro direct binding assay between FLAG-LUZP1 and GST-MLC. Representative immunoblots are shown. S, supernatant; P, pellet.   A representative plot in which the amount of bound GST-WT-, AA-, or DD-MLC/FLAG-LUZP1 was plotted with the Michaelis-Menten fitted curve.   Bar plots with dot density plots showing that the dissociation constant (Kd) of LUZP1 from DD-MLC was significantly lower than that from WT-MLC and AA-MLC [4.04 ± 1.05 μM (WT-MLC) vs. 4.88 ± 1.79 μM (AA-MLC) vs. 0.85 ± 0.39 μM (DD-MLC)]. n = 5 (WT-MLC and AA-MLC) and n = 4 (DD-MLC). *p < 0.05 [Kruskal-Wallis test followed by Steel-Dwass test]. Bars and error bars represent the mean ± SD.


Figure 4Representative immunoblot of WT , LUZP1 KO , and Venus - LUZP1 - expressing LUZP1 knockout ( REV ) Eph4 cells treated with 100 nM calyculin A for 30 min . Quantification of the ppMLC / MLC ratio relative to WT control , confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [ WT , 1 . 00 ( control ) vs . 1 . 40 ± 0 . 06 ( calyculin A ) vs . 1 . 14 ± 0 . 33 ( washout ) ; KO , 0 . 09 ± 0 . 04 ( control ) vs . 1 . 49 ± 0 . 06 ( calyculin A ) vs . 0 . 81 ± 0 . 99 ( washout ) ; REV , 2 . 06 ± 1 . 78 ( control ) vs . 1 . 82 ± 1 . 50 ( calyculin A ) vs . 1 . 80 ± 1 . 14 ( washout ) ] . n = 3 . Bars and error bars represent the mean ± SD .

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Figure 4Representative immunoblot of WT, LUZP1 KO, and Venus-LUZP1-expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A [WT, 1.00 (control) vs. 1.40 ± 0.06 (calyculin A) vs. 1.14 ± 0.33 (washout); KO, 0.09 ± 0.04 (control) vs. 1.49 ± 0.06 (calyculin A) vs. 0.81 ± 0.99 (washout); REV, 2.06 ± 1.78 (control) vs. 1.82 ± 1.50 (calyculin A) vs. 1.80 ± 1.14 (washout)]. n = 3. Bars and error bars represent the mean ± SD.


Figure 5In vitro MLC phosphorylation assay using 1 μg GST - PP1c β / δ in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , and 0 - 5 μg GST - LUZP1 . Quantification of the di - phosphorylated MLC ( ppMLC ) / MLC ratio relative to the control showed that LUZP1 up - regulated ppMLC / MLC levels in a dose - dependent manner [ 1 . 00 ( 1st lane , control ) vs . 1 . 27 ± 0 . 33 ( 2nd lane ) vs . 1 . 76 ± 0 . 68 ( 3rd lane ) vs . 2 . 53 ± 1 . 65 ( 4th lane ) vs . 2 . 93 ± 2 . 45 ( 5th lane ) ] . n = 3 or 6 . * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD . In vitro Merlin phosphorylation assay using 1 μg GST - PP1c β / δ , 100 ng GST - Merlin , 2 pg p21 - activated kinase 1 ( PAK1 ) , and 5 μg GST - LUZP1 . Quantification of the phosphorylated Merlin ( pMerlin ) / Merlin ratio relative to the control showed that LUZP1 up - regulated pMerlin / Merlin levels [ 0 . 23 ± 0 . 15 ( 1st lane ) vs . 1 . 00 ( 2nd lane , control ) vs . 0 . 32 ± 0 . 17 ( 3rd lane ) vs . 0 . 97 ± 0 . 42 ( 4th lane ) vs . 1 . 25 ± 0 . 39 ( 5th lane ) ] . n = 4 or 9 . * p < 0 . 05 , * * p < 0 . 01 [ Kruskal - Wallis test followed by Steel test ( compared with 3rd lane ) ] . Bars and error bars represent the mean ± SD .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5In vitro MLC phosphorylation assay using 1 μg GST-PP1c β/δ in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, and 0-5 μg GST-LUZP1. Quantification of the di-phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 up-regulated ppMLC/MLC levels in a dose-dependent manner [1.00 (1st lane, control) vs. 1.27 ± 0.33 (2nd lane) vs. 1.76 ± 0.68 (3rd lane) vs. 2.53 ± 1.65 (4th lane) vs. 2.93 ± 2.45 (5th lane)]. n = 3 or 6. **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.   In vitro Merlin phosphorylation assay using 1 μg GST-PP1c β/δ, 100 ng GST-Merlin, 2 pg p21-activated kinase 1 (PAK1), and 5 μg GST-LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 up-regulated pMerlin/Merlin levels [0.23 ± 0.15 (1st lane) vs. 1.00 (2nd lane, control) vs. 0.32 ± 0.17 (3rd lane) vs. 0.97 ± 0.42 (4th lane) vs. 1.25 ± 0.39 (5th lane)]. n = 4 or 9. *p < 0.05, **p < 0.01 [Kruskal-Wallis test followed by Steel test (compared with 3rd lane)]. Bars and error bars represent the mean ± SD.


Figure 6Box plots with dot density plots showing the ratio of the apical area / basal area in co - cultures of Venus - LUZP1 - expressing LUZP1 knockout ( REV ) and LUZP1 knockout ( LUZP1 KO ) Eph4 cells ; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [ REV , 0 . 65 ± 0 . 16 ( control ) vs . 0 . 90 ± 0 . 18 ( nocodazole ) vs . 0 . 64 ± 0 . 16 ( washout ) ; KO , 1 . 30 ± 0 . 17 ( control ) vs . 1 . 07 ± 0 . 13 ( nocodazole ) vs . 1 . 32 ± 0 . 19 ( washout ) ] . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . The solid lines represent the medians and the boxes represent the inter - quartile ranges . The error bars extending from the box represents the data within 1 . 5 times of the interquartile range . Representative confocal micrographs of co - cultures of LUZP1 - expressing wild - type ( WT ) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min . Nocodazole treatment partially reversed the difference in di - phosphorylated MLC ( ppMLC ) levels within circumferential rings ( CRs ) between WT and LUZP1 KO cells . Scale bar , 10 μm . Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down - regulated in WT Eph4 cells after nocodazole treatment . Importantly , ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [ WT , 21 . 43 ± 6 . 96 arbitrary units ( a . u . ) ( control ) vs . 17 . 67 ± 5 . 40 a . u . ( nocodazole ) vs . 20 . 84 ± 7 . 19 a . u . ( washout ) ; KO , 8 . 74 ± 1 . 71 a . u . ( control ) vs . 8 . 67 ± 1 . 89 a . u . ( nocodazole ) vs . 7 . 96 ± 2 . 35 a . u . ( washout ) ] . n = 3 . * * p < 0 . 01 ( Kruskal - Wallis test followed by Steel - Dwass test ) . Bars and error bars represent the mean ± standard deviation ( SD ) . In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST - MLC , 4 ng GST - ROCK1 catalytic domain , 1 mM ATP , 1 μg GST - protein phosphatase 1c β / δ ( PP1c β / δ ) , and 0 - 5 μg GST - LUZP1 . Quantification of the relative ppMLC / MLC ratio to the control showed that MTs promote LUZP1 - mediated inhibition of PP1c β / δ [ 1 . 00 ( 1st - lane , control ) vs . 1 . 42 ± 0 . 59 ( 2nd - lane ) vs . 1 . 72 ± 0 . 76 ( 3rd - lane ) vs . 1 . 99 ± 0 . 56 ( 4th - lane ) vs . 1 . 14 ± 0 . 37 ( 5th - lane ) vs . 2 . 87 ± 1 . 51 ( 6th - lane ) vs . 2 . 74 ± 1 . 19 ( 7th - lane ) vs . 2 . 50 ± 0 . 88 ( 8th - lane ) ] . n = 6 . * p < 0 . 05 [ Kruskal - Wallis test followed by Steel test ( compared with 1st lane ) ] . Bars and error bars represent the mean ± SD .

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

Figure 6Box plots with dot density plots showing the ratio of the apical area/basal area in co-cultures of Venus-LUZP1-expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells [REV, 0.65 ± 0.16 (control) vs. 0.90 ± 0.18 (nocodazole) vs. 0.64 ± 0.16 (washout); KO, 1.30 ± 0.17 (control) vs. 1.07 ± 0.13 (nocodazole) vs. 1.32 ± 0.19 (washout)]. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). The solid lines represent the medians and the boxes represent the inter-quartile ranges. The error bars extending from the box represents the data within 1.5 times of the interquartile range. Representative confocal micrographs of co-cultures of LUZP1-expressing wild-type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di-phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm.   Bar plots with dot density plots showing that ppMLC levels within CRs were significantly down-regulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment [WT, 21.43 ± 6.96 arbitrary units (a.u.) (control) vs. 17.67 ± 5.40 a.u. (nocodazole) vs. 20.84 ± 7.19 a.u. (washout); KO, 8.74 ± 1.71 a.u. (control) vs. 8.67 ± 1.89 a.u. (nocodazole) vs. 7.96 ± 2.35 a.u. (washout)]. n = 3. **p < 0.01 (Kruskal-Wallis test followed by Steel-Dwass test). Bars and error bars represent the mean ± standard deviation (SD).   In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST-MLC, 4 ng GST-ROCK1 catalytic domain, 1 mM ATP, 1 μg GST-protein phosphatase 1c β/δ (PP1c β/δ), and 0-5 μg GST-LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1-mediated inhibition of PP1c β/δ [1.00 (1st-lane, control) vs. 1.42 ± 0.59 (2nd-lane) vs. 1.72 ± 0.76 (3rd-lane) vs. 1.99 ± 0.56 (4th-lane) vs. 1.14 ± 0.37 (5th-lane) vs. 2.87 ± 1.51 (6th-lane) vs. 2.74 ± 1.19 (7th-lane) vs. 2.50 ± 0.88 (8th-lane)]. n = 6. *p < 0.05 [Kruskal-Wallis test followed by Steel test (compared with 1st lane)]. Bars and error bars represent the mean ± SD.


Figure 1A , B Tumor growth curve ( left ) and summary of end - point tumor masses ( right ) of 6 - 8 week - old wildtype ( WT ) and Peli1 - KO ( KO ) mice inoculated s . c . with B16F10 ( A ; WT , n = 7 ; KO , n = 6 ) or E . G7 ( B ; WT , n = 5 ; KO , n = 5 ) tumor cells . C , D Flow cytometry analysis of CD4 + and CD8 + T cells in CD45 . 2 + TILs and dLN cells from B16F10 tumor - bearing wildtype and Peli1 - KO mice , presented as a representative plot ( C ) and summary graph based on multiple mice ( D ; WT , n = 6 ; KO , n = 6 ) . E , F Flow cytometry analysis of IFNγ - producing CD4 + and CD8 + T cells in TILs , and dLN cells of B16F10 tumor - bearing ( day 19 ) wildtype and Peli1 - KO mice , presented as a representative plot ( E ) and summary graph ( F ) . ( WT , n = 6 ; KO , n = 6 ) G , H Flow cytometry analysis of IFNγ + CD8 + T cells in TILs of E . G7 tumor - bearing ( day 24 ) wildtype and Peli1 - KO mice , presented as a representative plot ( G ) and summary graph ( H ) . ( WT , n = 3 ; KO , n = 3 )

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A,B Tumor growth curve (left) and summary of end-point tumor masses (right) of 6-8 week-old wildtype (WT) and Peli1-KO (KO) mice inoculated s.c. with B16F10 (A; WT, n=7; KO, n=6) or E.G7 (B; WT, n=5; KO, n=5) tumor cells.C,D Flow cytometry analysis of CD4+ and CD8+ T cells in CD45.2+ TILs and dLN cells from B16F10 tumor-bearing wildtype and Peli1-KO mice, presented as a representative plot (C) and summary graph based on multiple mice (D; WT, n=6; KO, n=6).E,F Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs, and dLN cells of B16F10 tumor-bearing (day 19) wildtype and Peli1-KO mice, presented as a representative plot (E) and summary graph (F). (WT, n=6; KO, n=6)G,H Flow cytometry analysis of IFNγ+CD8+ T cells in TILs of E.G7 tumor-bearing (day 24) wildtype and Peli1-KO mice, presented as a representative plot (G) and summary graph (H). (WT, n=3; KO, n=3)


Figure 2A , B Tumor growth curve ( left ) and summary graph of end - point tumor masses ( right ) of wildtype ( WT ) and Peli1 - TKO ( TKO ) mice inoculated s . c . with B16 - OVA ( A ; WT , n = 5 ; KO , n = 5 ) or E . G7 ( B ; WT , n = 6 ; KO , n = 4 ) tumor cells . C Flow cytometry analysis of IFNγ - producing CD4 + and CD8 + T cells in TILs and dLN cells from B16OVA tumor - bearing wildtype and Peli1 - TKO mice , presented as a representative plot ( upper ) and summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 ) D Flow cytometry analysis of GZMB + CD8 + T cells in TILs from B16 - OVA tumor - bearing wildtype and Peli1 - TKO mice , presented as a representative plot ( upper ) and summary graph ( lower ) . ( WT , n = 5 ; KO , n = 5 ) E Tumor growth curve ( left ) and summary graph of end - point tumor masses ( right ) of B16 - OVA tumor - bearing wildtype and the indicated Peli1 conditional KO mice . ( Treg KO model : WT , n = 5 ; Treg KO , n = 6 ; MKO model : WT , n = 3 ; MKO , n = 5 ; BKO model : WT , n = 9 ; BKO , n = 9 )

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 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 O O O O O O O O O O

Figure 2A,B Tumor growth curve (left) and summary graph of end-point tumor masses (right) of wildtype (WT) and Peli1-TKO (TKO) mice inoculated s.c. with B16-OVA (A; WT, n=5; KO, n=5) or E.G7 (B; WT, n=6; KO, n=4) tumor cells.C Flow cytometry analysis of IFNγ-producing CD4+ and CD8+ T cells in TILs and dLN cells from B16OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)D Flow cytometry analysis of GZMB+CD8+ T cells in TILs from B16-OVA tumor-bearing wildtype and Peli1-TKO mice, presented as a representative plot (upper) and summary graph (lower). (WT, n=5; KO, n=5)E Tumor growth curve (left) and summary graph of end-point tumor masses (right) of B16-OVA tumor-bearing wildtype and the indicated Peli1 conditional KO mice. (Treg KO model: WT, n=5; Treg KO, n=6; MKO model: WT, n=3; MKO, n=5; BKO model: WT, n=9; BKO, n=9)


Figure 3A , B Seahorse analysis of basal ( glucose injection ) and maximal ( oligomycin injection ) ECAR or basal ( no treatment ) and maximal ( FCCP injection ) OCR in WT and Peli1 - KO OT - I CD8 T cells that were either untreated ( naïve ) or activated with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) for 16 h . Data are presented as a representative plot ( A ) or summary graph based on 6 wildtype and 6 KO mice ( B ) . qRT - PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T cells stimulated with anti - CD3 and anti - CD28 for 6 h . D qRT - PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1 - KO OT - I CD8 T cells stimulated with anti - CD3 and anti - CD28 for 6 h . E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1 - KO OT - I CD8 T cells that were stimulated with plate - bound anti - CD3 and anti - CD28 for 24 h .

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

Figure 3A,B Seahorse analysis of basal (glucose injection) and maximal (oligomycin injection) ECAR or basal (no treatment) and maximal (FCCP injection) OCR in WT and Peli1-KO OT-I CD8 T cells that were either untreated (naïve) or activated with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) for 16 h. Data are presented as a representative plot (A) or summary graph based on 6 wildtype and 6 KO mice (B).qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.D qRT-PCR analysis of the indicated genes using RNAs isolated from wildtype or Peli1-KO OT-I CD8 T cells stimulated with anti-CD3 and anti-CD28 for 6 h.E Immunoblot analysis of HK2 and Glut1 in wildtype or Peli1-KO OT-I CD8 T cells that were stimulated with plate-bound anti-CD3 and anti-CD28 for 24 h.


Figure 4A Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of freshly isolated CD8 T cells from wildtype ( WT ) or Peli1 KO ( KO ) mice ( 6 weeks old ) . Data are presented as a representative blot ( left ) and summary graphs of densitometric quantifications of the indicated proteins ( presented as phosphorylated / total protein ratio ) based on three independent experiments . B , C Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype or Peli1 - KO OT - I CD8 T cells that were first rested on ice for 15 min and then stimulated in low - serum ( 1 % FBS ) medium with anti - CD3 and anti - CD28 for the indicated time periods . D , E Seahorse analysis of basal and maximal ECAR ( D ) or OCR ( E ) using wildtype or Peli1 - KO OT - I CD8 T cells that were activated for 16 h with plate - bound anti - CD3 ( 1 μg / ml ) plus anti - CD28 ( 1 μg / ml ) in the presence of solvent control ( DMSO ) , rapamycin ( Rapa , 10 nM ) , or Torin 1 ( 100 nM ) . Data are presented as a representative plot ( left ) and summary graphs ( right ) . F , G Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype or Peli1 - KO OT - I CD8 T cells that were either not treated ( - ) or stimulated ( + ) for 1 h ( F ) or 24 h ( G ) with anti - CD3 ( 1 μg / ml ) and anti - CD28 ( 1 μg / ml ) in the presence ( + ) or absence ( - ) of rapamycin ( 10 nM ) and Torin 1 ( 100 nM ) . H qRT - PCR analysis of Ifng gene expression in naïve wildtype or Peli1 - KO OT - I CD8 T cells that were either not treated ( - ) or stimulated for 6 h ( + ) with anti - CD3 plus anti - CD28 in the presence ( + ) or absence ( - ) or rapamycin . I - K Schematic of experimental design ( I ) , tumor growth curve ( J ) , and summary of day 15 tumor weight ( K ) of B6 . SJL mice inoculated with B16 - OVA melanoma cells ( 2 x 105 ) and adoptively transferred on day 7 with in vitro activated and rapamycin - or DMSO - treated wildtype or Peli1 - KO OT - I CD8 T cells ( 4 x 105 ) .

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 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 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 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 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 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

Figure 4A Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of freshly isolated CD8 T cells from wildtype (WT) or Peli1 KO (KO) mice (6 weeks old). Data are presented as a representative blot (left) and summary graphs of densitometric quantifications of the indicated proteins (presented as phosphorylated/total protein ratio) based on three independent experiments.B,C Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were first rested on ice for 15 min and then stimulated in low-serum (1% FBS) medium with anti-CD3 and anti-CD28 for the indicated time periods.D,E Seahorse analysis of basal and maximal ECAR (D) or OCR (E) using wildtype or Peli1-KO OT-I CD8 T cells that were activated for 16 h with plate-bound anti-CD3 (1 μg/ml) plus anti-CD28 (1 μg/ml) in the presence of solvent control (DMSO), rapamycin (Rapa, 10 nM), or Torin 1 (100 nM). Data are presented as a representative plot (left) and summary graphs (right).F,G Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated (+) for 1 h (F) or 24 h (G) with anti-CD3 (1 μg/ml) and anti-CD28 (1 μg/ml) in the presence (+) or absence (-) of rapamycin (10 nM) and Torin 1 (100 nM).H qRT-PCR analysis of Ifng gene expression in naïve wildtype or Peli1-KO OT-I CD8 T cells that were either not treated (-) or stimulated for 6 h (+) with anti-CD3 plus anti-CD28 in the presence (+) or absence (-) or rapamycin.I-K Schematic of experimental design (I), tumor growth curve (J), and summary of day 15 tumor weight (K) of B6.SJL mice inoculated with B16-OVA melanoma cells (2 x 105) and adoptively transferred on day 7 with in vitro activated and rapamycin- or DMSO-treated wildtype or Peli1-KO OT-I CD8 T cells (4 x 105).


Figure 5Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T cells that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , and then treated for indicated time periods with FBS ( 10 % ) ( A ) Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) OT - I CD8 T cells that were activated for 16 h with plate - coated anti - CD3 ( 1 μg / ml ) plus anti - CD28 , starved for 6 h in serum - free RPMI medium , and then treated for indicated time periods with insulin ( B ) . Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with FBS ( C ) Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with insulin ( D ) Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in whole - cell lysates of wildtype ( WT ) or Peli1 - KO ( KO ) MEFs that were starved for 16 h in serum - free DMEM medium and then treated for the indicated time periods with EGF ( E ) .

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

Figure 5Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with FBS (10%) (A)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) OT-I CD8 T cells that were activated for 16 h with plate-coated anti-CD3 (1 μg/ml) plus anti-CD28, starved for 6 h in serum-free RPMI medium, and then treated for indicated time periods with insulin (B).Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with FBS (C)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with insulin (D)Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in whole-cell lysates of wildtype (WT) or Peli1-KO (KO) MEFs that were starved for 16 h in serum-free DMEM medium and then treated for the indicated time periods with EGF (E).


Figure 6A Immunoblot analysis of the indicated proteins in whole - cell lysates of wildtype ( WT ) and Peli1 - KO ( KO ) CD8 T cells that were either not treated ( NT ) or activated for 16 h with plate - bound anti - CD3 and anti - CD28 . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC1 ( middle panel ) and TSC2 ( right panel ) protein bands relative to the level of α - Tubulin . Immunoblot analysis of the indicated proteins in whole - cell lysates of wildtype and Peli1 - KO CD8 T cells that were activated for 1 h with anti - CD3 and anti - CD28 and then treated with cycloheximide ( CHX ) for the indicated time periods . Data are presented as a representative blot ( B ) a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated ( 0 min ) cells ( C ) . D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1 - KO CD8 T cells stimulated with anti - CD3 plus anti - CD28 for 1 h and then incubated with ( + ) or without ( - ) cycloheximide ( CHX ) and / or MG132 for 2 h . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC1 ( middle panel ) and TSC2 ( right panel ) protein bands relative to the level of α - Tubulin . Ubiquitination analysis of immunoprecipitated TSC2 ( E ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T cells by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains . Ubiquitination analysis of immunoprecipitated TSC1 ( F ) from activated ( anti - CD3 plus anti - CD28 for 2 h ) wildtype or Peli1 - KO CD8 T cells by immunobloting using anti - ubiquitin Abs detecting total ( Ub ) , K48 ( Ub - K48 ) , or K63 ( Ub - K63 ) polyubiquitin chains . Analysis of ubiquitin conjugation to TSC1 ( G , H ) in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors . Analysis of ubiquitin conjugation to TSC1 TSC2 ( I , J ) in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors . K Co - immunoprecipitaton analysis of TSC1 - TSC2 interaction in 293T cells transiently transfected ( for 48 h ) with the indicated expression vectors . L Co - immunoprecipitation analysis of endogenous TSC1 - TSC2 interaction in wildtype and Peli1 - KO CD8 T cells that were either not treated ( NT ) or stimulated for 2 h with anti - CD3 plus anti - CD28 . The right panel is a summary graph of TSC2 / TSC1 ratio based on quantification of the blot from anti - TSC1 IP ( the right four lanes of the top two panels ) .

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

Figure 6A Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype (WT) and Peli1-KO (KO) CD8 T cells that were either not treated (NT) or activated for 16 h with plate-bound anti-CD3 and anti-CD28. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.Immunoblot analysis of the indicated proteins in whole-cell lysates of wildtype and Peli1-KO CD8 T cells that were activated for 1 h with anti-CD3 and anti-CD28 and then treated with cycloheximide (CHX) for the indicated time periods. Data are presented as a representative blot (B)a summary graph of quantified TSC1 and TSC2 protein bands presented as percentage of the level in untreated (0 min) cells (C).D Immunoblot analysis of TSC1 and TSC2 in wildtype or Peli1-KO CD8 T cells stimulated with anti-CD3 plus anti-CD28 for 1 h and then incubated with (+) or without (-) cycloheximide (CHX) and/or MG132 for 2 h. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC1 (middle panel) and TSC2 (right panel) protein bands relative to the level of α-Tubulin.Ubiquitination analysis of immunoprecipitated TSC2 (E) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.Ubiquitination analysis of immunoprecipitated TSC1 (F) from activated (anti-CD3 plus anti-CD28 for 2 h) wildtype or Peli1-KO CD8 T cells by immunobloting using anti-ubiquitin Abs detecting total (Ub), K48 (Ub-K48), or K63 (Ub-K63) polyubiquitin chains.Analysis of ubiquitin conjugation to TSC1 (G, H) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.Analysis of ubiquitin conjugation to TSC1 TSC2 (I, J) in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.K Co-immunoprecipitaton analysis of TSC1-TSC2 interaction in 293T cells transiently transfected (for 48 h) with the indicated expression vectors.L Co-immunoprecipitation analysis of endogenous TSC1-TSC2 interaction in wildtype and Peli1-KO CD8 T cells that were either not treated (NT) or stimulated for 2 h with anti-CD3 plus anti-CD28. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the blot from anti-TSC1 IP (the right four lanes of the top two panels).


Figure 7A Analysis of ubiquitin conjugation to wildtype ( WT ) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag - tagged TSC1 and its mutants along with ( + ) or without ( - ) the indicated expression vectors . B Immunoblot analysis of the indicated phosphorylated ( p - ) and total proteins in lysates of TSC1 - KO MEFs transfected with ( + ) or without ( - ) TSC1 wildtype or K30A expression vectors . The cells were serum starved and then left either untreated ( NT ) or stimulated with 10 % FBS for 30 mins . Data are presented as a representative blot ( left panel ) and a summary graph of quantified TSC2 ( right panel ) protein bands relative to the level of β - Actin . C Analysis of TSC2 ubiquitination in 293 cells transfected ( for 48 h ) with ( + ) or without ( - ) the indicated expression vectors . D Co - immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) the indicated expression vectors . KO MEF cells were transfected with indicated expression plasmids . The right panel is a summary graph of TSC2 / TSC1 ratio based on quantification of the last four lanes of the anti - Flag IP blot in left panel . E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with ( + ) or without ( - ) TSC1 WT or K30A and treated with MG132 for 2 h before lysis . F Immunoblot analysis of endogenous TSC2 in TSC1 - KO MEFs transfected ( for 48 h ) with Myc - TSC1 wildtype or K30A and subsequently treated with cycloheximide ( 10 μg / ml ) for the indicated time periods before lysis . The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated ( 0 min ) lane .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 7A Analysis of ubiquitin conjugation to wildtype (WT) TSC1 and the indicated TSC1 mutants in 293T cells transiently transfected Flag-tagged TSC1 and its mutants along with (+) or without (-) the indicated expression vectors.B Immunoblot analysis of the indicated phosphorylated (p-) and total proteins in lysates of TSC1-KO MEFs transfected with (+) or without (-) TSC1 wildtype or K30A expression vectors. The cells were serum starved and then left either untreated (NT) or stimulated with 10% FBS for 30 mins. Data are presented as a representative blot (left panel) and a summary graph of quantified TSC2 (right panel) protein bands relative to the level of β-Actin.C Analysis of TSC2 ubiquitination in 293 cells transfected (for 48 h) with (+) or without (-) the indicated expression vectors.D Co-immunoprecipitation analysis of the interaction between endogenous TSC2 and exogenous TSC1 wildtype and K30A in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) the indicated expression vectors. KO MEF cells were transfected with indicated expression plasmids. The right panel is a summary graph of TSC2/TSC1 ratio based on quantification of the last four lanes of the anti-Flag IP blot in left panel.E Analysis of ubiquitin conjugation to endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with (+) or without (-) TSC1 WT or K30A and treated with MG132 for 2 h before lysis.F Immunoblot analysis of endogenous TSC2 in TSC1-KO MEFs transfected (for 48 h) with Myc-TSC1 wildtype or K30A and subsequently treated with cycloheximide (10 μg/ml) for the indicated time periods before lysis. The right panel is a summary graph of quantified TSC2 WT and K30A protein bands presented as percentages of the level in the untreated (0 min) lane.


Figure 2 ( A ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor before and after shockwave stimulation in HBSS Ca2 + medium with ( yellow line , n = 6 , N = 3 ) or without the Piezo1 expression ( black line , n = 6 , N = 3 ) . ( B ) The representative FRET / ECFP ratio images of calcium biosensor in Piezo1 - expressed HEK cells before ( top left ) and after ( top right ) LIS stimulation . Phase image with laser initiating point ( left ) and fluorescent image indicating Piezo1 expression ( right ) were also shown on the bottom panels ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( C ) The time course of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2 + medium ( n = 6 , N = 3 ) .

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

Figure 2(A) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor before and after shockwave stimulation in HBSS Ca2+ medium with (yellow line, n = 6, N = 3) or without the Piezo1 expression (black line, n = 6, N = 3). (B) The representative FRET/ECFP ratio images of calcium biosensor in Piezo1-expressed HEK cells before (top left) and after (top right) LIS stimulation. Phase image with laser initiating point (left) and fluorescent image indicating Piezo1 expression (right) were also shown on the bottom panels (Scale bar, 40 μm). Color scale bars in the figures are to show the FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (C) The time course of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells before and after multiple shockwave stimulations in HBSS Ca2+ medium (n = 6, N = 3).


Figure 3 ( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before ( left ) and after ( right ) shockwave stimulation ( Scale bar , 40 μm ) . Color scale bars in the figures are to show the ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized ECFP / FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2 + media with ( green line , n = 16 , N = 3 ) or without Piezo1 ( black line , n = 10 , N = 3 ) . ( C ) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2 + medium . * P < 0 . 05 from student two - tailed t - test .

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

Figure 3(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 overexpressed HEK cells before (left) and after (right) shockwave stimulation (Scale bar, 40 μm). Color scale bars in the figures are to show the ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized ECFP/FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after shockwave stimulation in HBSS Ca2+ media with (green line, n = 16, N = 3) or without Piezo1 (black line, n = 10, N = 3). (C) The percentage change of FRET ratio of the FAK FRET biosensor in HEK cells with or without Piezo1 after shockwave stimulation in HBSS Ca2+ medium. *P < 0.05 from student two-tailed t-test.


Figure 4 ( A ) The representative ECFP / FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate ECFP / FRET ratios , with cold and hot colors representing low and high ratios , respectively . ( B ) The time courses of normalized FRET ratio ( Mean ± SEM ) of FAK FRET biosensor before and after Yoda1 stimulation with ( pink line , n = 10 , N = 3 ) or without Piezo1 ( black line , n = 11 , N = 3 ) . The grey line represents the DMSO control group ( n = 8 , N = 3 ) . ( C ) The representative FRET / ECFP ratio images of the calcium biosensor in the Piezo1 - expressing HEK cells before and after 25 μM Yoda1 stimulation ( Scale bar , 10 μm ) . Color scale bars in the figures indicate FRET / ECFP ratios , with cold and hot colors representing low and high ratios , respectively . ( D ) The time courses of normalized FRET ratio ( Mean ± SEM ) of calcium biosensor in the Piezo1 - expressing HEK cells ( purple line , n = 9 , N = 3 ) before and after 25 μM Yoda1 stimulation in culture medium .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4(A) The representative ECFP/FRET ratio images of FAK biosensor in Piezo1 expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate ECFP/FRET ratios, with cold and hot colors representing low and high ratios, respectively. (B) The time courses of normalized FRET ratio (Mean ± SEM) of FAK FRET biosensor before and after Yoda1 stimulation with (pink line, n = 10, N = 3) or without Piezo1 (black line, n = 11, N = 3). The grey line represents the DMSO control group (n = 8, N = 3).(C) The representative FRET/ECFP ratio images of the calcium biosensor in the Piezo1-expressing HEK cells before and after 25 μM Yoda1 stimulation (Scale bar, 10 μm). Color scale bars in the figures indicate FRET/ECFP ratios, with cold and hot colors representing low and high ratios, respectively. (D) The time courses of normalized FRET ratio (Mean ± SEM) of calcium biosensor in the Piezo1-expressing HEK cells (purple line, n = 9, N = 3) before and after 25 μM Yoda1 stimulation in culture medium.


Figure 7 ( A ) & ( B ) , The time courses ( Mean ± SEM ) ( A ) and representative images ( Scale bar , 10 μm ) of FRET ratio of SHP2 biosensor ( Y542F ) before and after 25 μM or 0 . 5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression ( red bar , n = 5 , N = 3 ; blue bar , n = 5 , N = 3 ; purple bar , n = 11 , N = 3 ; green bar , n = 5 , N = 3 ) .

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

Figure 7(A)&(B), The time courses (Mean ± SEM) (A) and representative images (Scale bar, 10 μm) of FRET ratio of SHP2 biosensor (Y542F) before and after 25 μM or 0.5 μM Yoda1 stimulation in cells with or without Piezo1 overexpression (red bar, n = 5, N=3; blue bar, n = 5, N=3; purple bar, n = 11, N=3; green bar, n = 5, N=3).


Figure 1Survival rates of control and Cdc50a cKO mice after oil or 4 - OHT injection ( n = 5 for each group , P * * * = 0 . 0003 ) . Bar graphs showing audiogenic seizure scores following auditory stimuli ( 5 , 10 , 15 , 20 , 40 kHz or white noise ( WN ) at 70 , 80 , 90 , or 100 dB ) in control ( E ) and Cdc50a cKO ( F ) mice ( n = 8 for each group ) . Representative confocal z - stack images showing cFos ( magenta ) - positive neurons from control and Cdc50a cKO mice ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) . Scale bar , 20 μm . Bar graphs showing the relative area of cFos - positive cells in the brains of control and Cdc50a cKO mice ( n = 5 for each group , * * P = 0 . 0030 , n . s . , not significant ) .

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Figure 1Survival rates of control and Cdc50a cKO mice after oil or 4-OHT injection (n = 5 for each group, P*** = 0.0003).Bar graphs showing audiogenic seizure scores following auditory stimuli (5, 10, 15, 20, 40 kHz or white noise (WN) at 70, 80, 90, or 100 dB) in control (E) and Cdc50a cKO (F) mice (n = 8 for each group).Representative confocal z-stack images showing cFos (magenta)-positive neurons from control and Cdc50a cKO mice (CTX: cortex, HP: hippocampus, and IC: inferior colliculus). Scale bar, 20 μm. Bar graphs showing the relative area of cFos-positive cells in the brains of control and Cdc50a cKO mice (n = 5 for each group, **P = 0.0030, n.s., not significant).


Figure 2Schematic diagram of the experiment . Representative confocal z - stack images of PSD95 ( green ) - and VGLUT1 ( red ) - positive excitatory ( A ) and Gephyrin ( green ) - and VGAT ( magenta ) - positive inhibitory ( B ) synapses in the CTX , HP , and IC ( CTX : cortex , HP : hippocampus , and IC : inferior colliculus ) of control and Cdc50a cKO mice . The dashed line represents where the high magnified insert was taken from . Scale bar , 20 μm . Representative current clamp traces from the IC of control and Cdc50a cKO mice ; + 100 pA ( upper ) and + 400 pA ( lower ) currents were injected into control mice ( left ) and Cdc50a cKO mice ( right ) for 500 ms .

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Figure 2Schematic diagram of the experiment. Representative confocal z-stack images of PSD95 (green)- and VGLUT1 (red)-positive excitatory (A) and Gephyrin (green)- and VGAT (magenta)-positive inhibitory (B) synapses in the CTX, HP, and IC (CTX: cortex, HP: hippocampus, and IC: inferior colliculus) of control and Cdc50a cKO mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.Representative current clamp traces from the IC of control and Cdc50a cKO mice; +100 pA (upper) and +400 pA (lower) currents were injected into control mice (left) and Cdc50a cKO mice (right) for 500 ms.


Figure 3The bar graphs show the relative percentages of neurons showing PS exposure only in the soma ( left ) and overall PS exposure ( right ) ( n = 30 - 40 cells for each group ) in control and Cdc50a cKO mice . Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP . Representative confocal z - stack images showing Gephyrin - positive inhibitory post - synapses in the somas ( red ) and proximal ( green ) and distal dendrites ( cyan ) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice . Scale bar , 20 μm . Bar graphs showing the number of Gephyrin - positive inhibitory post - synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice ( n = 4 for each group , P * * * = 0 . 0002 , P * * * * < 0 . 0001 ) .

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Figure 3The bar graphs show the relative percentages of neurons showing PS exposure only in the soma (left) and overall PS exposure (right) (n = 30-40 cells for each group) in control and Cdc50a cKO mice.Schematic diagram of imaging of the neuronal somas and proximal and distal dendrites of neurons in the CA1 region of the HP. Representative confocal z-stack images showing Gephyrin-positive inhibitory post-synapses in the somas (red) and proximal (green) and distal dendrites (cyan) of neurons in the CA1 region of the HP in control and Cdc50a cKO mice. Scale bar, 20 μm. Bar graphs showing the number of Gephyrin-positive inhibitory post-synapses in the somas and proximal and distal dendrites of neurons in the CA1 of the HP in control and Cdc50a cKO mice (n = 4 for each group, P*** = 0.0002, P**** < 0.0001).