Figure 4B , C , OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13 . 1 copy number alteration . Pearson correlation of OTUB1 copy number ( log2 ratio ) with OTUB1 mRNA levels ( RNAseq normalized read counts , log2 transformed ) was analyzed . D , E , F , OTUB1 expression in TCGA lung adenocarcinoma ( LUAD ) and lung squamous cell carcinoma ( SCC ) patients . Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis . p - values were determined by two - sided t - Test . Total number of patients , n . G , Gain of 11q13 . 1 locus is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test . H , OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development . TCGA lung adenocarcinoma patients were stratified by tumor stages ( T1 - T4 ) and OTUB1 expression levels as described in Materials and Methods . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test . I , KRAS mutation is a late event in lung adenocarcinoma progression . TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages ( T1 - T4 ) . Total numbers of patients , n . Statistical comparison of the sample distributions were compared using Chi - square test .

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

Figure 4B,C, OTUB1 overexpression in TCGA lung carcinomas is associated with 11q13.1 copy number alteration. Pearson correlation of OTUB1 copy number (log2 ratio) with OTUB1 mRNA levels (RNAseq normalized read counts, log2 transformed) was analyzed.D,E,F, OTUB1 expression in TCGA lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (SCC) patients. Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Box whisker plots represent OTUB1 mRNA expression levels in TCGA lung carcinoma patients determined by RNAseq analysis. p-values were determined by two-sided t-Test. Total number of patients, n.G, Gain of 11q13.1 locus is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients with diploid or gain of the OTUB1 locus were stratified according tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.H, OTUB1 mRNA overexpression is an early event in lung adenocarcinoma development. TCGA lung adenocarcinoma patients were stratified by tumor stages (T1-T4) and OTUB1 expression levels as described in Materials and Methods. Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.I, KRAS mutation is a late event in lung adenocarcinoma progression. TCGA lung adenocarcinoma patients with different KRAS mutation status were stratified by tumor stages (T1-T4). Total numbers of patients, n. Statistical comparison of the sample distributions were compared using Chi-square test.


Figure 5 , Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1 . B , OTUB1 suppression decreases serum - induced ERK1 / 2 phosphorylation . Serum - starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10 % serum for the indicated time periods . Levels of phosphorylated ERK1 / 2 and total ERK1 / 2 were analyzed by Meso Scale assay . The results are expressed as a mean of pERK1 / 2 levels relative to total ERK1 / 2 ± s . e . m ; p - values were determined by two - sided t - Test , n = 2 . C , D , OTUB1 suppression impairs the anchorage - independent growth of Western blot cancer cell lines . Representative images of soft - agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 . C , D , OTUB1 suppression impairs the anchorage - independent growth of lung cancer cell lines . Representative images of soft - agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP . The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 . E , Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice . p - value was determined by 2 - way ANOVA .

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

Figure 5, Suppression of OTUB1 expression in NSCLC cell lines by specific shRNAs against OTUB1 was confirmed by immunoblotting using antibody against OTUB1.B, OTUB1 suppression decreases serum-induced ERK1/2 phosphorylation. Serum-starved A549 cells expressing shRNAs against OTUB1 or GFP were stimulated with 10% serum for the indicated time periods. Levels of phosphorylated ERK1/2 and total ERK1/2 were analyzed by Meso Scale assay. The results are expressed as a mean of pERK1/2 levels relative to total ERK1/2 ± s.e.m; p-values were determined by two-sided t-Test, n=2.C, D, OTUB1 suppression impairs the anchorage-independent growth of Western blot cancer cell lines. Representative images of soft-agar colonies formed by Western blot cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.C, D, OTUB1 suppression impairs the anchorage-independent growth of lung cancer cell lines. Representative images of soft-agar colonies formed by lung cells expressing shRNAs against OTUB1 or GFP. The number of soft agar colonies formed by cells expressing shOTUB1 compared to cells expressing shGFP. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.E, Xenograft tumor growth of A549 cells expressing shRNAs against GFP or OTUB1 subcutaneously injected into nude mice. p-value was determined by 2-way ANOVA.


Figure 6A , OTUB1 overexpression promotes serum - induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells . B , OTUB1 overexpression does not affect serum - induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells . A , B , Serum - starved lung cells expressing Flag - OTUB1 or an empty vector ( V ) were stimulated with 10 % serum for the indicated time periods . Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1 / 2 , ERK1 / 2 , Flag and vinculin . C , Representative immunoblots of Flag - tagged OTUB1 overexpression in lung adenocarcinoma cell linesD , E , OTUB1 overexpression affects anchorage - independent growth of lung adenocarcinoma cell lines . Representative images of soft - agar colonies formed by the indicated cells expressing Flag - OTUB1 or empty vector ( V ) . The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 . D , E , OTUB1 overexpression affects anchorage - independent growth of lung adenocarcinoma cell lines . Representative images of soft - agar colonies formed by the indicated cells expressing Flag - OTUB1 or empty vector ( V ) . The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector . Data are presented as mean ± s . e . m . p - values were determined by two - sided t - Test , n = 3 . F , G , Xenograft tumor growth of A549 , H1993 or H1437 cells expressing Flag - OTUB1 or an empty vector ( V ) subcutaneously injected into nude mice . p - values were determined by 2 - way ANOVA .

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 O O 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 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 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

Figure 6A, OTUB1 overexpression promotes serum-induced MAPK activation in wt KRAS H1993 lung adenocarcinoma cells.B, OTUB1 overexpression does not affect serum-induced MAPK activation in mutant KRAS A549 lung adenocarcinoma cells. A,B, Serum-starved lung cells expressing Flag-OTUB1 or an empty vector (V) were stimulated with 10% serum for the indicated time periods. Whole cell lysates were analyzed by immunoblotting using antibodies against pERK1/2, ERK1/2, Flag and vinculin.C, Representative immunoblots of Flag-tagged OTUB1 overexpression in lung adenocarcinoma cell linesD, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.D, E, OTUB1 overexpression affects anchorage-independent growth of lung adenocarcinoma cell lines. Representative images of soft-agar colonies formed by the indicated cells expressing Flag-OTUB1 or empty vector (V). The number of soft agar colonies formed by OTUB1 expressing cells compared to cells expressing an empty vector. Data are presented as mean ± s.e.m. p-values were determined by two-sided t-Test, n=3.F,G, Xenograft tumor growth of A549, H1993 or H1437 cells expressing Flag-OTUB1 or an empty vector (V) subcutaneously injected into nude mice. p-values were determined by 2-way ANOVA.


Figure 7A , Representative examples of OTUB1 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Materials and Methods . Scale bar , 100 micron . B , OTUB1 expression in lung non - small cell carcinomas with different KRAS mutation status . OTUB1 expression was assessed in TMA samples by IHC . C , OTUB1 up - regulation is an early event in lung adenocarcinoma development . Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages ( T1 - T3 ) . Total number of patients , n . D , Representative examples of phosphorylated - ERK1 / 2 immunostaining in lung adenocarcinomas . Samples were scored as negative / low , medium or high staining as described in Material and Methods . Scale bar , 100 micron . E , OTUB1 levels correlates with increased levels of phosphorylated - ERK1 / 2 in wt KRAS lung adenocarcinomas . Association between OTUB1 and pERK1 / 2 expression levels was assessed in the same tumor samples by IHC . F , Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1 . Ki67 expression was assessed by IHC . The percentage of Ki67 - positive cells was counted , and the samples were scored as negative / low , < 10 % ; medium , between 10 and 40 % ; high , > 40 % . See Figure EV4 for representative images of Ki67 staining . G , Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival . H , OTUB1 expression levels are not associated with survival rate of KRAS - mutant lung adenocarcinoma TCGA patients . G , H , Patients were stratified according to their OTUB1 mRNA levels and / or their KRAS status as described in Materials and Methods . Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan - Meier curves . p - values were determined by Log Rank Test .

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

Figure 7A, Representative examples of OTUB1 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Materials and Methods. Scale bar, 100 micron.B, OTUB1 expression in lung non-small cell carcinomas with different KRAS mutation status. OTUB1 expression was assessed in TMA samples by IHC.C, OTUB1 up-regulation is an early event in lung adenocarcinoma development. Lung adenocarcinoma patients expressing different levels of OTUB1 were stratified by tumor stages (T1-T3). Total number of patients, n.D, Representative examples of phosphorylated-ERK1/2 immunostaining in lung adenocarcinomas. Samples were scored as negative/low, medium or high staining as described in Material and Methods. Scale bar, 100 micron.E, OTUB1 levels correlates with increased levels of phosphorylated-ERK1/2 in wt KRAS lung adenocarcinomas. Association between OTUB1 and pERK1/2 expression levels was assessed in the same tumor samples by IHC.F, Ki67 expression in lung adenocarcinomas expressing different levels of OTUB1. Ki67 expression was assessed by IHC. The percentage of Ki67-positive cells was counted, and the samples were scored as negative/low, <10%; medium, between 10 and 40%; high, > 40%. See Figure EV4 for representative images of Ki67 staining.G, Wt KRAS lung adenocarcinoma TCGA patients with medium OTUB1 expression have poorer overall survival. H, OTUB1 expression levels are not associated with survival rate of KRAS-mutant lung adenocarcinoma TCGA patients. G,H, Patients were stratified according to their OTUB1 mRNA levels and/or their KRAS status as described in Materials and Methods. Overall survival of lung adenocarcinoma patients expressing different levels of OTUB1 mRNA as measured by Kaplan-Meier curves. p-values were determined by Log Rank Test.


Figure 2 ( A ) Identified acetylated histone peptides plotted against their log2 SILAC ratio ( 1h after 16 J / m2 UV / mock ) , ranked by SILAC ratio ( C ) Western blot of acid extracted histones and WCE from HeLa cells , treated with HATi ( CTK7A , 100 μM and CPTH2 , 50 μM ) or HDACi ( TSA , 1 μM ) for 4h or mock treated as indicated ( D ) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV - irradiation ( 16 J / m2 ) . Blots were stained with α - acetyl - lysine ( top panels , high and low exposure ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel ) ( F ) Representative western blots of HeLa WCE from different time points after UV - C irradiation ( 16J / m2 ) using the indicated histone modification specific antibodies . Histone H4 and tubulin were used as loading

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 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

Figure 2(A) Identified acetylated histone peptides plotted against their log2 SILAC ratio (1h after 16 J/m2 UV/mock), ranked by SILAC ratio(C) Western blot of acid extracted histones and WCE from HeLa cells, treated with HATi (CTK7A, 100 μM and CPTH2, 50 μM) or HDACi (TSA, 1 μM) for 4h or mock treated as indicated(D) A representative western blot of histone acetylation levels of HeLa cells lysed at indicated time points after UV-irradiation (16 J/m2). Blots were stained with α-acetyl-lysine (top panels, high and low exposure), α-histone H4 (middle panel) and α-tubulin (bottom panel)(F) Representative western blots of HeLa WCE from different time points after UV-C irradiation (16J/m2) using the indicated histone modification specific antibodies. Histone H4 and tubulin were used as loading controls


Figure 4 ( A ) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation ( 16 J / m2 , left panel ) , α - amanitin ( second panel , 100 µg / ml ) , THZ1 ( third panel , 1 μM ) or flavopiridol ( right panel , 1 μM ) treatment . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel ) ( C ) Representative western blots of histone acetylation levels of HeLa cells pre - treated with transcription inhibitors ( mock ( left ) , THZ1 ( middle , 1 μM ) or flavopiridol ( right , 1 μM ) an hour before UV irradiation and lysed at the indicated time points after UV ( 16J / m2 ) . Blots were stained with the indicated

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 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 4(A) Representative western blots of histone acetylation levels of HeLa cells lysed at indicated time points after UV irradiation (16 J/m2, left panel), α-amanitin (second panel, 100 µg/ml), THZ1 (third panel, 1 μM) or flavopiridol (right panel, 1 μM) treatment. Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)(C) Representative western blots of histone acetylation levels of HeLa cells pre-treated with transcription inhibitors (mock (left), THZ1 (middle, 1 μM) or flavopiridol (right, 1 μM) an hour before UV irradiation and lysed at the indicated time points after UV (16J/m2). Blots were stained with the indicated antibodies


Figure 5 ( A ) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation ( 16 J / m2 ) , HU / AraC treatment ( 100 mM / 10 μM ) or MMC treatment ( 10 µg / ml ) . Blots were stained with α - acetyl - lysine ( top panel ) , α - histone H4 ( middle panel ) and α - tubulin ( bottom panel ) ( C ) Representative Western blots , stained with the indicated antibodies of HeLa cells pre - treated o / n with an S - phase inhibitor ( PHA 767491 hydrochloride , 10 μM ) and lysed at indicated time points after UV ( 16 J / m2 )

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 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

Figure 5(A) Representative western blots of histone acetylation levels of HeLa cells obtained at the indicated time points after UV irradiation (16 J/m2), HU/AraC treatment (100 mM/10 μM) or MMC treatment (10 µg/ml). Blots were stained with α-acetyl-lysine (top panel), α-histone H4 (middle panel) and α-tubulin (bottom panel)(C) Representative Western blots, stained with the indicated antibodies of HeLa cells pre-treated o/n with an S-phase inhibitor (PHA 767491 hydrochloride, 10 μM) and lysed at indicated time points after UV (16 J/m2)


Figure 1C Top : Schematic illustration of the miR - 17 - 92 cluster with colours marking miRNAs belonging to the same miRNA family . Bottom : TaqMan analysis of the mature miR - 17 - 92 miRNAs in Srsf3 knockout ( KO ) and control ( Ctrl ) iPSCs ( * P < 0 . 05 , * * * P < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological 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

Figure 1C Top: Schematic illustration of the miR-17-92 cluster with colours marking miRNAs belonging to the same miRNA family. Bottom: TaqMan analysis of the mature miR-17-92 miRNAs in Srsf3 knockout (KO) and control (Ctrl) iPSCs (*P<0.05, ***P<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).


Figure 2B TaqMan analysis of the six miR - 17 - 92 miRNAs in HEK293T cells overexpressing the WT - miR - 17 - 92 , 17 / 20a - ∆ CNNC or total - ∆ CNNC construct ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One - way ANOVA , mean as ± SEM , n = 4 , biological replicates ) . C Western blot analysis of HA - tagged SRSF3 - FL or SRSF3 - ∆ RS expression in LIM1215 cells ( detection with a HA - antibody ) . D TaqMan analysis of the six miR - 17 - 92 miRNAs in LIM1215 cells overexpressing empty pCGN vector , SRSF3 - FL or SRSF3 - ∆ RS constructs ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , One - way ANOVA , data as mean ± SEM , n = 3 , biological 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2B TaqMan analysis of the six miR-17-92 miRNAs in HEK293T cells overexpressing the WT-miR-17-92, 17/20a-∆CNNC or total-∆CNNC construct (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, mean as ± SEM, n=4, biological replicates).C Western blot analysis of HA-tagged SRSF3-FL or SRSF3-∆RS expression in LIM1215 cells (detection with a HA-antibody).D TaqMan analysis of the six miR-17-92 miRNAs in LIM1215 cells overexpressing empty pCGN vector, SRSF3-FL or SRSF3-∆RS constructs (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).


Figure 3B Visualization and comparison of WT miR - 17 - 92 SHAPE reactivities in control and SRSF3 - KD cells . First panel : Mean control ( blue ) and SRSF3 - KD ( red ) SHAPE reactivities , calculated over 11nt sliding windows . Second panel : ∆ SHAPE reactivity differences between control and SRSF3 - KD . Regions with higher reactivity in control are marked with blue shading and regions with higher reactivity in SRSF3 - KD with red shading . Third panel : The SRSF3 - KD to control reactivity ratios . The black boxes mark the CNNC sites . G Visualization and comparison of total - ∆ CNNC miR - 17 - 92 SHAPE reactivities in control and SRSF3 - KD cells . First panel : Mean control ( grey ) and SRSF3 - KD ( orange ) SHAPE reactivities , calculated over 11nt sliding windows . Second panel : ∆ SHAPE reactivity differences between control and SRSF3 - KD . Regions with higher reactivity in control are marked with grey shading and regions with higher reactivity in SRSF3 - KD with orange shading . Third panel : The SRSF3 - KD to control reactivity ratios . The red boxes mark the mutated CNNC sites .

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 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

Figure 3B Visualization and comparison of WT miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (blue) and SRSF3-KD (red) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with blue shading and regions with higher reactivity in SRSF3-KD with red shading. Third panel: The SRSF3-KD to control reactivity ratios. The black boxes mark the CNNC sites.G Visualization and comparison of total-∆CNNC miR-17-92 SHAPE reactivities in control and SRSF3-KD cells. First panel: Mean control (grey) and SRSF3-KD (orange) SHAPE reactivities, calculated over 11nt sliding windows. Second panel: ∆SHAPE reactivity differences between control and SRSF3-KD. Regions with higher reactivity in control are marked with grey shading and regions with higher reactivity in SRSF3-KD with orange shading. Third panel: The SRSF3-KD to control reactivity ratios. The red boxes mark the mutated CNNC sites.


Figure 4B RT - qPCR quantification of Srsf3 and Cdkn1a ( p21 ) mRNA levels in Srsf3 - knockout ( KO ) and control ( Ctrl ) iPSCs ( * p < 0 . 05 , * * * < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological replicates ) . C Quantification of Cdkn1a mRNA expression by RT - qPCR during reprogramming in Srsf3 - KO and control cells ( * * * p < 0 . 001 , * * * * p < 0 . 0001 , Two - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) . D RT - qPCR quantification of CDKN1A ( p21 ) levels in HEK293T cells overexpressing empty vector , WT - miR - 17 - 92 , 17 / 20a - ∆ CNNC and total - CNNC ∆ expression constructs ( * p < 0 . 05 , * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) . E RT - qPCR quantification of CDKN1A ( p21 ) levels in LIM1215 cells over expressing empty vector , SRSF3 - FL and SRSF3 - ∆ RS constructs ( * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological replicates ) . F RT - qPCR quantification of CDKN1A ( p21 ) levels in LIM1215 cells overexpressing scrambled control , miR - 17 , miR - 20a or miR - 17 + miR - 20a mimics ( * * p < 0 . 01 , One - way ANOVA , data as mean ± SEM , n = 3 , biological 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 4B RT-qPCR quantification of Srsf3 and Cdkn1a (p21) mRNA levels in Srsf3-knockout (KO) and control (Ctrl) iPSCs (*p<0.05, ***<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).C Quantification of Cdkn1a mRNA expression by RT-qPCR during reprogramming in Srsf3-KO and control cells (***p<0.001, ****p<0.0001, Two-way ANOVA, data as mean ± SEM, n=3, biological replicates).D RT-qPCR quantification of CDKN1A (p21) levels in HEK293T cells overexpressing empty vector, WT-miR-17-92, 17/20a-∆CNNC and total-CNNC∆ expression constructs (*p<0.05, **p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).E RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells over expressing empty vector, SRSF3-FL and SRSF3-∆RS constructs (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates). F RT-qPCR quantification of CDKN1A (p21) levels in LIM1215 cells overexpressing scrambled control, miR-17, miR-20a or miR-17+miR-20a mimics (**p<0.01, One-way ANOVA, data as mean ± SEM, n=3, biological replicates).


Figure 5A Western blot analysis of SRSF3 and CDKN1A expression in SRSF3 - T2A - GFP overexpressing ( SRSF3 ) and control ( GFP ) LIM1215 cells . GAPDH served as a loading control . B TaqMan analysis of miR - 17 - 92 miRNAs in SRSF3 - T2A - GFP overexpressing ( SRSF3 - GFP ) and GFP - only control ( GFP ) LIM1215 cells ( * p < 0 . 05 , * * * * p < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological replicates ) . C RT - qPCR quantification of CDKN1A ( p21 ) mRNA levels in SRSF3 - T2A - GFP overexpressing ( SRSF3 - GFP ) and control ( GFP ) LIM1215 cells ( * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 3 , biological replicates ) . D Left : Schematic of the Firefly luciferase reporter constructs Right : Luciferase activity in SRSF3 overexpressing ( SRSF3 - GFP ) and control ( GFP ) LIM1215 cells transfected with the reporter constructs ( * * p < 0 . 01 , * * * p < 0 . 001 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological replicates ) . F Quantification of fraction of cells in G1 , S and G2 phases in SRSF3 overexpressing cells ( SRSF3 - GFP ) relative to control ( GFP ) LIM1215 cells ( * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 4 , biological 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A Western blot analysis of SRSF3 and CDKN1A expression in SRSF3-T2A-GFP overexpressing (SRSF3) and control (GFP) LIM1215 cells. GAPDH served as a loading control.B TaqMan analysis of miR-17-92 miRNAs in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and GFP-only control (GFP) LIM1215 cells (*p<0.05, ****p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).C RT-qPCR quantification of CDKN1A (p21) mRNA levels in SRSF3-T2A-GFP overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells (**p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=3, biological replicates).D Left: Schematic of the Firefly luciferase reporter constructs Right: Luciferase activity in SRSF3 overexpressing (SRSF3-GFP) and control (GFP) LIM1215 cells transfected with the reporter constructs (**p<0.01, ***p<0.001, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).F Quantification of fraction of cells in G1, S and G2 phases in SRSF3 overexpressing cells (SRSF3-GFP) relative to control (GFP) LIM1215 cells (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=4, biological replicates).


Figure 6A , B RT - qPCR quantification of SRSF3 ( A ) and CDKN1A ( B ) mRNA levels in colorectal tumours and their paired normal samples ( * p < 0 . 05 , * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , data as mean ± SEM , n = 25 , biological replicates ) . N = normal , T = tumour . D Association between SRSF3 expression and tumour ( T ) stage . T1 ( n = 3 ) , T2 ( n = 5 ) , T3 ( n = 11 ) and T4 ( n = 3 ) tumours , SRSF3 fold change as the dependent variable . Linear regression analysis ( central band = median , boxes = 25th and 75th percentile and whiskers = min and max , n = biological replicates ) . E Association between SRSF3 expression and tumour differentiation status . Moderately ( Mod . ) differentiated tumours ( n = 19 ) and poorly differentiated tumours ( n = 5 ) , SRSF3 fold change as the dependent variable . Linear regression analysis ( central band = median , boxes = 25th and 75th percentile and whiskers = min and max ; n = biological replicates ) . G SRSF3 and CDKN1A expression in TCGA COAD data ( version 2016 _ 01 _ 28 ; tumour n = 459 and normal n = 41 ( biological replicates ) ; RSEM , RNA - seq by Expectation Maximation ; * * p < 0 . 01 , two - tailed unpaired Student ' s t - test , central band = median , dotted lines = 25th and 75th percentile ) .

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

Figure 6A, B RT-qPCR quantification of SRSF3 (A) and CDKN1A (B) mRNA levels in colorectal tumours and their paired normal samples (*p<0.05, **p<0.01, two-tailed unpaired Student's t-test, data as mean ± SEM, n=25, biological replicates). N=normal, T=tumour.D Association between SRSF3 expression and tumour (T) stage. T1 (n=3), T2 (n=5), T3 (n=11) and T4 (n=3) tumours, SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max, n=biological replicates). E Association between SRSF3 expression and tumour differentiation status. Moderately (Mod.) differentiated tumours (n=19) and poorly differentiated tumours (n=5), SRSF3 fold change as the dependent variable. Linear regression analysis (central band=median, boxes =25th and 75th percentile and whiskers=min and max; n=biological replicates).G SRSF3 and CDKN1A expression in TCGA COAD data (version 2016_01_28; tumour n=459 and normal n=41 (biological replicates); RSEM, RNA-seq by Expectation Maximation; **p<0.01, two-tailed unpaired Student's t-test, central band=median, dotted lines =25th and 75th percentile).


Figure 1A . Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 ( Patient 2 ) and 19 ( Patient 3 ) years of age . B . Mid - sagittal ( I ) , coronal ( II ) and transversal ( III ) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants . Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles . Arrows indicate thin corpus callosum , red arrowheads point to enlarged extra - axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss . C . EEG of Patient 3 . Left panel : 1 - 2 Hz left fronto - temporal spike and multi - spike slow wave discharges ( Fp1 - F7 and T3 - T5 ) spread to the homologous same hemisphere and opposite hemisphere , predominantly seen on bipolar montage EEG . Right panel : Spike - and multi - spike slow wave activities together with fast rhythms originating from bilateral fronto - temporal - frontocentral regions indicate electrographic seizure activity . Described changes are marked by circles .

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

Figure 1A. Anterior and lateral photographs of two patients with ELP6 variants showing dysmorphic features at 20 (Patient 2) and 19 (Patient 3) years of age.B. Mid-sagittal (I), coronal (II) and transversal (III) MRI scans of Patient 1 with newly identified ELP4 variants and Patient 2 and Patient 3 with ELP6 variants. Atrophy of the cerebellum with prominence of the cerebellar folia is marked by circles. Arrows indicate thin corpus callosum, red arrowheads point to enlarged extra-axial CSF spaces and black arrowheads to cerebral sulci in keeping with cerebral and cerebellar volume loss.C. EEG of Patient 3. Left panel: 1-2 Hz left fronto-temporal spike and multi-spike slow wave discharges (Fp1-F7 and T3-T5) spread to the homologous same hemisphere and opposite hemisphere, predominantly seen on bipolar montage EEG. Right panel: Spike-and multi-spike slow wave activities together with fast rhythms originating from bilateral fronto-temporal-frontocentral regions indicate electrographic seizure activity. Described changes are marked by circles.


Figure 3C . Pull - down experiments of the assembled mElp123456 complexes carrying patients - derived mutations . The mElp123WT subcomplexes were immobilized on the Dynabeads ( strep ) via Elp3 protein and incubated with the Elp456 variants . The inputs and pull downs were loaded on the SDS - PAGE gels and visualized by Coomassie staining . The molecular mass marker is indicated on the left ( M ) . D . Co - immunoprecipitation analyses of Elongator complexes in patient - derived fibroblast cell . Whole cell lysates ( left ) and eluates after immune - precipitation ( right ) are shown for control fibroblasts ( ctrl ) and cultured patient fibroblasts ( Elp4Y91C / L296I ( patient 1 ) ; Elp6L118W / L118W ( patient 2 ) , Elp6L118W / L118W ( patient 3 ) . Used antibodies for immobilization ( ELP1 ) and detection ( ELP1 , ELP3 , ELP4 , ELP5 and α - tubulin ) are indicated . n = 4 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 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

Figure 3C. Pull-down experiments of the assembled mElp123456 complexes carrying patients-derived mutations. The mElp123WT subcomplexes were immobilized on the Dynabeads (strep) via Elp3 protein and incubated with the Elp456 variants. The inputs and pull downs were loaded on the SDS-PAGE gels and visualized by Coomassie staining. The molecular mass marker is indicated on the left (M).D. Co-immunoprecipitation analyses of Elongator complexes in patient-derived fibroblast cell. Whole cell lysates (left) and eluates after immune-precipitation (right) are shown for control fibroblasts (ctrl) and cultured patient fibroblasts (Elp4Y91C/L296I (patient 1); Elp6L118W/L118W (patient 2), Elp6L118W/L118W (patient 3). Used antibodies for immobilization (ELP1) and detection (ELP1, ELP3, ELP4 , ELP5 and α-tubulin) are indicated. n = 4 independent experiments.


Figure 4A . High - performance liquid chromatography ( HPLC ) coupled to mass spectrometry ( MS ) used to quantify the Elongator - dependent tRNA modification 5 - carbamoylmethyluridine ( ncm5U ) in human fibroblasts . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . B . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxy - carbonylmethyluridine ( mcm5U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . C . HPLC - MS quantification of Elongator - dependent tRNA modification 5 - methoxycarbonylmethyl - 2 - thiouridine ( mcm5s2U ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM . D . HPLC - MS quantification of Elongator - independent tRNA modification 1 - methyladenosine ( m1A ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . E . HPLC - MS quantification of Elongator - independent tRNA modification 7 - methylguanosine ( m7G ) . Pseudouridine ( Ψ ) was used as an internal normalization standard . n = 3 technical repeats per genotype . Data information : Statistical analysis for data one - way ANOVA ( α = 0 . 05 ) with a Dunnett ' s post - hoc test . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; ns - not significant ) . Data represent mean ± SEM . F . Differential expression ( DE ) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control ( n = 3 technical repeats per genotype ) . Down - ( red ) and upregulated ( blue ) peptides are depicted on volcano plots .

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 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 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

Figure 4A. High-performance liquid chromatography (HPLC) coupled to mass spectrometry (MS) used to quantify the Elongator-dependent tRNA modification 5-carbamoylmethyluridine (ncm5U) in human fibroblasts. Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. B. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxy-carbonylmethyluridine (mcm5U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. C. HPLC-MS quantification of Elongator-dependent tRNA modification 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.D. HPLC-MS quantification of Elongator-independent tRNA modification 1-methyladenosine (m1A). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. E. HPLC-MS quantification of Elongator-independent tRNA modification 7-methylguanosine (m7G). Pseudouridine (Ψ) was used as an internal normalization standard. n = 3 technical repeats per genotype. Data information: Statistical analysis for data one-way ANOVA (α = 0.05) with a Dunnett's post-hoc test. Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ns - not significant). Data represent mean ± SEM.F. Differential expression (DE) of peptides in fibroblasts obtained from the individuals with ELP4 and ELP6 variants relative to control (n = 3 technical repeats per genotype). Down- (red) and upregulated (blue) peptides are depicted on volcano plots.


Figure 6A . Representative images of Golgi - Cox - stained brain sections in the hippocampal area of adult ( 2 - months - old ) wild - type and mutant mice . Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses . Scale bar : 100 μm . Abbreviations : DG - dentate gyrus , CA1 - cornu ammonis 1 , CA2 - cornu ammonis 2C . Quantification of total dendritic length from dendritic tree reconstructions shown in B . n = 15 ; 5 neurons per animal and 3 animals per genotype . D . Sholl analysis of the complexity of basal dendritic arbors . n = 15 ; 5 neurons per animal and 3 animals per genotype . Data information : Statistical analysis : unpaired two - tailed t - test ( α = 0 . 05 ) with Welch ' s correction . Holm - Sidak correction was applied to adjust for multiple comparisons ( D ) . Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ± SEM ( with error bars in D removed when smaller than the corresponding data point ) . E . Representative images of dendritic spines on basal dendrites of the cortical neurons . Quantification of spine density per 10 μm on secondary dendrites is shown . n = 15 ; 5 neurons per animal and 3 animals per genotype . Scale bar : 10 μm . Data information : Statistically significant differences are indicated ( * p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; ns - not significant ) . Data represent mean ±

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

Figure 6A. Representative images of Golgi-Cox-stained brain sections in the hippocampal area of adult (2-months-old) wild-type and mutant mice. Rectangles represent CA1 region containing pyramidal neurons selected for neuron reconstruction and subsequent analyses. Scale bar: 100 μm. Abbreviations: DG - dentate gyrus, CA1 - cornu ammonis 1, CA2 - cornu ammonis 2C. Quantification of total dendritic length from dendritic tree reconstructions shown in B. n = 15; 5 neurons per animal and 3 animals per genotype. D. Sholl analysis of the complexity of basal dendritic arbors. n = 15; 5 neurons per animal and 3 animals per genotype. Data information: Statistical analysis: unpaired two-tailed t-test (α = 0.05) with Welch's correction. Holm-Sidak correction was applied to adjust for multiple comparisons (D). Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM (with error bars in D removed when smaller than the corresponding data point).E. Representative images of dendritic spines on basal dendrites of the cortical neurons. Quantification of spine density per 10 μm on secondary dendrites is shown. n = 15; 5 neurons per animal and 3 animals per genotype. Scale bar: 10 μm. Data information: Statistically significant differences are indicated (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ns - not significant). Data represent mean ± SEM


Figure 1Hydrolysis of fluorogenic substrates ( Suc - LLVY - AMC ) for the chymotrypsin - like activity of human ( B ) or mouse ( C ) 20S constitutive proteasome ( CP ) or 20S immunoproteasome ( IP ) at various PRN1126 concentrations . Data are presented as the means ± s . d . from quadruplicate assays . The highest fluorescence value was set to 100 % . The experiments were repeated twice with similar results .

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

Figure 1Hydrolysis of fluorogenic substrates (Suc-LLVY-AMC) for the chymotrypsin-like activity of human (B) or mouse (C) 20S constitutive proteasome (CP) or 20S immunoproteasome (IP) at various PRN1126 concentrations. Data are presented as the means ± s.d. from quadruplicate assays. The highest fluorescence value was set to 100%. The experiments were repeated twice with similar results.


Figure 2Presentation of UTY246 - 254 on splenocytes after exposure to indicated concentrations of PRN1126 . Data are presented as the mean absorbance ± s . d . of three replicate cultures . The experiment has been performed twice , yielding similar results . Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 mice treated with the indicated concentrations of PRN1126 overnight . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM . Splenocytes from C57BL / 6 mice ( C ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . ( C ) IL - 6 concentrations are presented as mean and individual data points from triplicate wells . The experiment has been performed twice , yielding similar results . Splenocytes from human PBMCs ( D ) were exposed ( continuous treatment ) to 300 nM PRN1126 or vehicle ( DMSO ) or 300 nM ONX 0914 and stimulated with LPS overnight . IL - 6 concentrations in the supernatant were analyzed by ELISA . Data are presented as single dots from three independent donors . The highest cytokine concentration was set to 100 % . * P < 0 . 05 . Unpaired student ´ s t - test . Colitis was induced by oral administration of 3 % DSS for 5 d . Mice were treated daily ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . Data points represent mean ± SEM of 15 mice pooled from three independent experiments . Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated three times a week ( s . c . ) with either PRN1126 ( 40 mg / kg ) , or ONX 0914 ( 10 mg / kg ) , or vehicle . All data were statistically compared to the vehicle treated group . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Two - way ANOVA . Shown are the means of the clinical scores ± SEM ( n = 6 per group ) .

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

Figure 2Presentation of UTY246-254 on splenocytes after exposure to indicated concentrations of PRN1126. Data are presented as the mean absorbance ± s.d. of three replicate cultures. The experiment has been performed twice, yielding similar results.Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 mice treated with the indicated concentrations of PRN1126 overnight. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM.Splenocytes from C57BL/6 mice (C) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. (C) IL-6 concentrations are presented as mean and individual data points from triplicate wells. The experiment has been performed twice, yielding similar results.Splenocytes from human PBMCs (D) were exposed (continuous treatment) to 300 nM PRN1126 or vehicle (DMSO) or 300 nM ONX 0914 and stimulated with LPS overnight. IL-6 concentrations in the supernatant were analyzed by ELISA. Data are presented as single dots from three independent donors. The highest cytokine concentration was set to 100%. *P < 0.05. Unpaired student´s t-test.Colitis was induced by oral administration of 3 % DSS for 5 d. Mice were treated daily (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. Data points represent mean ± SEM of 15 mice pooled from three independent experiments.Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated three times a week (s.c.) with either PRN1126 (40 mg/kg), or ONX 0914 (10 mg/kg), or vehicle. All data were statistically compared to the vehicle treated group. *P < 0.05, **P < 0.01, ***P < 0.001. Two-way ANOVA. Shown are the means of the clinical scores ± SEM (n = 6 per group).


Figure 3Hydrolysis of fluorogenic substrates ( Ac - PAL - AMC ) for LMP2 - activity of human ( upper panel ) or mouse ( lower panel ) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914 . Data are presented as the means of fluorescence ± s . d . from quadruplicate assays . The experiments were repeated three times with similar results . Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914 . Ficoll enriched lymphocytes from C57BL / 6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro . SDS - PAGE and immunoblotting against indicated proteins was performed . Shown are representative western blots out of three independent experiments with similar outcome .

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

Figure 3Hydrolysis of fluorogenic substrates (Ac-PAL-AMC) for LMP2-activity of human (upper panel) or mouse (lower panel) 20S immunoproteasome at various concentrations of PRN1126 or ONX 0914. Data are presented as the means of fluorescence ± s.d. from quadruplicate assays. The experiments were repeated three times with similar results.Altered electrophoretic mobility of IP subunits by covalent modification with ONX 0914. Ficoll enriched lymphocytes from C57BL/6 mice were treated with DMSO or 300 nM ONX 0914 for 2 h in vitro. SDS-PAGE and immunoblotting against indicated proteins was performed. Shown are representative western blots out of three independent experiments with similar outcome.


Figure 4Flow cytometry analysis of H - 2Kb surface expression on splenocytes derived from C57BL / 6 wild type ( left side ) or LMP7 - / - ( right side ) mice treated with vehicle ( DMSO ) , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) overnight . Unstained splenocytes were used as negative control . Pooled data from three independent experiments ( n = 9 ) are shown as the means of median fluorescent intensity ± SEM . All data were statistically compared to the DMSO treated group . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Flow cytometry analysis of H-2Kb surface expression on splenocytes derived from C57BL/6 wild type (left side) or LMP7-/- (right side) mice treated with vehicle (DMSO), ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) overnight. Unstained splenocytes were used as negative control. Pooled data from three independent experiments (n=9) are shown as the means of median fluorescent intensity ± SEM. All data were statistically compared to the DMSO treated group. **P < 0.01, ***P < 0.001. One-way ANOVA.


Figure 5Splenocytes from C57BL / 6 mice ( A ) or human PBMCs ( B ) were incubated ( continuous treatment ) with DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and stimulated with LPS overnight . The IL - 6 concentration in the supernatant was analyzed by ELISA . The highest IL - 6 concentration from each experiment was set to 100 % . ( A ) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates . ( B ) Data are presented as the mean ± SEM of 6 different healthy donors . All data were statistically compared to the DMSO treated group . The differentiation of CD4 + T cells isolated from the spleens of C57BL / 6 mice that were exposed ( continuous treatment ) to DMSO , ONX 0914 ( 300 nM ) , PRN1126 ( 300 nM ) , ML604440 ( 300 nM ) , LU - 001i ( 300 nM ) , PRN1126 + ML604440 ( 300 nM each ) , or PRN1126 + LU - 001i ( 300 nM each ) and that were stimulated with plate bound antibodies to CD3 / CD28 in the presence of TGF - β and IL - 6 and scavenging antibodies neutralising IL - 4 and IFN - γ was measured in 3 days cultures . IL - 17 expression was detected by intracellular cytokine staining after a short restimulation with PMA / Ionomycin . IL - 17 expression in activated T cells cultured in the absence of Th17 - polarizing conditions is shown as a comparison ( no cocktail ) . Values reflect the percentage of CD4 + cells that are IL - 17A + . Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates . All data were statistically compared to the DMSO treated group .

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 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

Figure 5Splenocytes from C57BL/6 mice (A) or human PBMCs (B) were incubated (continuous treatment) with DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+LU-001i (300 nM each) and stimulated with LPS overnight. The IL-6 concentration in the supernatant was analyzed by ELISA. The highest IL-6 concentration from each experiment was set to 100%. (A) Data are presented as the mean ± SEM of 4 independent experiments each measured in triplicates. (B) Data are presented as the mean ± SEM of 6 different healthy donors. All data were statistically compared to the DMSO treated group.The differentiation of CD4+ T cells isolated from the spleens of C57BL/6 mice that were exposed (continuous treatment) to DMSO, ONX 0914 (300 nM), PRN1126 (300 nM), ML604440 (300 nM), LU-001i (300 nM), PRN1126+ML604440 (300 nM each), or PRN1126+ LU-001i (300 nM each) and that were stimulated with plate bound antibodies to CD3/CD28 in the presence of TGF-β and IL-6 and scavenging antibodies neutralising IL-4 and IFN-γ was measured in 3 days cultures. IL-17 expression was detected by intracellular cytokine staining after a short restimulation with PMA/Ionomycin. IL-17 expression in activated T cells cultured in the absence of Th17-polarizing conditions is shown as a comparison (no cocktail). Values reflect the percentage of CD4+ cells that are IL-17A+. Data are presented as the means ± SEM of 3 independent experiments each measured in triplicates. All data were statistically compared to the DMSO treated group.


Figure 6Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . ( A ) The body weight of individual mice was monitored daily and the percent weight loss ( y - axis ) was plotted versus time ( x - axis ) . All data were statistically compared to the vehicle treated group . * Colitis was induced by oral administration of 3 % DSS . Mice were treated daily ( s . c . ) with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the begin of the experiment . Data points represent means ± SEM of 15 mice pooled from three independent experiments . On day 9 after initiation of DSS treatment , colon lengths were measured ( n = 15 ) . Naïve mice ( n = 5 ) were used as healthy controls . All data were statistically compared to the vehicle treated group . Mice were immunized with MOG35 - 55 peptide and were monitored daily for clinical symptoms of EAE . Mice were treated intermittently with three times a week ( s . c . ) schedule with LU - 001i ( 15 mg / kg ) , PRN1126 ( 40 mg / kg ) , PRN1126 + LU - 001i ( 40 mg / kg + 15 mg / kg ) , or vehicle starting from the start of the experiment . Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments . All data were statistically compared to the vehicle treated group .

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 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 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

Figure 6Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. (A) The body weight of individual mice was monitored daily and the percent weight loss (y-axis) was plotted versus time (x-axis). All data were statistically compared to the vehicle treated group. *Colitis was induced by oral administration of 3% DSS. Mice were treated daily (s.c.) with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the begin of the experiment. Data points represent means ± SEM of 15 mice pooled from three independent experiments. On day 9 after initiation of DSS treatment, colon lengths were measured (n=15). Naïve mice (n=5) were used as healthy controls. All data were statistically compared to the vehicle treated group.Mice were immunized with MOG35-55 peptide and were monitored daily for clinical symptoms of EAE. Mice were treated intermittently with three times a week (s.c.) schedule with LU-001i (15 mg/kg), PRN1126 (40 mg/kg), PRN1126+ LU-001i (40 mg/kg+15 mg/kg), or vehicle starting from the start of the experiment. Data points represent the means of the clinical scores ± SEM of 12 mice pooled from two independent experiments. All data were statistically compared to the vehicle treated group.


Figure 1C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice were fasted overnight and then re - fed for 6 ( A - C ) Levels of the indicated proteins detected by IB in ( A ) liver and intestinal extracts from C57BL / 6 mice and in intestinal extracts from ( B ) C57BL / 6 or SHP - KO mice or ( C ) FGF15 - KO mice or littermates . Band quantitation was done using Image J , and the LC3 II / I ratio and p62 / actin and lamp1 / actin ratios are shown at the right . ( A - C ) The mean and standard deviation ( SD ) are plotted . Statistical significance was determined by ( A ) two - tailed Student ' s t - tests or ( B , C ) two - way ANOVA with the Tukey post - test . P - values are indicated ( n = 3 mice ) .

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 1C57BL/6, SHP-KO, and FGF15-KO or littermate mice were fasted overnight and then re-fed for 6 (A-C) Levels of the indicated proteins detected by IB in (A) liver and intestinal extracts from C57BL/6 mice and in intestinal extracts from (B) C57BL/6 or SHP-KO mice or (C) FGF15-KO mice or littermates. Band quantitation was done using Image J, and the LC3 II/I ratio and p62/actin and lamp1/actin ratios are shown at the right. (A-C) The mean and standard deviation (SD) are plotted. Statistical significance was determined by (A) two-tailed Student's t-tests or (B, C) two-way ANOVA with the Tukey post-test. P-values are indicated (n=3 mice).


Figure 4C57BL / 6 , SHP - KO , and FGF15 - KO or littermate mice as indicated were fasted for 12 h and then , re - fed for 6 h . levels of the indicated proteins in intestinal extracts from ( B ) C57BL / 6 and SHP - KO mice or ( D ) FGF15 - KO mice and littermates with quantitation of the bands at right . , ( B , D ) n = 3 mice . The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are indicated ( G ) HT29 cells were transfected with GFP - LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 , control plasmid DNA , and scrambled siRNA . The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 10 μm . G , The mean and SD are plotted . Statistical significance was determined by ( G ) one - way ANOVA with the Tukey post - test . P - values are indicated ( H ) HT29 cells were treated with 50 ng / ml FGF19 with or without 100 nM bafilomycin A1 for 12 h . LC3 and actin levels in cell extracts were determined by IB . Quantification of bands is shown at the right ( n = 3 culture plates ) . H ) The mean and SD are plotted . Statistical significance was determined by two - way ANOVA with the Tukey post - test P - values are indicated ( I ) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for the indicated times . ApoB48 and actin levels were determined by IB ( n = 2 culture plates ) .

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

Figure 4C57BL/6, SHP-KO, and FGF15-KO or littermate mice as indicated were fasted for 12 h and then, re-fed for 6 h. levels of the indicated proteins in intestinal extracts from (B) C57BL/6 and SHP-KO mice or (D) FGF15-KO mice and littermates with quantitation of the bands at right. , (B, D) n=3 mice. The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated(G) HT29 cells were transfected with GFP-LC3 and with SHP siRNA or SHP expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19, control plasmid DNA, and scrambled siRNA. The FGF19 groups were treated with control DNA or SHP expression plasmid or SHP siRNA or control scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 10 μm. G, The mean and SD are plotted. Statistical significance was determined by (G) one-way ANOVA with the Tukey post-test. P-values are indicated(H) HT29 cells were treated with 50 ng/ml FGF19 with or without 100 nM bafilomycin A1 for 12 h. LC3 and actin levels in cell extracts were determined by IB. Quantification of bands is shown at the right (n=3 culture plates). H) The mean and SD are plotted. Statistical significance was determined by two-way ANOVA with the Tukey post-test P-values are indicated(I) HT29 cells were transfected with Lamp1 siRNA or scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for the indicated times. ApoB48 and actin levels were determined by IB (n=2 culture plates).


Figure 5 ( A ) HT29 cells were transfected with GFP - LC3 and with Atg7 siRNA or control scrambled RNA ( siCtl ) and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and control siRNA . The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 ) . Scale bar , 20 μm . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated . ( B ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng / ml FGF19 for 12 h . LC3 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) . D ) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA ( siCtl ) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 12 h . ( D ) ApoB48 and actin levels in cell extracts determined by IB . Bands were quantified and the LC3 - II / LC3 - I ratio is shown at the right ( n = 3 culture plates ) . The mean and SD are plotted . Statistical significance was determined by one - way ANOVA with the Tukey post - test . P - values are indicated .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HT29 cells were transfected with GFP-LC3 and with Atg7 siRNA or control scrambled RNA (siCtl) and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and control siRNA. The FGF19 groups were treated with Atg7 siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20). Scale bar, 20 μm. The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.(B) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA and 48 h later were treated with 50 ng/ml FGF19 for 12 h. LC3 and actin levels in cell extracts determined by IB (n=3 culture plates).D) HT29 cells were transfected with Atg7 siRNA or control scrambled siRNA (siCtl) for 48 h and then treated with oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 12 h. (D) ApoB48 and actin levels in cell extracts determined by IB. Bands were quantified and the LC3-II/LC3-I ratio is shown at the right (n=3 culture plates). The mean and SD are plotted. Statistical significance was determined by one-way ANOVA with the Tukey post-test. P-values are indicated.


Figure 6Mice were re - fed for 2 h ( B ) after fasting overnight . ( B ) CoIP of TFEB with SHP . Upper panel : Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples . Lower panel : levels of TFEB and SHP in input samples detected by IB . Similar results were obtained in a second experiment . ( F , G ) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from ( F ) C57BL / 6 and SHP - KO mice or ( G ) FGF15 - KO mice or littermates re - fed for 6 h after fasting overnight ( n = 3 mice ) .

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 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

Figure 6Mice were re-fed for 2 h (B) after fasting overnight. (B) CoIP of TFEB with SHP. Upper panel: Levels of TFEB detected by IB in SHP and IgG immunoprecipitated samples. Lower panel: levels of TFEB and SHP in input samples detected by IB. Similar results were obtained in a second experiment.(F, G) Protein levels detected by IB of ULK1 and ATGL in intestinal extracts from (F) C57BL/6 and SHP-KO mice or (G) FGF15-KO mice or littermates re-fed for 6 h after fasting overnight (n=3 mice).


Figure 7 ( A ) HT29 cells were transfected with GFP - LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum - free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng / ml FGF19 for 6 h The control group ( Veh ) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA . The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated . Cells were imaged by confocal microscopy ( Zeiss , LSM700 ) . GFP - LC3 puncta were counted and the number of puncta / cell is shown at right ( n = 20 cells ) . Scale bar , 10 μm . ( A , The mean and SD are plotted . Statistical significance was determined by ( A ) one - way ANOVA with the Tukey post - test , P - values are indicated . ( B ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or control scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 ( BAF ) for 12 h . Levels of the indicated proteins determined by IB . LC3 II / I and p62 / actin ratios are shown at the right ( n = 3 - 4 culture plates ) . B , The mean and SD are plotted . Statistical significance was determined by ( B ) two - way ANOVA with the Tukey post - test , P - values are indicated . ( C ) HT29 cells were transfected with siRNA for TFEB ( siTFEB ) or control RNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . Protein levels detected by IB of ULK1 and ATGL ( n = 3 culture plates ) . ( E ) HT29 cells were transfected with siRNA for Tfeb ( siTFEB ) or scrambled siRNA ( siCtl ) , followed 48 h later by treatment with FGF19 or vehicle for 12 h . ApoB48 and actin levels in cell extracts determined by IB ( n = 3 culture plates ) .

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 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 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 O O O 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) HT29 cells were transfected with GFP-LC3 and with Tfeb siRNA or Tfeb expression plasmids and incubated in serum-free medium containing 200 μM oleic acid for 6 h followed by treatment with 50 ng/ml FGF19 for 6 h The control group (Veh) was treated with vehicle for FGF19 and transfected with control plasmid DNA and scrambled siRNA. The FGF19 groups were treated with control DNA or TFEB expression plasmid or with Tfeb siRNA or scrambled siRNA as indicated. Cells were imaged by confocal microscopy (Zeiss, LSM700). GFP-LC3 puncta were counted and the number of puncta/cell is shown at right (n = 20 cells). Scale bar, 10 μm. (A, The mean and SD are plotted. Statistical significance was determined by (A) one-way ANOVA with the Tukey post-test, P-values are indicated.(B) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or control scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle with or without 100 nM bafilomycin A1 (BAF) for 12 h. Levels of the indicated proteins determined by IB. LC3 II/I and p62/actin ratios are shown at the right (n=3-4 culture plates). B, The mean and SD are plotted. Statistical significance was determined by (B) two-way ANOVA with the Tukey post-test, P-values are indicated.(C) HT29 cells were transfected with siRNA for TFEB (siTFEB) or control RNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. Protein levels detected by IB of ULK1 and ATGL (n=3 culture plates).(E) HT29 cells were transfected with siRNA for Tfeb (siTFEB) or scrambled siRNA (siCtl), followed 48 h later by treatment with FGF19 or vehicle for 12 h. ApoB48 and actin levels in cell extracts determined by IB (n=3 culture plates).


Figure 8 ( A , C57BL / 6 ( A ) were re - fed for 6 h after fasting overnight . The cytosolic and nuclear fractions were isolated from intestine or liver , and the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin . B ) FGF15 - KO mice or littermates ( B ) were re - fed for 6 h after fasting overnight . the indicated proteins were detected by IB ( n = 2 mice ) . TBLN indicates tubulin . FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( C ) The nuclear fraction was isolated and the nuclear levels of TFEB , SHP , and LAMIN were determined by IB . FGF15 - KO mice or littermates were fasted overnight and re - fed for the indicated times . ( D ) Levels of LC3 , p62 , and actin in cell extracts were determined by IB . ( E ) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng / ml FGF19 for 30 min . The indicated proteins were detected by IB ( n = 3 culture plates ) . ( F ) FGF15 - KO mice or littermates were re - fed for 0 , 2 , or 3 h after fasting overnight . The indicated proteins in intestinal extracts were detected by IB ( n = 2 mice ) . ( G ) HT29 cells were transfected with expression plasmids for flag - tagged TFEB or its mutants as indicated and 24 h later , cells were treated with FGF19 ( 50 ng / ml ) for 12 h . ∆ CT indicates the TFEB mutant with a C - terminal deletion of 15 amino acids . The indicated proteins were detected by IB ( n = 3 culture plates ) .

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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 8(A, C57BL/6 (A) were re-fed for 6 h after fasting overnight. The cytosolic and nuclear fractions were isolated from intestine or liver, and the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.B) FGF15-KO mice or littermates (B) were re-fed for 6 h after fasting overnight. the indicated proteins were detected by IB (n=2 mice). TBLN indicates tubulin.FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (C) The nuclear fraction was isolated and the nuclear levels of TFEB, SHP, and LAMIN were determined by IB.FGF15-KO mice or littermates were fasted overnight and re-fed for the indicated times. (D) Levels of LC3, p62, and actin in cell extracts were determined by IB.(E) HT29 cells were cultured overnight in serum free media and treated with vehicle or 50 ng/ml FGF19 for 30 min. The indicated proteins were detected by IB (n=3 culture plates).(F) FGF15-KO mice or littermates were re-fed for 0, 2, or 3 h after fasting overnight. The indicated proteins in intestinal extracts were detected by IB (n=2 mice).(G) HT29 cells were transfected with expression plasmids for flag-tagged TFEB or its mutants as indicated and 24 h later, cells were treated with FGF19 (50 ng/ml) for 12 h. ∆CT indicates the TFEB mutant with a C-terminal deletion of 15 amino acids. The indicated proteins were detected by IB (n=3 culture plates).


Figure 1A - B Representative image of a proliferative cell in the DG labeled with an anti - PH3 antibody ( green ) ( A ) and quantification of the number of PH3 + cells in the DG of WT and Tau - / - mice ( B ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) . C - D Representative image of an apoptotic cell in the DG labeled with an anti - fractin antibody ( red ) ( C ) and quantification of the number of fractin + cells in the DG of WT and Tau - / - mice ( D ) ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) . E - F Schematic diagram of the cell survival experimental design using thymidine analogs ( F ) and quantification of the percentage of surviving cells in Tau - / - mice as compared to WT mice at each cell age ( 1 - , 2 - , 4 - , 6 - and 8 - week - old cells ) ( F ) ( mean ± SEM ( normalized data ) ; Student ´ s t - test ) . H Quantification of the percentage of 1 - , 4 - and 8 - week - old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau - / - mice ( χ2 test ) . I - J Representative images of progenitor cells in the DG labeled with anti - Sox2 ( red ) and anti - BLBP ( green ) antibodies ( I ) , and neuroblasts and immature neurons labeled with anti - DCX ( red ) and anti - calretinin ( green ) antibodies , respectively ( J ) . K - N Quantification of the number of Sox2 + ( K ) , BLBP + ( L ) , DCX + ( M ) and calretinin + ( N ) cells in the DG of WT and Tau - / - mice ( mean ± SEM ; n = 10 mice WT , n = 8 mice Tau - / - ; Student ´ s t - test ) .

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

Figure 1A-B Representative image of a proliferative cell in the DG labeled with an anti-PH3 antibody (green) (A) and quantification of the number of PH3+ cells in the DG of WT and Tau-/- mice (B) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).C-D Representative image of an apoptotic cell in the DG labeled with an anti-fractin antibody (red) (C) and quantification of the number of fractin+ cells in the DG of WT and Tau-/- mice (D) (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).E-F Schematic diagram of the cell survival experimental design using thymidine analogs (F) and quantification of the percentage of surviving cells in Tau-/- mice as compared to WT mice at each cell age (1-, 2-, 4-, 6- and 8-week-old cells) (F) (mean ± SEM (normalized data); Student´s t-test).H Quantification of the percentage of 1-, 4- and 8-week-old newborn neurons that express the DCX neuroblast marker and the NeuN mature neuron marker in WT and Tau-/- mice (χ2 test).I-J Representative images of progenitor cells in the DG labeled with anti-Sox2 (red) and anti-BLBP (green) antibodies (I), and neuroblasts and immature neurons labeled with anti-DCX (red) and anti-calretinin (green) antibodies, respectively (J).K-N Quantification of the number of Sox2+ (K), BLBP+ (L), DCX+ (M) and calretinin+ (N) cells in the DG of WT and Tau-/- mice (mean ± SEM; n= 10 mice WT, n= 8 mice Tau-/-; Student´s t-test).


Figure 2A Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . A schematic representation of Sholl ´ s analysis is shown in the WT image . B - C Quantification of total dendritic length ( B ) and Sholl ´ s analysis ( C ) of 4 - week - old newborn granule neurons in WT and Tau - / - mice . D Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus . E - F Quantification of total dendritic length ( E ) and Sholl ´ s analysis ( F ) of 8 - week - old newborn granule neurons in WT and Tau - / - mice . G Representative images of 4 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . H - IQuantification of the number of PSDs / µm ( H ) and PSD area ( I ) in each dendritic branching order of 4 - week - old newborn granule neurons in WT and Tau - / - mice . J Representative images of 8 - week - old newborn granule neurons of WT and Tau - / - mice infected by a PSD95 - GFP - expressing retrovirus and their corresponding high - power magnifications showing PSDs ( green ) . K - L Quantification of the number of PSDs / µm ( K ) and PSD area ( L ) in each dendritic branching order of 8 - week - old newborn granule neurons in WT and Tau - / - mice .

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 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 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

Figure 2A Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus. A schematic representation of Sholl´s analysis is shown in the WT image.B-C Quantification of total dendritic length (B) and Sholl´s analysis (C) of 4-week-old newborn granule neurons in WT and Tau-/- mice.D Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus.E-F Quantification of total dendritic length (E) and Sholl´s analysis (F) of 8-week-old newborn granule neurons in WT and Tau-/- mice.G Representative images of 4-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).H-IQuantification of the number of PSDs/µm (H) and PSD area (I) in each dendritic branching order of 4-week-old newborn granule neurons in WT and Tau-/- mice.J Representative images of 8-week-old newborn granule neurons of WT and Tau-/- mice infected by a PSD95-GFP-expressing retrovirus and their corresponding high-power magnifications showing PSDs (green).K-L Quantification of the number of PSDs/µm (K) and PSD area (L) in each dendritic branching order of 8-week-old newborn granule neurons in WT and Tau-/- mice.


Figure 3A Representative traces of mEPSCs recorded at a holding potential of − 65 mV from WT ( black traces ) and Tau - / - ( red traces ) mice . B Exemplary traces of mEPSCs from one cell from WT ( black trace ) and Tau - / - ( red trace ) mice . C Resting membrane potential of WT and Tau - / - mice ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; * 0 . 05 > p ≥ 0 . 01 , Student ´ s t - test ) . D - E mEPSC amplitude ( D ) and mEPSC frequency ( E ) of WT and Tau - / - animals ( mean ± SEM ; n = 13 cells WT , n = 10 cells Tau - / - ; Student ´ s t test or Mann - Whitney ´ s U test ) . F Cumulative probability plot of the total mEPSC amplitudes , small mEPSCs ( i ) and big mEPSCs ( ii ) from both WT and Tau - / - mice ( black and red traces , respectively ) ( * * * p < 0 . 001 ; Kolmogorov - Smirnov Z test ) .

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

Figure 3A Representative traces of mEPSCs recorded at a holding potential of −65 mV from WT (black traces) and Tau-/- (red traces) mice.B Exemplary traces of mEPSCs from one cell from WT (black trace) and Tau-/- (red trace) mice.C Resting membrane potential of WT and Tau-/- mice (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; *0.05 > p ≥ 0.01, Student´s t-test).D-E mEPSC amplitude (D) and mEPSC frequency (E) of WT and Tau-/- animals (mean ± SEM; n= 13 cells WT, n= 10 cells Tau-/-; Student´s t test or Mann-Whitney´s U test).F Cumulative probability plot of the total mEPSC amplitudes, small mEPSCs (i) and big mEPSCs (ii) from both WT and Tau-/- mice (black and red traces, respectively) (***p < 0.001; Kolmogorov-Smirnov Z test).


Figure 4A Representative image of a mouse subjected to forced swimming in the Porsolt test . C Representative images of 1 - week - old newborn granule neurons labeled with CldU ( green ) belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice . Cell nuclei were labeled with DAPI ( blue ) . Scale bar 50 µm . D - E Quantification of the number of 1 - week - old CldU + cells ( D ) and apoptotic fractin + cells ( E ) in each experimental group .

O B-PANEL_START 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

Figure 4A Representative image of a mouse subjected to forced swimming in the Porsolt test.C Representative images of 1-week-old newborn granule neurons labeled with CldU (green) belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice. Cell nuclei were labeled with DAPI (blue). Scale bar 50 µm.D-E Quantification of the number of 1-week-old CldU+ cells (D) and apoptotic fractin+ cells (E) in each experimental group.


Figure 5B Representative images of 8 - week - old newborn granule neurons of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice infected with a PSD95 - GFP - expressing retrovirus . In the WT CNP image a schematic representation of Sholl ´ s analysis is shown . C - D Quantification of total dendritic length ( C ) and Sholl ´ s analysis ( D ) of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) . E - F Representative images of an 8 - week - old newborn granule neuron , in which proximal dendrites are highlighted ( E ) , and of PSDs ( green ) in proximal dendrites belonging to WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( F ) . G - H Quantification of the number of PSDs / µm ( G ) and PSD area ( H ) in each dendritic branching order of 8 - week - old newborn granule neurons ( n = 3 mice per experimental condition ) . I - J Representative Western Blot ( I ) and quantification ( J ) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau - / - mice subjected or not to the Porsolt test ( n = 6 mice per experimental condition ) . K Representative image showing 8 - week - old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field ( indicated by a white square ) . L - M Representative images ( L ) and quantification of the area ( M ) of MFTs of 8 - week - old newborn neurons in the CA3 region of WT CNP , WT P , Tau - / - CNP , and Tau - / - P mice ( n = 3 mice per experimental condition ) . Arrows indicate MFTs .

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

Figure 5B Representative images of 8-week-old newborn granule neurons of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice infected with a PSD95-GFP-expressing retrovirus. In the WT CNP image a schematic representation of Sholl´s analysis is shown.C-D Quantification of total dendritic length (C) and Sholl´s analysis (D) of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).E-F Representative images of an 8-week-old newborn granule neuron, in which proximal dendrites are highlighted (E), and of PSDs (green) in proximal dendrites belonging to WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (F).G-H Quantification of the number of PSDs/µm (G) and PSD area (H) in each dendritic branching order of 8-week-old newborn granule neurons (n= 3 mice per experimental condition).I-J Representative Western Blot (I) and quantification (J) of the levels of GluR1 subunit of AMPA receptors in hippocampi of WT and Tau-/- mice subjected or not to the Porsolt test (n= 6 mice per experimental condition).K Representative image showing 8-week-old newborn granule neurons in the DG projecting axons through the mossy fiber tract into the CA3 field (indicated by a white square).L-M Representative images (L) and quantification of the area (M) of MFTs of 8-week-old newborn neurons in the CA3 region of WT CNP, WT P, Tau-/- CNP, and Tau-/- P mice (n= 3 mice per experimental condition). Arrows indicate MFTs.