Figure 6 ( A ) RCC1 . 24 ‐ NeoR ( endogenous ) , and RCC1 . 24 cells to which NeoR protein had been added ( exogenous ) , were treated with different concentrations of 3 ‐ MA and cell surface presentation of the NeoR ‐ derived peptide on HLA ‐ DP3 monitored with the T cell clone 20 - 4 / A4 ‐ specific T cell clone 20 - 4 / A4 . Inhibition of autophagy by 3 ‐ MA causes an almost complete inhibition of endogenous antigen presentation , while presentation of exogenous NeoR is not affected . ( B ) EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( endogenous ) , or incubated with recombinant NeoR protein ( exogenous ) , were treated with the inhibitors of autophagy , wortmannin and 3 ‐ MA , and HLA ‐ DP3 ‐ restricted presentation of the NeoR ‐ derived peptide determined . Inhibition of antigen presentation by leupeptin served as control . ( C ) Presentation of the HLA ‐ A2 ‐ restricted tyrosinase peptide in EBV ‐ B1 . 11 cells transfected with pINCO ‐ Tyrosinase is not affected by these inhibitors of autophagy . FACS analysis of HLA ‐ class I and HLA ‐ DP expression on RCC1 . 24 ( D ) and EBV ‐ B1 . 11 cells ( E ) As compared to untreated cells ( thick ‐ lined histogram ) , treatment of cells with 7 . 5 mM 3 ‐ MA ( shaded histogram ) does not cause down ‐ regulation of HLA ‐ class I or HLA ‐ DP . Isotype control is shown as thin ‐ lined histogram .

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 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 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 6(A) RCC1.24‐NeoR (endogenous), and RCC1.24 cells to which NeoR protein had been added (exogenous), were treated with different concentrations of 3‐MA and cell surface presentation of the NeoR‐derived peptide on HLA‐DP3 monitored with the T cell clone 20-4/A4‐specific T cell clone 20-4/A4. Inhibition of autophagy by 3‐MA causes an almost complete inhibition of endogenous antigen presentation, while presentation of exogenous NeoR is not affected.(B) EBV‐B1.11 cells transfected with pINCO‐NeoR (endogenous), or incubated with recombinant NeoR protein (exogenous), were treated with the inhibitors of autophagy, wortmannin and 3‐MA, and HLA‐DP3‐restricted presentation of the NeoR‐derived peptide determined. Inhibition of antigen presentation by leupeptin served as control.(C) Presentation of the HLA‐A2‐restricted tyrosinase peptide in EBV‐B1.11 cells transfected with pINCO‐Tyrosinase is not affected by these inhibitors of autophagy.FACS analysis of HLA‐class I and HLA‐DP expression on RCC1.24 (D) and EBV‐B1.11 cells (E) As compared to untreated cells (thick‐lined histogram), treatment of cells with 7.5 mM 3‐MA (shaded histogram) does not cause down‐regulation of HLA‐class I or HLA‐DP. Isotype control is shown as thin‐lined histogram.


Figure 1Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . All treatment groups were then analyzed by qPCR for gene expression ( A ) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7 - 54 and FA - TLR7 - 54 ( n = 3 , technical replicates ) . Human monocytic ( THP - 1 ) cells were induced to acquire an M2 - like phenotype ( see Methods ) and treated with different concentrations of TLR7 - 54 or FA - TLR7 - 54 for either 48h or 2h In the latter case , after the 2h incubation , culture medium was replaced with drug - free medium and incubation was continued for 46h . cell supernatants were analyzed for secreted cytokines by ELISA . ( B - C ) Changes in CCL18 and IL - 1β in the culture media induced upon treatment with TLR7 - 54 and FA - TLR7 - 54 for the treatment regimens ( n = 3 , technical replicates ) .

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

Figure 1Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. All treatment groups were then analyzed by qPCR for gene expression (A) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7-54 and FA-TLR7-54 (n=3, technical replicates).Human monocytic (THP-1) cells were induced to acquire an M2-like phenotype (see Methods) and treated with different concentrations of TLR7-54 or FA-TLR7-54 for either 48h or 2h In the latter case, after the 2h incubation, culture medium was replaced with drug-free medium and incubation was continued for 46h. cell supernatants were analyzed for secreted cytokines by ELISA. (B-C) Changes in CCL18 and IL-1β in the culture media induced upon treatment with TLR7-54 and FA-TLR7-54 for the treatment regimens (n=3, technical replicates).


Figure 2M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . mRNA levels of profibrotic markers , Arg1 ( A ) , CD206 ( B ) and CD163 ( C ) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) . M2 - induced human monocyte - derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h , or initially for 2h in the presence or absence of FA - glucosamine ( competition ) followed by 46h in the absence of drug ( 2 + 46h ) , as described in Fig 1 . protein levels of secreted profibrotic CCL18 ( D ) and antifibrotic cytokines , CXCL10 ( E ) and IL - 6 ( F ) ( n = 3 , technical replicates ) were then determined . Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA - glucosamine ( 2 + 46h , competition ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B) and CD163 (C) Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).M2-induced human monocyte-derived macrophages were treated with 100 nM of the indicated drug either continuously for 48h, or initially for 2h in the presence or absence of FA-glucosamine (competition) followed by 46h in the absence of drug (2+46h), as described in Fig 1. protein levels of secreted profibrotic CCL18 (D) and antifibrotic cytokines, CXCL10 (E) and IL-6 (F) (n=3, technical replicates) were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA-glucosamine (2+46h, competition).


Figure 3 ( A ) Healthy mice or BLM - induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200 - fold excess FA - glucosamine to block all folate receptors . After 2h , mice were euthanized , resected and imaged for fluorescence intensity ( n = 5 ) . ( B ) Alternatively , lungs from mice injected with 100 nmol OTL38 in the present or absence of 200 - fold excess FA - glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4 / 80 prior to FACS analysis ( n = 3 ) . Representative plots showing the gating strategy yielding live macrophages ( 7AAD - CD11b + F4 / 80 + ) and OTL38 positive macrophages are shown . ( C ) Percentages of live macrophages in BLM - induced mice ( n = 3 ) . ( D ) Percentages of lung macrophages that accumulated OTL38 in vivo ( 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 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 B-PANEL_START 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

Figure 3(A) Healthy mice or BLM-induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200-fold excess FA-glucosamine to block all folate receptors. After 2h, mice were euthanized, resected and imaged for fluorescence intensity (n=5).(B) Alternatively, lungs from mice injected with 100 nmol OTL38 in the present or absence of 200-fold excess FA-glucosamine were collagenase digested and stained with 7AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n=3). Representative plots showing the gating strategy yielding live macrophages (7AAD- CD11b+ F4/80+) and OTL38 positive macrophages are shown.(C) Percentages of live macrophages in BLM-induced mice (n=3).(D) Percentages of lung macrophages that accumulated OTL38 in vivo (n=3).


Figure 4Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( A ) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR ( n = 3 ) . Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( B ) BALF cells were pelleted and similarly analyzed for the indicated mRNAs ( n = 3 ) . Healthy mice or BLM - induced mice were injected intravenously on day 10 with either vehicle ( 3 % DMSO in PBS ) , or 10 nmol TLR7 - 54 or FA - TLR7 - 54 dissolved in vehicle , and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid ( BALF ) . ( C ) BALF supernatant was also collected and analyzed by ELISA for IL - 6 , IFNα and TNFα ( 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 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 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

Figure 4Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (A) Lungs were digested and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n=3).Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n=3).Healthy mice or BLM-induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7-54 or FA-TLR7-54 dissolved in vehicle, and 1 or 4h later sacrificed to collect both lungs and bronchioalveolar lavage fluid (BALF). (C) BALF supernatant was also collected and analyzed by ELISA for IL-6, IFNα and TNFα (n=3).


Figure 5 ( B ) Survival analysis of the treatment groups ( Healthy , n = 5 ; others , n = 10 ) . BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for Arg1 ( C ) , MMP9 ( D ) , TIMP3 ( E ) BALF was collected on day 21 and centrifuged to isolate cell pellets . Cells ( primarily macrophages ) were analyzed by qPCR for CD86 ( F ) and IRAK4 ( G ) ( n = 5 ) . BALF supernatant was analyzed by ELISA for IFNγ ( H ) ( n = 5 ) . ( I ) Lungs were resected and subjected to hematoxylin - eosin ( H & E ) , trichome ( collagen ) and α - SMA IHC staining ( scale bars , 200 μm ) , Lungs were ( J ) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content ( Healthy , n = 5 ; others , n = 10 ) , ( K ) Ashcroft score quantitation of fibrosis ( n = 5 ) .

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

Figure 5(B) Survival analysis of the treatment groups (Healthy, n=5; others, n=10).BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for Arg1 (C), MMP9 (D), TIMP3 (E)BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for CD86 (F) and IRAK4 (G) (n=5).BALF supernatant was analyzed by ELISA for IFNγ (H) (n=5).(I) Lungs were resected and subjected to hematoxylin-eosin (H&E), trichome (collagen) and α-SMA IHC staining (scale bars, 200 μm),Lungs were (J) hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content (Healthy, n=5; others, n=10),(K) Ashcroft score quantitation of fibrosis (n=5).


Figure 6Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( A ) Analysis of body weight change versus time ( n = 10 ) . Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( B ) Analysis of the number of cells per milliliter of BALF ( n = 5 ) . Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( C ) Quantitation of total hydroxyproline content per right lung ( Healthy control , n = 5 ; others , n = 7 - 9 ) . Healthy control ( filled circles ) or BLM - induced mice were treated with vehicle ( filled squares ) , 1 nmol ( empty circles ) , 3 nmol ( empty squares ) , or 10 nmol ( filled triangles ) FA - TLR7 - 54 and then sacrificed on day 21 for analysis . ( D ) H & E staining and trichrome staining of lung tissue ( scale bars , 200 μm ) .

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 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 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 O O O O O O O O O O

Figure 6Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (A) Analysis of body weight change versus time (n=10).Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (B) Analysis of the number of cells per milliliter of BALF (n=5).Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (C) Quantitation of total hydroxyproline content per right lung (Healthy control, n=5; others, n=7-9).Healthy control (filled circles) or BLM-induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA-TLR7-54 and then sacrificed on day 21 for analysis. (D) H&E staining and trichrome staining of lung tissue (scale bars, 200 μm).


Figure 7Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( A - C ) Measurement of plasma IL - 6 ( A ) , IFNα ( B ) and TNFα ( C ) ( n = 3 ) . Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( D - F ) Effect of drug concentration on plasma levels of IL - 6 ( D ) , IFNα ( E ) , and TNFα ( F ) at 1 . 5h , 1h , or 1h after treatment , respectively ( n = 2 ) . Healthy mice were tail vein injected with 10 nmol TLR7 - 54 ( circles ) or FA - TLR7 - 54 ( squares ) , and peripheral blood was collected at indicated time points after drug injection . ( G ) Change in body weight as a measure of systemic toxicity during alternate day dosing ( n = 2 ) .

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 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 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 7Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (A-C) Measurement of plasma IL-6 (A), IFNα (B) and TNFα (C) (n=3).Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (D-F) Effect of drug concentration on plasma levels of IL-6 (D), IFNα (E), and TNFα (F) at 1.5h, 1h, or 1h after treatment, respectively (n=2).Healthy mice were tail vein injected with 10 nmol TLR7-54 (circles) or FA-TLR7-54 (squares), and peripheral blood was collected at indicated time points after drug injection. (G) Change in body weight as a measure of systemic toxicity during alternate day dosing (n=2).


Figure 1 ( A ) Classical , ( B ) Rolled , and ( C ) Rotated subpopulations of the 80S * HCVIRES complex . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) . ( D ) Subpopulations of the 80S * HCVIRES complex containing P - site tRNA . The 80S is in the classical configuration and the head tilt is not present . Left column shows an overview of the final cryo - EM reconstructions with the HCVIRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCVIRES ( pink ) , tRNA ( green ) . ( E ) The cryo - EM map was filtered according to resolution . 40S * HCV IRES . Left column shows an overview of the final cryo - EM reconstructions with the HCV IRES ( pink ) , 40S subunit ( yellow ) , 60S subunit ( blue ) and tRNA ( green ) . Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts , respectively . 28S - 5S - 5 . 8S rRNA ( blue ) , 60S proteins ( gold ) , 18S rRNA ( yellow ) , 40S proteins ( dark gray ) , HCV IRES ( pink ) , tRNA ( green ) .

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 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 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

Figure 1(A) Classical, (B) Rolled, and (C) Rotated subpopulations of the 80S*HCVIRES complex. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCVIRES (pink), tRNA (green).(D) Subpopulations of the 80S*HCVIRES complex containing P-site tRNA. The 80S is in the classical configuration and the head tilt is not present. Left column shows an overview of the final cryo-EM reconstructions with the HCVIRES (pink),40S subunit (yellow),60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the60S and40S parts, respectively.28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray),HCVIRES (pink), tRNA (green).(E) The cryo-EM map was filtered according to resolution. 40S*HCV IRES. Left column shows an overview of the final cryo-EM reconstructions with the HCV IRES (pink), 40S subunit (yellow), 60S subunit (blue) and tRNA (green). Middle and right column shows mesh representation of the maps and docked models of the 60S and 40S parts, respectively. 28S-5S-5.8S rRNA (blue), 60S proteins (gold), 18S rRNA (yellow), 40S proteins (dark gray), HCV IRES (pink), tRNA (green).


Figure 2 ( A ) Comparison of 40S subunit positions in Rolled , Rotated , Classical / no head tilt ( orange ) and Classical 80S * HCVIRES complex ( yellow ) in common 60S alignment . The cryo - EM map of the Classical 80S * HCVIRES was filtered to 6 Å . Arrows indicate the direction of movement during transition between two different states . The distance changes in the 40S subunit positions resulting from the rigid body transformation are color - coded in Å units . ( B ) Comparison of the HCVIRES in Classical 80S * HCVIRES complex ( gray ) and upon P - tRNA binding ( Classical , no head tilt ; purple ) in a common 40S body alignment . The distance of the movement of domain II was indicated with an arrow and Å

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

Figure 2(A) Comparison of 40S subunit positions in Rolled, Rotated, Classical/no head tilt (orange) and Classical 80S*HCVIRES complex (yellow) in common 60S alignment. The cryo-EM map of the Classical 80S*HCVIRES was filtered to 6 Å. Arrows indicate the direction of movement during transition between two different states. The distance changes in the 40S subunit positions resulting from the rigid body transformation are color-coded in Å units.(B) Comparison of the HCVIRES in Classical 80S*HCVIRES complex (gray) and upon P-tRNA binding (Classical, no head tilt; purple) in a common 40S body alignment. The distance of the movement of domain II was indicated with an arrow and Å units


Figure 3 ( A ) Adopted secondary structure of the IRES representing the tertiary fold . ( B ) Atomic model of the HCV IRES RNA . IRES domains are presented as coloured ribbons , overlaid with experimental IRES density ( gray , semi - transparent ) . ( C ) . Molecular model of the HCV IRES ( pink ) with ribosomal interaction sites ( coloured proteins and helices ) .

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

Figure 3(A) Adopted secondary structure of the IRES representing the tertiary fold.(B) Atomic model of the HCV IRES RNA. IRES domains are presented as coloured ribbons, overlaid with experimental IRES density (gray, semi-transparent).(C). Molecular model of the HCV IRES (pink) with ribosomal interaction sites (coloured proteins and helices).


Figure 4 ( A ) The secondary structure of the HCVIRES domain III ( hIII1 , hIII2 , IIIe and IIId ) and helix 26 ( h26 ) from the 18S rRNA . Colors of HCVIRES Domains are the same as in Fig 3 . Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line . ( B ) The interaction core between IIId , IIIe , hIII1 of HCVIRES ( colored as in ( A ) ) and Expansion segment 7 ( ES7 ) of h26 ( dark gray ) . ( C ) The rearrangement of h26 ( red ) upon binding of HCVIRES . The 80S * HCVIRES complex ( yellow ) is compared to the elongating POST state ( gray ; PDB 5Aj0 ) ( Behrmann et al , 2015 ) . The loop closing the helix is highlighted in red and dark gray . ( E ) Close up on the interactions between GGG266 - 268 of IIId ( cyan ) and CCC1116 - 1118 of ES7 ( gray ) . ( F ) Close up on the interactions between G295 , A296 and U297 of IIIe ( green ) , A136 and A288 of hIII1 ( yellow ) and U1114 , U1115 of ES7 ( gray ) .

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 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

Figure 4(A) The secondary structure of the HCVIRES domain III (hIII1, hIII2, IIIe and IIId) and helix 26 (h26) from the 18S rRNA. Colors of HCVIRES Domains are the same as in Fig 3. Interaction between the HCVIRES and 18S rRNA are indicated as red lines and a tertiary IRES interaction is indicated as black line.(B) The interaction core between IIId, IIIe, hIII1 of HCVIRES (colored as in (A)) and Expansion segment 7 (ES7) of h26 (dark gray).(C) The rearrangement of h26 (red) upon binding of HCVIRES. The 80S*HCVIRES complex (yellow) is compared to the elongating POST state (gray; PDB 5Aj0) (Behrmann et al, 2015). The loop closing the helix is highlighted in red and dark gray.(E) Close up on the interactions between GGG266-268 of IIId (cyan) and CCC1116-1118 of ES7 (gray).(F) Close up on the interactions between G295, A296 and U297 of IIIe (green), A136 and A288 of hIII1 (yellow) and U1114, U1115 of ES7 (gray).


Figure 5 ( A ) Comparison of the 40S subunit from Classical 80S * HCVIRES complex ( yellow ) and canonical POST state ( gray ; EMD 2875 ) ( Behrmann et al , 2015 ) in common 40S body alignment . The arrow indicates the direction of the head tilt movement . Ribosomal proteins of the 40S head ( uS7 , eS25 and eS28 ) that interact with the HCVIRES domains II and IV ( pink ) on the 40S head are illuminated with blue , green , and purple , respectively . The positions of the proteins in the canonical POST state are illuminated in lighter color . The numbers indicate interaction site of HCVIRES and the 40S subunit . ( B ) Close up view on the interaction site of HCVIRES and the 40S head region . Numbers correspond to red labels in ( A ) . The rearrangement of the uS7 loop ( blue ) compared to the Classical 80S * HCVIRES complex ( gray ) is indicated . The arrow indicates the direction of the rearrangement of the uS7 loop . ( B - 1 and B - 4 ) inter subunit view from 40S subunit . ( B - 2 and - 3 ) inter subunit view from 60S subunit .

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

Figure 5(A) Comparison of the 40S subunit from Classical 80S*HCVIRES complex (yellow) and canonical POST state (gray; EMD 2875) (Behrmann et al, 2015) in common 40S body alignment. The arrow indicates the direction of the head tilt movement. Ribosomal proteins of the 40S head (uS7, eS25 and eS28) that interact with the HCVIRES domains II and IV (pink) on the 40S head are illuminated with blue, green, and purple, respectively. The positions of the proteins in the canonical POST state are illuminated in lighter color. The numbers indicate interaction site of HCVIRES and the 40S subunit.(B) Close up view on the interaction site of HCVIRES and the 40S head region. Numbers correspond to red labels in (A). The rearrangement of the uS7 loop (blue) compared to the Classical 80S*HCVIRES complex (gray) is indicated. The arrow indicates the direction of the rearrangement of the uS7 loop. (B-1 and B-4) inter subunit view from 40S subunit. (B-2 and -3) inter subunit view from 60S subunit.


Figure 6 ( A , B ) Comparative solvent side views of the small subunit with the bacterial SD - chamber on the 30S from the 70S POST state ( PDB 2HGR ) ( Yusupova et al , 2006 ) and the corresponding region of the eukaryotic 40S in the classical 80S * HCV IRES complex . Analogous proteins and rRNA helices are highlighted in the same colours , respectively . ( A ) Crystal structure of the 30S subunit with mRNA . The SD - helix is established between the mRNA ( orange ) and the 3 ' end of the 16S rRNA ( blue ) . ( B ) Atomic model of the 40S subunit with the HCV IRES model ( pink ) . The mRNA - part and S2 of the IRES are shown in orange and cyan , the 3 ' - end of the 18S rRNA is shown in blue . ( C ) Overview and zoomed view of Classical 80S * HCVIRES . The cryo - EM map of the Classical 80S * HCVIRES was filtered according to resolution . ( D and E ) mRNA exit channel from in Classical 80S * HCVIRES and POST state ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) . The two structures are aligned at the 40S body . We note eS28 as part of the 40S head is differently located because of the head tilt . The surface representation was created using the pdb models . Shown are the 18S rRNA ( yellow ) , ribosomal proteins ( gray ) , eS26 ( cyan ) eS28 ( purple ) , HCVIRES ( pink ) .

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 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

Figure 6(A, B) Comparative solvent side views of the small subunit with the bacterial SD-chamber on the 30S from the 70S POST state (PDB 2HGR) (Yusupova et al, 2006) and the corresponding region of the eukaryotic 40S in the classical 80S*HCV IRES complex. Analogous proteins and rRNA helices are highlighted in the same colours, respectively. (A) Crystal structure of the 30S subunit with mRNA. The SD-helix is established between the mRNA (orange) and the 3' end of the 16S rRNA (blue).(B) Atomic model of the 40S subunit with the HCV IRES model (pink). The mRNA-part and S2 of the IRES are shown in orange and cyan, the 3'-end of the 18S rRNA is shown in blue.(C) Overview and zoomed view of Classical 80S*HCVIRES. The cryo-EM map of the Classical 80S*HCVIRES was filtered according to resolution.(D and E) mRNA exit channel from in Classical 80S*HCVIRES and POST state (PDB 5AJ0) (Behrmann et al, 2015). The two structures are aligned at the 40S body. We note eS28 as part of the 40S head is differently located because of the head tilt. The surface representation was created using the pdb models. Shown are the 18S rRNA (yellow), ribosomal proteins (gray), eS26 (cyan) eS28 (purple), HCVIRES (pink).


Figure 7 ( A ) Comparison of HCV IRES from 80S * HCV IRES Classical ( pink ) and tRNAs from POST state ( gray ) ( PDB 5AJ0 ) ( Behrmann et al , 2015 ) in a common 40S body alignment of the cryo - EM maps . ( B ) Same as above but in a common 40S head alignment . ( C ) Comparison of the 40S subunit and bound ligands of the classical 80S * HCVIRES complex ( orange ) and 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( yellow ) ( EMD 2683 ) ( Yamamoto et al , 2014 ) in common 60S alignment . ( D ) The models of the 80S * HCVIRES Classical complex ( gray ) , 80S * HCVIRES * Met - tRNA * eIF5B * GMPPNP complex ( POST - like state ) ( green ) ( PDB 4UPZ ) ( Yamamoto et al , 2014 ) and 80S * HCVIRES Classical / no head tilt complex ( pink ) are rigid body fitted into the corresponding densities in a common 40S body alignment . The Helix 68 from the 28S rRNA is depicted as well ( blue ) .

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 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

Figure 7(A) Comparison of HCV IRES from 80S*HCV IRES Classical (pink) and tRNAs from POST state (gray)(PDB 5AJ0) (Behrmann et al, 2015) in a common 40S body alignment of the cryo-EM maps.(B) Same as above but in a common 40S head alignment.(C) Comparison of the 40S subunit and bound ligands of the classical 80S*HCVIRES complex (orange) and 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (yellow)(EMD 2683) (Yamamoto et al, 2014) in common 60S alignment.(D) The models of the 80S*HCVIRES Classical complex (gray), 80S*HCVIRES*Met-tRNA*eIF5B*GMPPNP complex (POST-like state) (green)(PDB 4UPZ) (Yamamoto et al, 2014) and 80S*HCVIRES Classical/no head tilt complex (pink) are rigid body fitted into the corresponding densities in a common 40S body alignment. The Helix 68 from the 28S rRNA is depicted as well (blue).


Figure 8 ( A and B ) Comparison of the 80S * HCV IRES Classical complex and Classical / no head tilt complex ( 40S yellow , 60S blue , IRES pink , P - tRNA green ) at intersubunit view . Inter subunit bridge 1b / c and 1a are indicated as B1b / c and B1a , respectively . The cryo - EM map of classical 80S * HCV IRES classical complex was filtered to 6 Å . ( C and D ) Close up of the mRNA tunnel . The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å

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

Figure 8(A and B) Comparison of the 80S*HCV IRES Classical complex and Classical/no head tilt complex (40S yellow, 60S blue, IRES pink, P-tRNA green) at intersubunit view. Inter subunit bridge 1b/c and 1a are indicated as B1b/c and B1a, respectively. The cryo-EM map of classical 80S*HCV IRES classical complex was filtered to 6 Å.(C and D) Close up of the mRNA tunnel. The distance between helix 29 and 44 of the 18S rRNA are indicated with an arrow and Å units


Figure 1 , Total protein levels of Munc18 - 1 . HEK293T cells transfected with WT or mutant Munc18 - 1 variants and either GFP , syntaxin - 11 - 264 or syntaxin - 11 - 180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins , normalized to β - actin ( Synt - 1A = syntaxin - 1A ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test , followed by Dunn ' s multiple comparison test ; n = 11 independent experiments ; exact p values are shown in Appendix Table S1 ) . Solubility of Munc18 - 1 . HEK293T cells transfected as in ( C ) were solubilized in 0 . 1 % Triton X - 100 ( TX ) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting . TX - soluble Munc18 - 1 was measured as percent of total Munc18 - 1 by quantitative immunoblotting . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test followed by Dunn ' s multiple comparison test , or by one - way ANOVA followed by Bonferroni post - hoc test ; n = 5 - 9 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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 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

Figure 1, Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1).Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).


Figure 3Same as in ( A , B ) except that compounds 9 , 10 , 11 , 13 , 14 and 16 were added at 0 . 25 , 1 , 5 , 20 , 100 , or 250 µM . Note , that the data for 250 µM were not plotted in ( D ) because of causing neuronal death . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 by Kruskal - Wallis test with Dunn ' s multiple comparison test , or one - way ANOVA with Dunnett ' s post - hoc test ; n = 6 - 15 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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

Figure 3Same as in (A, B) except that compounds 9, 10, 11, 13, 14 and 16 were added at 0.25, 1, 5, 20, 100, or 250 µM. Note, that the data for 250 µM were not plotted in (D) because of causing neuronal death. Data are means ± SEM (*p < 0.05, **p < 0.01 by Kruskal-Wallis test with Dunn's multiple comparison test, or one-way ANOVA with Dunnett's post-hoc test; n = 6-15 independent experiments; exact n and p values are shown in Appendix Table S1).


Figure 4Limited proteolysis . Recombinant purified proteins were incubated with increasing concentrations of trypsin . Remaining protein levels were analyzed by quantitative immunoblotting . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 , by 2 - way ANOVA and Dunnett ' s multiple - comparison test ; n = 4 independent experiments ; exact p values are shown in Appendix Table S1 ) .

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

Figure 4Limited proteolysis. Recombinant purified proteins were incubated with increasing concentrations of trypsin. Remaining protein levels were analyzed by quantitative immunoblotting. Data are means ± SEM (* p < 0.05, **p < 0.01, ****p < 0.0001, by 2-way ANOVA and Dunnett's multiple-comparison test; n = 4 independent experiments; exact p values are shown in Appendix Table S1).


Figure 5Munc18 - 1 knockout neurons expressing G544D Munc18 - 1 were subjected to analysis of mean firing rate before addition of compounds ( 0 h ) or 48 hours after vehicle ( DMSO ) or compound addition . 16 electrodes per well were analyzed for neuronal firing ( A ; purple boxes indicate network activity ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA and Dunnett ' s multiple comparison test , or Kruskal - Wallis test followed by Dunn ' s post - hoc test ; n = 11 - 16 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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

Figure 5Munc18-1 knockout neurons expressing G544D Munc18-1 were subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).


Figure 6Uptake of synaptotagmin - 1 antibody during high K + stimulation . Neurons expressing cre recombinase and / or WT , R406H or G544D Munc18 - 1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay . Endocytosed synaptotagmin - 1 antibody was quantified by immunostaining ( A - C ; scale bar = 30 µm ; 4 - PBA = 4 - phenylbutyrate ) , via counting the number of pixels > intensity of 15 ( D ) . Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 10 independent experiments ; exact n and p values are shown in Appendix Table S1 ) .

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 6Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).


Figure 7Plot of change in fluorescence from baseline to peak fluorescence for Munc18 - 1 G544D ( H ) Data are means ± SEM ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by one - way one - way ANOVA and Dunnett ' s multiple comparison test ; n = 7 - 23 ; exact n and p values are shown in Appendix Table S1 ) . Note , that compound 10 is fluorescent at a similar wavelength than pHluorin , so experiments at concentrations higher than 1 µM were not possible .

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

Figure 7Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.


Figure 8Heat - induced paralysis . Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 ° C over a period of 120 min , and paralysis was scored at indicated time points . Data are means ± SEM ( n = 3 - 4 independent experiments on ten worms per experiment ; exact n and p values are shown in Appendix Table S1 ) . ( B , D , F , H , J , L ) Quantification of paralysis at 20 minutes . Data are means ± SEM ( * , # p < 0 . 05 , * * , # # p < 0 . 01 , * * * , # # # p < 0 . 001 , # as compared to WT , * as compared to mutant by two - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 4 independent experiments on ten worms per experiment ; exact n and p values are shown in Appendix Table S1 ) . Rescue of the subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP were immobilized , and the ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger puncta , broken arrowheads to single , smaller puncta ( N , O ) . Scale bar in ( N ) and ( O ) = 10 µm . 4 - PBA = 4 - phenylbutyrate . Rescue of the subcellular localization of UNC - 18 in worms expressing G544D UNC - 18 . C . elegans expressing WT : : GFP or G544D : : GFP at 1 , 5 , 20 or 100 µM compound were immobilized , and the ventral nerve cord was imaged . Solid arrowheads point to pairs of bigger puncta , broken arrowheads to single , smaller punctaQuantification of soluble and aggregated Munc18 - 1 . Data are means ± SEM ( * * p < 0 . 01 , * * * p < 0 . 001 by one - way ANOVA and Dunnett ' s multiple comparison test ; n = 3 - 4 worms ; exact n and p values are shown in Appendix Table S1 ) .

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 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 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 O O O O O

Figure 8Heat-induced paralysis. Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 °C over a period of 120 min, and paralysis was scored at indicated time points. Data are means ± SEM (n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1). (B, D, F, H, J, L) Quantification of paralysis at 20 minutes. Data are means ± SEM (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, # as compared to WT, * as compared to mutant by two-way ANOVA and Dunnett's multiple comparison test; n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1).Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP at 1, 5, 20 or 100 µM compound were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller punctaQuantification of soluble and aggregated Munc18-1. Data are means ± SEM (**p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-4 worms; exact n and p values are shown in Appendix Table S1).


Figure 1 ( A ) The hair coats of Sirt7 − / − and Sirt7 + / + mice were clipped on P60 in the mid second telogen phase . Images were captured at P60 , P120 and P150 . ( B ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 − / − and Sirt7 + / + mice . n = 6 mice / genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( C ) H & E staining of the skin in unshaved Sirt7 − / − and Sirt7 + / + mice on P90 and P130 . ( D ) Representative immunofluorescence images showing Ki67 and p - cadherin ( Pcad , labeling HG cells ) expression in Sirt7 − / − and Sirt7 + / + HFs on P74 and P90 . The HF boundary and dermal papilla ( DP ) is denoted by the dotted and dashed lines , respectively . ( E ) Percent Ki67 positive cells in each hair germ in D ; n = 3 mice and 10 HGs were counted in each mouse . ( F ) Telogen duration in Sirt7 − / − and Sirt7 + / + mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( G ) The hair coats of Sirt7 - TG and WT mice were clipped on P45 and images were captured on P56 and P90 . ( H ) The postnatal day when the hair coat recovered by 25 % , 50 % , 75 % and 100 % in shaved Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( I ) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7 - TG and WT HFs at P60 and P65 . The HF boundary and DP is noted by the dotted and dashed lines , respectively . ( J ) The percentage of Ki67 positive cells in each HG ; n = 3 mice and 10 HGs were counted in each mouse . ( K ) H & E staining of skin tissues in unshaved Sirt7 - TG and WT mice on P65 . ( L ) Telogen duration in Sirt7 - TG and WT mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum .

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

Figure 1(A) The hair coats of Sirt7−/− and Sirt7+/+ mice were clipped on P60 in the mid second telogen phase. Images were captured at P60, P120 and P150.(B) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7−/− and Sirt7+/+ mice. n = 6 mice/genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(C) H&E staining of the skin in unshaved Sirt7−/− and Sirt7+/+ mice on P90 and P130.(D) Representative immunofluorescence images showing Ki67 and p-cadherin (Pcad, labeling HG cells) expression in Sirt7−/− and Sirt7+/+ HFs on P74 and P90. The HF boundary and dermal papilla (DP) is denoted by the dotted and dashed lines, respectively.(E) Percent Ki67 positive cells in each hair germ in D; n = 3 mice and 10 HGs were counted in each mouse.(F) Telogen duration in Sirt7−/− and Sirt7+/+ mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(G) The hair coats of Sirt7-TG and WT mice were clipped on P45 and images were captured on P56 and P90.(H) The postnatal day when the hair coat recovered by 25%, 50%, 75% and 100% in shaved Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(I) Representative immunofluorescence images showing Ki67 and Pcad expression in Sirt7-TG and WT HFs at P60 and P65. The HF boundary and DP is noted by the dotted and dashed lines, respectively.(J) The percentage of Ki67 positive cells in each HG; n = 3 mice and 10 HGs were counted in each mouse.(K) H&E staining of skin tissues in unshaved Sirt7-TG and WT mice on P65.(L) Telogen duration in Sirt7-TG and WT mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.


Figure 2 ( A - B ) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation - induced hair cycle in WT mice . P56 , P69 , P74 and P84 indicate the postnatal day in the second hair cycle . Pd , post depilation . Dashed line indicates bulge ( Bu ) of a hair follicle and solid line indicates the Ki - 67 positive staining region . ( C ) The hair coats of Sirt7f / f and Sirt7f / f ; K15 - Cre mice were clipped at P60 and images were captured on P60 , P125 and P150 . ( D ) The postnatal day of hair coat recovery by 25 % , 50 % 75 % and 100 % in shaved Sirt7f / f and Sirt7f / f ; K15 - Cre mice . n = 6 mice per genotype . Box - and - whisker plots : mid - line , median ; box , 25th and 75th percentiles ; whiskers , minimum and maximum . ( E ) H & E staining of the skin in Sirt7f / f and Sirt7f / f ; K15 - Cre mice at P100 . ( F ) Representative immunofluorescence images showing Ki67 / Pcad expression ( upper ) and Cd34 / Ki67 expression ( lower ) in Sirt7fl / fl and Sirt7f / f ; K15 - Cre HFs on P56 , P74 and P85 . The white dashed line indicates bulge of hair follicle . ( G ) The percentage of Ki67 - positive cells per HG ; n = 3 mice and 10 HGs were counted in each mouse . ( H ) Ki67 - positive cells per bulge ; n = 3 mice and 10 bulges were counted in each mouse .

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

Figure 2(A-B) IHC labeling of Sirt7 and Ki67 expression in the skin at different HF phases in the second hair cycle and in the depilation-induced hair cycle in WT mice. P56, P69, P74 and P84 indicate the postnatal day in the second hair cycle. Pd, post depilation. Dashed line indicates bulge (Bu) of a hair follicle and solid line indicates the Ki-67 positive staining region.(C) The hair coats of Sirt7f/f and Sirt7f/f;K15-Cre mice were clipped at P60 and images were captured on P60, P125 and P150.(D) The postnatal day of hair coat recovery by 25%, 50% 75% and 100% in shaved Sirt7f/f and Sirt7f/f;K15-Cre mice. n = 6 mice per genotype. Box-and-whisker plots: mid-line, median; box, 25th and 75th percentiles; whiskers, minimum and maximum.(E) H&E staining of the skin in Sirt7f/f and Sirt7f/f;K15-Cre mice at P100.(F) Representative immunofluorescence images showing Ki67/Pcad expression (upper) and Cd34/Ki67 expression (lower) in Sirt7fl/fl and Sirt7f/f;K15-Cre HFs on P56, P74 and P85. The white dashed line indicates bulge of hair follicle.(G) The percentage of Ki67-positive cells per HG; n = 3 mice and 10 HGs were counted in each mouse.(H) Ki67-positive cells per bulge; n = 3 mice and 10 bulges were counted in each mouse.


Figure 3 ( A ) Representative colonies formed by Sirt7 + / + , Sirt7 - / - and Sirt7 - TG bulge SCs that were puriﬁed from telogen - phase HFs and allowed to grow for 2 weeks . ( B - C ) Quantiﬁcations of colony number and size in ( A ) ; n = 3 mice per genotype . Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice ( D ) proteins in HFSC colonies from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice Nfatc1 protein levels were quantified using Image J Plus 6 . 0 ( E , n = 3 mice for each group ) . ( F ) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs . ( G ) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells . ( H ) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments ( n = 3 ) . The relative luciferase activity was determined by dual luciferase reporter assay . ( I - J ) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f / f , Sirt7f / f ; K15 - Cre and Sirt7 - TG mice at the indicted time points . The black dashed line indicates the bulge ( Bu ) of hair follicle . ( K - L ) qPCR analysis of Nfatc1 - targeted genes in HFSCs colonies cultured from Sirt7 + / + , Sirt7 - / - and Sirt7 - TG mice , n = 3 for each genotypes .

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

Figure 3(A) Representative colonies formed by Sirt7+/+, Sirt7-/- and Sirt7-TG bulge SCs that were puriﬁed from telogen-phase HFs and allowed to grow for 2 weeks. (B-C) Quantiﬁcations of colony number and size in (A); n = 3 mice per genotype. Immunoblot analysis of the indicated proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice (D)proteins in HFSC colonies from Sirt7+/+, Sirt7-/- and Sirt7-TG mice Nfatc1 protein levels were quantified using Image J Plus 6.0 (E, n = 3 mice for each group).(F) Immunoblot analysis of the NFATc1 expression level in human keratinocyte HaCaT cells treated with SIRT7 siRNAs.(G) Immunoblot analysis of the NFATc1 expression in the presence of ectopic SIRT7 expression in HaCaT cells.(H) NFATc1 luciferase activity was monitored in HEK293T cells after the indicated treatments (n=3). The relative luciferase activity was determined by dual luciferase reporter assay.(I-J) IHC analyses of Nfatc1 expression in hair follicles of Sirt7f/f, Sirt7f/f ;K15-Cre and Sirt7-TG mice at the indicted time points. The black dashed line indicates the bulge (Bu) of hair follicle.(K-L) qPCR analysis of Nfatc1-targeted genes in HFSCs colonies cultured from Sirt7+/+, Sirt7-/-and Sirt7-TG mice, n=3 for each genotypes.


Figure 4 ( A - B ) Immunoblot analysis of the interactions between ectopic HA - SIRT7 and FLAG - NFATc1in HEK293T cells . ( C ) Co - immunoprecipitation ( CoIP ) between endogenous NFATc1 and SIRT7 in HEK293T cells . ( D ) Immunoblots showing endogenous NFATc1 pull - down by GST - SIRT7 in HEK293T cells . ( E ) Immunoblot analysis of HA - SIRT7 WT and various mutants in the anti His - NFATc1 immunoprecipitates . ∆ N indicates N - terminal deletion ; ∆ C indicates C - terminal deletion ; AC indicates N - terminal and C - terminal deletion . ( F ) Immunoblot analysis of HA - SIRT7 levels in truncated FLAG - NFATc1 immunoprecipitates . N indicates the N - terminal domain ; C indicates C - terminal domain . ( G - H ) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells . ( I - J ) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments . Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vitro ( K ) Immunoblot analysis of NFATc1 - WT / K612R acetylation based on SIRT7 deacetylation assays in vivo ( L ) . Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) and acetylation levels under the indicated treatments . Immunoblots showing NFATc1 pan phosphorylation ( probing to anti S / T antibody ) and acetylation levels under the indicated treatments . ( O ) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants , noting that K612R represents deacetylation while K612Q hyperacetylation .

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

Figure 4(A-B) Immunoblot analysis of the interactions between ectopic HA-SIRT7 and FLAG-NFATc1in HEK293T cells.(C) Co-immunoprecipitation (CoIP) between endogenous NFATc1 and SIRT7 in HEK293T cells.(D) Immunoblots showing endogenous NFATc1 pull-down by GST-SIRT7 in HEK293T cells.(E) Immunoblot analysis of HA-SIRT7 WT and various mutants in the anti His-NFATc1 immunoprecipitates. ∆N indicates N-terminal deletion; ∆C indicates C-terminal deletion; AC indicates N-terminal and C-terminal deletion.(F) Immunoblot analysis of HA-SIRT7 levels in truncated FLAG-NFATc1 immunoprecipitates. N indicates the N-terminal domain; C indicates C-terminal domain.(G-H) Immunoblot analysis of the interactions between endogenous NFATc1 and SIRT7 in keratinocyte HaCaT cells.(I-J) Immunoblot analysis of NFATc1 acetylation levels in response to the indicated treatments.Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vitro (K)Immunoblot analysis of NFATc1-WT/K612R acetylation based on SIRT7 deacetylation assays in vivo (L).Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.Immunoblots showing NFATc1 pan phosphorylation (probing to anti S/T antibody) and acetylation levels under the indicated treatments.(O) Immunoblot analysis of NFATc1 phosphorylation among K612 mutants, noting that K612R represents deacetylation while K612Q hyperacetylation.


Figure 5 ( A ) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide ( CHX ) , with or without SIRT7 overexpression . ( B ) Quantification of NFATc1 levels in ( A ) from 3 independent experiments . ( C ) Immunoblots of Nfatc1 protein levels in Sirt7 + / + and Sirt7 - / - keratinocytes . ( D ) Quantification of Nfatc1 levels in ( C ) from 3 independent experiments . ( E - F ) Immunoblots ( E ) and related quantitative data ( F , n = 3 ) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA - SIRT7 or a SIRT7 enzyme - dead mutant ( H187Y ) . ( G - H ) Immunoblots ( G ) and quantitative data ( H , n = 3 ) showing NFATc1 levels in HEK293T cells transfected with NFATc1 - CA , with or without ectopic SIRT7 , treated with MG132 . ( I - J ) Immunoblots ( I ) and related quantitative data ( J , n = 3 ) showing NFATc1 - WT and NFATc1 - K612R protein turnover in HEK293T cells . Immunoblots showing the interaction ( by co - immunoprecipitation ) between ectopic NFATc1 / PA28γ in HEK293T cells ( K ) ( L ) after manipulating SIRT7 levels . Immunoblots showing the interaction ( by co - immunoprecipitation ) between endogenous NFATc1 / PA28γ in HaCaT cells ( L ) after manipulating SIRT7 levels . ( M - N ) Immunoblot ( M ) and quantitative data ( N , n = 3 ) analysis of NFATc1 levels upon overexpression of HA - SIRT7 with or without PA28γ oligo siRNAs treatment . ( O ) Co - immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA - SIRT7 or an enzyme - dead mutant ( H187Y ) .

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

Figure 5(A) Representative immunoblots showing NFATc1 degradation in HEK293T cells stably overexpressing NFATc1 in the presence of cycloheximide (CHX), with or without SIRT7 overexpression. (B) Quantification of NFATc1 levels in (A) from 3 independent experiments. (C) Immunoblots of Nfatc1 protein levels in Sirt7+/+ and Sirt7-/- keratinocytes. (D) Quantification of Nfatc1 levels in (C) from 3 independent experiments. (E-F) Immunoblots (E) and related quantitative data (F, n=3) showing NFATc1 protein levels in HEK293T cells overexpressing wildtype HA-SIRT7 or a SIRT7 enzyme-dead mutant (H187Y).(G-H) Immunoblots (G) and quantitative data (H, n=3) showing NFATc1 levels in HEK293T cells transfected with NFATc1-CA, with or without ectopic SIRT7, treated with MG132.(I-J) Immunoblots (I) and related quantitative data (J, n=3) showing NFATc1-WT and NFATc1-K612R protein turnover in HEK293T cells.Immunoblots showing the interaction (by co-immunoprecipitation) between ectopic NFATc1/PA28γ in HEK293T cells (K) (L) after manipulating SIRT7 levels.Immunoblots showing the interaction (by co-immunoprecipitation) between endogenous NFATc1/PA28γ in HaCaT cells (L) after manipulating SIRT7 levels.(M-N) Immunoblot (M) and quantitative data (N, n=3) analysis of NFATc1 levels upon overexpression of HA-SIRT7 with or without PA28γ oligo siRNAs treatment.(O) Co-immunoprecipitation between NFATc1 and PA28γ in HEK293T cells cotransfected with WT HA-SIRT7 or an enzyme-dead mutant (H187Y).


Figure 6 ( A ) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7 + / + and Sirt7 - / - mice with or without Cyclosporin A ( CsA ) treatment . The black dashed line indicates bulge ( Bu ) of hair follicle . ( B ) Quantitative analysis of Nfatc1 levels in ( A , n = 10 hair follicles ) using ImageJ plus 6 . 0 . ( C ) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7 + / + and Sirt7 - / - HFs with or without CsA treatment . ( D ) The percentage of Ki67 - positive cells per HG in ( C , n = 10 hair follicles ) . ( E ) H & E staining of the skin in the indicated treatment groups at P100 . ( F ) Representative images showing haircoat recovery after the indicated treatments . The dashed circle indicates the shaved area ; the dotted line indicates the hair growth boundary .

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 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 B-PANEL_START 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

Figure 6(A) IHC staining of Nfatc1 expression in HFs on P84 in Sirt7+/+ and Sirt7-/- mice with or without Cyclosporin A (CsA) treatment. The black dashed line indicates bulge (Bu) of hair follicle. (B) Quantitative analysis of Nfatc1 levels in (A, n=10 hair follicles) using ImageJ plus 6.0. (C) Immunofluorescent staining of Ki67 and Pcad at P84 in Sirt7+/+ and Sirt7-/- HFs with or without CsA treatment.(D) The percentage of Ki67-positive cells per HG in (C, n=10 hair follicles).(E) H&E staining of the skin in the indicated treatment groups at P100.(F) Representative images showing haircoat recovery after the indicated treatments. The dashed circle indicates the shaved area; the dotted line indicates the hair growth boundary.


Figure 7 ( A ) Immunoblot analysis of Sirt7 expression in young ( 3M ) and aged ( 18M ) HFSCs . # 1 , # 2 , # 3 represented individual mice . Below , quantification of Sirt7 protein levels ; n = 3 mice for each group ; the Y - axis represents the relative band intensity ( measured with Image J ® ) normalized to the signal in young mice . ( B - D ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young ( 3 months ) and old ( 18 months ) mice ( B ) , and the relative levels of Sirt7 and Nfatc1 - positive cells in the bulge ( C - D ) . The white / black dashed line indicates bulge ( Bu ) of hair follicle . ( E ) Images were captured of the hair coats of Sirt7 + / + and Sirt7 - / - mice at 15 months . The yellow dashed lines indicate hair thinning or hair loss . ( F ) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15 - month - old Sirt7 + / + and Sirt7 - / - mice . The white / black dashed line indicates bulge ( Bu ) of hair follicle . ( G ) Quantification of Nfatc1 - positive cells per bulge in ( F , n = 10 hair follicles ) . ( H ) Images captured of the skin in the plucked regions ( blue dotted lines ) of WT ( 3 months ) , aged WT ( 20 months ) and aged Sirt7 - TG mice ( 20 months ) , 14 days after depilation . Pigmentation of the skin indicates hair regrowth . n = 3 mice per genotype . ( I ) Hair re - growth scores were analyzed for young WT , old WT and old Sirt7 - TG mice in ( H ) , n = 3 mice for each group . ( J ) The hair coat recovery score ( described in methods ) after the back hair of old WT and old Sirt7 - TG mice was shaved . n = 3 mice per genotype . ( K ) Images captured of the hair coats of WT and Sirt7 - TG mice at 28 months - of - age . The yellow dashed lines indicate the hair thinning or hair loss regions .

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 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 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 B-PANEL_START 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 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 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

Figure 7(A) Immunoblot analysis of Sirt7 expression in young (3M) and aged (18M) HFSCs. #1, #2, #3 represented individual mice. Below, quantification of Sirt7 protein levels; n=3 mice for each group; the Y-axis represents the relative band intensity (measured with Image J®) normalized to the signal in young mice.(B-D) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of young (3 months) and old (18 months) mice (B), and the relative levels of Sirt7 and Nfatc1-positive cells in the bulge (C-D). The white/black dashed line indicates bulge (Bu) of hair follicle.(E) Images were captured of the hair coats of Sirt7+/+ and Sirt7-/- mice at 15 months. The yellow dashed lines indicate hair thinning or hair loss.(F) Immunofluorescence and IHC staining of Nfatc1 and Sirt7 in HFs of 15-month-old Sirt7+/+ and Sirt7-/- mice. The white/black dashed line indicates bulge (Bu) of hair follicle.(G) Quantification of Nfatc1-positive cells per bulge in (F, n=10 hair follicles).(H) Images captured of the skin in the plucked regions (blue dotted lines) of WT (3 months), aged WT (20 months) and aged Sirt7-TG mice (20 months), 14 days after depilation. Pigmentation of the skin indicates hair regrowth. n = 3 mice per genotype.(I) Hair re-growth scores were analyzed for young WT, old WT and old Sirt7-TG mice in (H), n=3 mice for each group.(J) The hair coat recovery score (described in methods) after the back hair of old WT and old Sirt7-TG mice was shaved. n = 3 mice per genotype.(K) Images captured of the hair coats of WT and Sirt7-TG mice at 28 months-of-age. The yellow dashed lines indicate the hair thinning or hair loss regions.


Figure 1Speed distribution profile of PMN treated with 0 . 5 µM E4 or DMSO for 30 min . Graph shows an increase in the rate of axonal transport upon E4 treatment ( DMSO , 153 endosomes , 13 axons ; E4 , 165 endosomes , 13 axons ; N = 4 independent experiments , data shown are mean ± SEM ) . PMN treated with 0 . 5 µM E4 for 30 min showed a significant decrease in phospho - IGF1R levels compared to controls . Protein levels were normalised to β - actin ( * * * P < 0 . 001 , Students t - test , N = 3 independent experiments , data shown are mean ± SEM ) .

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 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 1Speed distribution profile of PMN treated with 0.5 µM E4 or DMSO for 30 min. Graph shows an increase in the rate of axonal transport upon E4 treatment (DMSO, 153 endosomes, 13 axons; E4, 165 endosomes, 13 axons; N=4 independent experiments, data shown are mean ± SEM).PMN treated with 0.5 µM E4 for 30 min showed a significant decrease in phospho-IGF1R levels compared to controls. Protein levels were normalised to β-actin (*** P< 0.001, Students t-test, N=3 independent experiments, data shown are mean ± SEM).


Figure 2PMN were treated with 1 µM PPP ( blue ) or 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Example images of DIV6 PMN treated with 1 µM PPP or 50 ng / ml IGF1 . PMN were stained with 30 nM AlexaFluor555 - HcT . The scale bar is 50 µm . PMN were treated with 1 µM PPP ( blue ) or 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Example of HcT - containing organelle tracking in the axon of PMN . Each colour denotes a single endosome tracked over time . The scale bar is 10 µm . PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows the average velocity of HcT - containing organelles , after PPP treatment compared to controls ( DMSO : 138 endosomes , 24 axons , 6 , 433 movements ; PPP : 130 endosomes , 23 axons , 5 , 222 movements ) ( * * P = 0 . 0061 , Student ' s t - test , N = 4 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles pausing per axon . There was no difference in pausing between PPP - treated neurons and controls ( P = 0 . 45 , Student ' s T - test , N = 4 independent experiments data shown are mean ± SEM ) . PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Graph shows the average velocity of HcT - containing organelles after IGF1 treatment compared to control ( water : 138 endosomes , 22 axons , 5 , 119 movements ; IGF1 : 106 endosomes , 20 axons , 5 , 202 movements ; * * * P = 2 . 83x10 - 5 , Student ' s T - test , N = 3 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles spent pausing per axon . IGF1 significantly increased the pausing time of HcT - containing organelles ( * P = 0 . 011 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) . PMN were treated with 1 µM PPP ( blue ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of PPP - treated neurons and controls . PPP caused an increase in instantaneous velocities of HcT - containing organelles ( N = 3 independent experiments , data shown are mean ± SEM ) . PMN were treated with 50 ng / ml IGF1 ( green ) for 30 min before assessing axonal transport using 30 nM AlexaFluor555 - HcT . Speed distribution profile of IGF1 - treated neurons and controls . IGF1 caused a decrease in instantaneous velocities of HcT - containing organelles compared to controls ( N = 3 independent experiments , data shown are mean ± SEM ) .

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 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 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 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 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

Figure 2PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example images of DIV6 PMN treated with 1 µM PPP or 50 ng/ml IGF1. PMN were stained with 30 nM AlexaFluor555-HcT. The scale bar is 50 µm.PMN were treated with 1 µM PPP (blue) or 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Example of HcT-containing organelle tracking in the axon of PMN. Each colour denotes a single endosome tracked over time. The scale bar is 10 µm.PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles, after PPP treatment compared to controls (DMSO: 138 endosomes, 24 axons, 6,433 movements; PPP: 130 endosomes, 23 axons, 5,222 movements) (** P= 0.0061, Student's t-test, N=4 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. There was no difference in pausing between PPP-treated neurons and controls (P=0.45, Student's T-test, N=4 independent experiments data shown are mean ± SEM).PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Graph shows the average velocity of HcT-containing organelles after IGF1 treatment compared to control (water: 138 endosomes, 22 axons, 5,119 movements; IGF1: 106 endosomes, 20 axons, 5,202 movements; *** P=2.83x10-5, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles spent pausing per axon. IGF1 significantly increased the pausing time of HcT-containing organelles (* P=0.011, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).PMN were treated with 1 µM PPP (blue) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of PPP-treated neurons and controls. PPP caused an increase in instantaneous velocities of HcT-containing organelles (N=3 independent experiments, data shown are mean ± SEM).PMN were treated with 50 ng/ml IGF1 (green) for 30 min before assessing axonal transport using 30 nM AlexaFluor555-HcT. Speed distribution profile of IGF1-treated neurons and controls. IGF1 caused a decrease in instantaneous velocities of HcT-containing organelles compared to controls (N=3 independent experiments , data shown are mean ± SEM).


Figure 3Western blot showing the levels of pAKT ( Ser473 ) , total Akt , pErk1 / 2 ( T202 / Y204 ) and total Erk1 / 2 after treatment with 1 µM PPP or 50 ng / ml IGF1 for 60 min . Protein was normalised to GAPDH . Western blot quantification . PMN treated with PPP showed a significant decrease in Akt activation ( * P = 0 . 049 , N = 5 independent experiments , Student ' s T - test ) , but no significant difference was seen on Erk1 / 2 activation ( P = 0 . 07 , Student ' s T - test , N = 5 independent experiments ) . 50 ng / ml IGF1 treated neurons showed a significant increase in Akt activation ( * * P = 0 . 008 , Student ' s T - test , N = 3 independent experiments ) . In contrast , no change in Erk1 / 2 activation ( P = 0 . 88 , Student ' s T - test , N = 3 independent experiments ) was observed ( data shown are mean ± SEM ) . Graph shows the average velocity of HcT - containing organelles treated with Capivasertib or Ipatasertib ( 10 nM ) for 30 min compared to controls ( DMSO : 483 endosomes , 34 axons , 41 , 897 movements ; Capivasertib : 658 endosomes , 43 axons , 49 , 841 movements ; Ipatasertib : 605 endosomes , 32 axons , 41 , 485 movements ) ( * * P = 2 x10 - 16 , One - way ANOVA , Tukey ' s post - hoc test : DMSO - Capivasertib and DMSO - Ipatasertib * * * P < 0 . 0001 ; N = 6 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Percent of time HcT - containing organelles pausing per axon . Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing ( * P = 1 . 14 x10 - 7 , One - way ANOVA , Tukey ' s post - hoc test : DMSO - Capivasertib P = 9 . 37x10 - 5 , DMSO - Ipatasertib P = 2 x10 - 7 ; data shown are mean ± SEM , N = 6 independent experiments ) .

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 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

Figure 3Western blot showing the levels of pAKT (Ser473), total Akt, pErk1/2 (T202/Y204) and total Erk1/2 after treatment with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Protein was normalised to GAPDH. Western blot quantification. PMN treated with PPP showed a significant decrease in Akt activation (* P=0.049, N=5 independent experiments, Student's T-test), but no significant difference was seen on Erk1/2 activation (P=0.07, Student's T-test, N=5 independent experiments). 50 ng/ml IGF1 treated neurons showed a significant increase in Akt activation (** P=0.008, Student's T-test, N=3 independent experiments). In contrast, no change in Erk1/2 activation (P=0.88, Student's T-test, N=3 independent experiments) was observed (data shown are mean ± SEM).Graph shows the average velocity of HcT-containing organelles treated with Capivasertib or Ipatasertib (10 nM) for 30 min compared to controls (DMSO: 483 endosomes, 34 axons, 41,897 movements; Capivasertib: 658 endosomes, 43 axons, 49,841 movements; Ipatasertib: 605 endosomes, 32 axons, 41,485 movements) (** P=2 x10-16, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib and DMSO-Ipatasertib *** P <0.0001; N=6 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Percent of time HcT-containing organelles pausing per axon. Treatment with Ipatasertib and Capivasertib reduced the amount of time organelles spent pausing (* P=1.14 x10-7, One-way ANOVA, Tukey's post-hoc test: DMSO-Capivasertib P= 9.37x10-5, DMSO-Ipatasertib P= 2 x10-7; data shown are mean ± SEM, N=6 independent experiments).


Figure 4PMN were treated with either an shRNA targeting IGF1R ( 1 µl ) or a scrambled control 6 h after plating . At DIV 6 - 7 , retrograde axonal transport was analysed using 30 nM AlexaFluor555 - HcT . Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls ( IGF1R shRNA : 108 endosomes , 21 axons , 801 movements ; scrambled shRNA : 94 endosomes , 20 axons , 777 movements , * P = 0 . 011 , Student ' s T - test , N = 3 independent experiments , boxplot shows median , first and third quartiles . Upper / lower whiskers extend to 1 . 5 * the interquartile range ) . Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control . IGF1R knockdown caused an increase in instantaneous velocities of HcT - containing organelles and decreased the amount of pausing ( N = 3 independent experiments , data shown are mean ± SEM ) .

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

Figure 4PMN were treated with either an shRNA targeting IGF1R (1 µl) or a scrambled control 6 h after plating. At DIV 6-7, retrograde axonal transport was analysed using 30 nM AlexaFluor555-HcT. Knockdown of IGF1R caused a significant increase in the average velocity of signalling endosomes compared to controls (IGF1R shRNA: 108 endosomes, 21 axons, 801 movements; scrambled shRNA: 94 endosomes, 20 axons, 777 movements, * P=0.011, Student's T-test, N=3 independent experiments, boxplot shows median, first and third quartiles. Upper/lower whiskers extend to 1.5 * the interquartile range). Speed distribution profile including pausing events of PMN treated with a shRNA targeting IGF1R or a scrambled control. IGF1R knockdown caused an increase in instantaneous velocities of HcT-containing organelles and decreased the amount of pausing (N=3 independent experiments, data shown are mean ± SEM).


Figure 5Western blot of PMN treated with 1 µM PPP or 50 ng / ml IGF1 for 60 min . Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins , all proteins were assessed from the same blot . Hook3 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 102 . 16 ± 4 . 3 % , P = 0 . 67 , Student ' s T - test , N = 3 independent experiments ; water , 100 % ; IGF1 , 93 . 48 ± 4 . 2 % , P = 0 . 26 , N = 3 independent experiments , data shown are mean ± SEM ) . However , BICD1 expression was significantly increased in PMN treated with PPP compared to control ( DMSO , 100 % ; PPP , 137 . 1 ± 7 . 3 % , * * P = 0 . 002 , Student ' s T - test , N = 7 independent experiments ) . In contrast , IGF1R stimulation by IGF1 had no effect on BICD1 expression ( water , 100 % ; IGF1 , 88 . 1 ± 14 . 0 % , P = 0 . 49 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) . BICD2 expression didn ' t change after treatment with PPP or IGF1 compared to controls ( DMSO , 100 % ; PPP , 104 . 44 ± 6 . 5 % , P = 0 . 51 , N = 3 independent experiments : water , 100 % ; IGF1 , 106 ± 7 . 5 % , P = 0 . 51 , Student ' s T - test , N = 3 independent experiments , data shown are mean ± SEM ) .

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

Figure 5Western blot of PMN treated with 1 µM PPP or 50 ng/ml IGF1 for 60 min. Cells were lysed and the extracts assessed for expression levels of dynein adaptor proteins, all proteins were assessed from the same blot. Hook3 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 102.16 ± 4.3%, P=0.67, Student's T-test, N=3 independent experiments; water, 100%; IGF1, 93.48 ± 4.2%, P=0.26, N=3 independent experiments, data shown are mean ± SEM). However, BICD1 expression was significantly increased in PMN treated with PPP compared to control (DMSO, 100%; PPP, 137.1 ± 7.3%, **P=0.002, Student's T-test, N=7 independent experiments). In contrast, IGF1R stimulation by IGF1 had no effect on BICD1 expression (water, 100%; IGF1, 88.1 ± 14.0%, P=0.49, Student's T-test, N=3 independent experiments, data shown are mean ± SEM). BICD2 expression didn't change after treatment with PPP or IGF1 compared to controls (DMSO, 100%; PPP, 104.44 ± 6.5%, P=0.51, N=3 independent experiments: water, 100%; IGF1, 106 ± 7.5%, P=0.51, Student's T-test, N=3 independent experiments, data shown are mean ± SEM).


Figure 6Western blot of PMN pre - treatment with cycloheximide ( 50 µg / ml ) or MG - 132 ( 10 µM ) for 30 min before incubation with 1 µM PPP for 60 min . Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells . ( DMSO , grey : 100 % , N = 7 independent experiments ; PPP , blue : 137 . 1 ± 7 . 3 % , N = 7 independent experiments ; CHX , pink : 89 . 5 ± 12 . 7 % , N = 5 independent experiments ; MG - 132 , teal : 124 ± 28 . 7 , N = 3 independent experiments ; P = 0 . 009 , one - way ANOVA ; Tukey ' s post - hoc test : DMSO - PPP * P = 0 . 04 , PPP - PPP + CHX * P = 0 . 01 ; data shown are mean ± SEM ) . Puromycin - PLA signal in PMN grown in microfluidics devices . Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment . The scale bars are 10 µm . White arrows point to puromycin - PLA puncta within the β3 - tubulin mask ; the empty arrow shows a PLA puncta not associated with neurons . Quantification of puromycin - PLA signal . PPP causes an increase in PLA puncta in the axon of PMN , which is abolished by treatment with 40 µM anisomycin ( DMSO , grey : 100 % ; PPP , blue : 139 . 3 ± 7 . 7 % ; PPP + anisomycin , purple : 86 . 3 ± 10 . 0 ; P = 0 . 005 , one - way ANOVA , Tukey ' s post - hoc test ; DMSO vs PPP , * P = 0 . 02 ; PPP vs PPP + anisomycin , * * 0 . 005 , N = 3 independent experiments , 27 - 57 images , data shown are mean ± SEM ) .

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 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 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

Figure 6Western blot of PMN pre-treatment with cycloheximide (50 µg/ml) or MG-132 (10 µM) for 30 min before incubation with 1 µM PPP for 60 min. Cycloheximide causes a significant decrease in BICD1 levels compared to PPP treated cells. (DMSO, grey: 100%, N=7 independent experiments; PPP, blue: 137.1 ± 7.3%, N=7 independent experiments; CHX, pink: 89.5 ± 12.7%, N=5 independent experiments; MG-132, teal: 124 ± 28.7, N=3 independent experiments; P=0.009, one-way ANOVA; Tukey's post-hoc test: DMSO-PPP * P=0.04, PPP-PPP+CHX * P=0.01; data shown are mean ± SEM).Puromycin-PLA signal in PMN grown in microfluidics devices. Images are magnifications from the white boxes in Appendix Fig S6 taken from the axonal compartment. The scale bars are 10 µm. White arrows point to puromycin-PLA puncta within the β3-tubulin mask; the empty arrow shows a PLA puncta not associated with neurons.Quantification of puromycin-PLA signal. PPP causes an increase in PLA puncta in the axon of PMN, which is abolished by treatment with 40 µM anisomycin (DMSO, grey: 100%; PPP, blue: 139.3 ± 7.7%; PPP + anisomycin, purple: 86.3 ± 10.0; P=0.005, one-way ANOVA, Tukey's post-hoc test; DMSO vs PPP, * P=0.02; PPP vs PPP + anisomycin, ** 0.005, N=3 independent experiments, 27-57 images, data shown are mean ± SEM).