293T cells were transfected with Flag - REV7 and GFP - SHLD1 ( Left ) or Flag - SHLD2 and GFP - SHLD1 ( Right ) expression vectors as indicated . 24h post - transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU - 60019 for 1 h prior to irradiation . 1h post - irradiation ( 10 Gy ) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti - Flag ( M2 ) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies .

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

293T cells were transfected with Flag-REV7 and GFP-SHLD1 (Left) or Flag-SHLD2 and GFP-SHLD1 (Right) expression vectors as indicated. 24h post-transfection cells were treated with DMSO or with 10uM of ATM inhibitor KU-60019 for 1 h prior to irradiation. 1h post-irradiation (10 Gy) nuclear extracts were prepared and REV7 or SHLD2 complexes were immunoprecipitated using anti-Flag (M2) Resin and then analyzed by immunoblotting using GFP and REV7 antibodies.


U2OS EJ5 - GFP cells were transfected with either siCTRL , siSHLD1 # 1 or siSHLD1 # 2 . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

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

U2OS EJ5-GFP cells were transfected with either siCTRL, siSHLD1 #1 or siSHLD1 #2. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


CH12F3 - 2 cells stably expressing either shCTRL , shSHLD1 # 1 or shSHLD1 # 2 were stimulated with a cocktail of cytokines ( CIT ) to induce class switching to IgA . The percentage of IgA + cells was monitored 24 and 48h post - stimulation by staining with an anti - IgA antibody followed by flow cytometry analysis .

O O O O O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O

CH12F3-2 cells stably expressing either shCTRL, shSHLD1#1 or shSHLD1#2 were stimulated with a cocktail of cytokines (CIT) to induce class switching to IgA. The percentage of IgA+ cells was monitored 24 and 48h post-stimulation by staining with an anti-IgA antibody followed by flow cytometry analysis.


U2OS DR - GFP cells were transfected with the indicated siRNAs . At 24hr post - transfection , cells were transfected with the I - SceI expression plasmid , and the GFP + population was analyzed 48 hr post - plasmid transfection . The percentage of GFP + cells was determined for each individual condition and subsequently normalized to the non - targeting condition ( siCTRL ) .

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

U2OS DR-GFP cells were transfected with the indicated siRNAs. At 24hr post-transfection, cells were transfected with the I-SceI expression plasmid, and the GFP+ population was analyzed 48 hr post-plasmid transfection. The percentage of GFP+ cells was determined for each individual condition and subsequently normalized to the non-targeting condition (siCTRL).


Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer ( TNBC ) . Data are represented as Kaplan - Meyer curves with expression classified as low and high .

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

Survival analysis of low and high expressers of SHLD2 in a cohort of 24 patients affected by Triple Negative Breast cancer (TNBC). Data are represented as Kaplan-Meyer curves with expression classified as low and high.


Relapse - free Survival ( Basal Subtype ) of low and high expressers of SHLD2 obtained from the KM - plotter database . Data are represented as Kaplan - Meyer curves with expression classified as low and high .

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

Relapse-free Survival (Basal Subtype) of low and high expressers of SHLD2 obtained from the KM-plotter database. Data are represented as Kaplan-Meyer curves with expression classified as low and high.


( A ) The transcript level of solo LTR in the wild type and the epcr1 mutant . Error bars are standard deviations of three technical replicates . The experiment was independently performed three times and similar results were obtained .

(A) The transcript level of solo LTR in the wild type and the epcr1 mutant. Error bars are standard deviations of three technical replicates. The experiment was independently performed three times and similar results were obtained.


( B ) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1 / 2 double mutant relative to the wild type . Two - week - old seedlings grown on vertical MS medium plates are shown .

(B) The developmental phenotype of the epcr1 and epcr2 single mutants and the epcr1/2 double mutant relative to the wild type. Two-week-old seedlings grown on vertical MS medium plates are shown.


( C ) The transcript levels of solo LTR , SDC , AtSN1 , AtGP1 and AtCOPIA28 in the wild type , epcr1 and epcr2 single mutants , epcr1 / 2 double mutant , and two individual EPCR1 transgenic lines in the epcr1 / 2 double mutant background . Error bars are standard deviations of three biological replicates .

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

(C) The transcript levels of solo LTR, SDC, AtSN1, AtGP1 and AtCOPIA28 in the wild type, epcr1 and epcr2 single mutants, epcr1/2 double mutant, and two individual EPCR1 transgenic lines in the epcr1/2 double mutant background. Error bars are standard deviations of three biological replicates.


( A ) The interaction between EPCR1 and ARID2 or ARID3 . Arabidopsis plants carrying EPCR1 - Flag and ARID2 - Myc or ARID3 - Myc transgenes were used for co - IP .

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

(A) The interaction between EPCR1 and ARID2 or ARID3. Arabidopsis plants carrying EPCR1-Flag and ARID2-Myc or ARID3-Myc transgenes were used for co-IP.


( B ) The interaction between TRB1 and ARID2 . Arabidopsis plants carrying TRB1 - Flag and / or ARID2 - Myc transgenes were used for co - IP .

O O O O O O 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) The interaction between TRB1 and ARID2. Arabidopsis plants carrying TRB1-Flag and/or ARID2-Myc transgenes were used for co-IP.


( C ) The interaction between TRB1 and EPCR1 . Arabidopsis plants carrying TRB1 - Flag and / or EPCR1 - Myc transgenes were used for co - IP .

(C) The interaction between TRB1 and EPCR1. Arabidopsis plants carrying TRB1-Flag and/or EPCR1-Myc transgenes were used for co-IP.


( E ) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull - down assay .

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(E) The interaction between TRB1 and PWWP2 or ARID2 was detected by an GST pull-down assay.


( F ) The interaction between EPCR1 , PWWP2 , ARID2 , and TRB1 was detected by an HIS pull - down assay . The full - length PWWP2 , ARID2 , and TRB1 , and the EPCR1 - N terminal ( 1 - 500 aa ) were used for the interaction assay .

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

(F) The interaction between EPCR1, PWWP2, ARID2, and TRB1 was detected by an HIS pull-down assay. The full-length PWWP2, ARID2, and TRB1, and the EPCR1-N terminal (1-500 aa) were used for the interaction assay.


( A ) The developmental phenotype of the arid2 / 3 / 4 mutant as compared to the wild type . Two - week - old seedlings grown on MS medium are shown .

(A) The developmental phenotype of the arid2/3/4 mutant as compared to the wild type. Two-week-old seedlings grown on MS medium are shown.


( B ) The developmental phenotype of the pwwp1 / 2 / 3 + / - mutant as compared to the wild type . One - month - old plants grown in soil are shown .

O O O O O O O O O O O O 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) The developmental phenotype of the pwwp1/2/3+/- mutant as compared to the wild type. One-month-old plants grown in soil are shown.


The effect of the arid2 , arid3 , and arid4 mutations on the silencing of solo LTR , SDC , and FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations .

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

The effect of the arid2, arid3, and arid4 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.


The effect of the pwwp1 , pwwp2 , and pwwp3 mutations on the silencing of solo LTR , SDC , and FWA as determined by qPCR . Showing are results of three biological replicates with standard deviations .

The effect of the pwwp1, pwwp2, and pwwp3 mutations on the silencing of solo LTR, SDC, and FWA as determined by qPCR. Showing are results of three biological replicates with standard deviations.


( E ) The effect of the trb1 mutation and the TRB2 knockdown ( TRB2 - KD ) on the silencing of solo LTR , as determined by qPCR ( right panel ) . The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown ( left panel ) . Error bars represent standard deviations of three biological replicates .

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

(E) The effect of the trb1 mutation and the TRB2 knockdown (TRB2-KD) on the silencing of solo LTR, as determined by qPCR (right panel). The transcript level of TRB2 was evaluated to confirm the TRB2 knockdown (left panel). Error bars represent standard deviations of three biological replicates.


( B ) Heat maps showing differentially transcribed genes and TEs in the arid2 / 3 / 4 and epcr1 / 2 mutants relative to the wild type .

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(B) Heat maps showing differentially transcribed genes and TEs in the arid2/3/4 and epcr1/2 mutants relative to the wild type.


( D ) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2 / 3 / 4 and epcr1 / 2 relative to the wild type .

(D) Genome browser snapshots showing the transcript patterns of ROS1 and IBM1 in arid2/3/4 and epcr1/2 relative to the wild type.


( E ) Effects of arid2 / 3 / 4 epcr1 / 2 on the transcript levels of ROS1 and IBM1 - L as determined by qPCR . The expression of ROS1 and the longer version of IBM1 ( IBM1 - L ) was evaluated by qPCR . ACT7 was amplified as an internal control . Bars represent SD from two independent experiments , each with three technical replications .

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

(E) Effects of arid2/3/4 epcr1/2 on the transcript levels of ROS1 and IBM1-L as determined by qPCR. The expression of ROS1 and the longer version of IBM1 (IBM1-L) was evaluated by qPCR. ACT7 was amplified as an internal control. Bars represent SD from two independent experiments, each with three technical replications.


( A ) The interaction of EPCR1 with HAM1 and HAM2 . EPCR1 - Flag transgenic plants were crossed to HAM1 - Myc and HAM2 - Myc transgenic plants . The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co - IP .

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

(A) The interaction of EPCR1 with HAM1 and HAM2. EPCR1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants. The progeny were used to evaluate the interaction between EPCR1 with HAM1 and HAM2 by co-IP.


( B ) The interaction of ARID2 , ARID3 , and ARID4 with HAM1 and HAM2 . ARID2 - Myc , ARID3 - Myc , and ARID4 - Myc transgenic plants were crossed to HAM1 - Flag and HAM2 - Flag transgenic plants and their progeny were used for co - IP .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The interaction of ARID2, ARID3, and ARID4 with HAM1 and HAM2. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HAM1-Flag and HAM2-Flag transgenic plants and their progeny were used for co-IP.


( C ) The interaction of TRB1 with HAM1 and HAM2 . TRB1 - Flag transgenic plants were crossed to HAM1 - Myc and HAM2 - Myc transgenic plants and their progeny were used for co - IP .

(C) The interaction of TRB1 with HAM1 and HAM2. TRB1-Flag transgenic plants were crossed to HAM1-Myc and HAM2-Myc transgenic plants and their progeny were used for co-IP.


( D ) The interaction of HDA9 with ARID2 , ARID3 , and ARID4 . ARID2 - Myc , ARID3 - Myc , and ARID4 - Myc transgenic plants were crossed to HDA9 - Flag transgenic plants and their progeny were used for co - IP .

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

(D) The interaction of HDA9 with ARID2, ARID3, and ARID4. ARID2-Myc, ARID3-Myc, and ARID4-Myc transgenic plants were crossed to HDA9-Flag transgenic plants and their progeny were used for co-IP.


( E ) The interaction of HDA6 with EPCR1 and ARID2 . EPCR1 - Flag and ARID2 - Flag transgenic plants were crossed to HDA6 - Myc transgenic plants and their progeny were used for co - IP .

(E) The interaction of HDA6 with EPCR1 and ARID2. EPCR1-Flag and ARID2-Flag transgenic plants were crossed to HDA6-Myc transgenic plants and their progeny were used for co-IP.


( A ) Overlap among H4K5 hyperacetylated regions in the arid2 / 3 / 4 and epcr1 / 2 mutants relative to the wild type . The overlap is significant ( P = 4 . 410 - 78 ) as determined by hypergeometric test . ( B ) Metaplot of histone H4K5 acetylation levels of TEs in wild type , arid2 / 3 / 4 , and epcr1 / 2 plants . Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP - seq analysis . TSS represents transcription start site and TTS represents transcription termination site . ( C ) Boxplot of histone H4K5 acetylation levels of TEs in wild type , arid2 / 3 / 4 , and epcr1 / 2 plants . Asterisks indicate that H4K5Ac levels is significantly higher in the arid2 / 3 / 4 ( P = 2 . 4210 - 13 ) and epcr1 / 2 ( P = 1 . 5210 - 8 ) mutants than in the wild type as determined by Welch ' s Two - Sample t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range .

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

(A) Overlap among H4K5 hyperacetylated regions in the arid2/3/4 and epcr1/2 mutants relative to the wild type. The overlap is significant (P=4.410-78) as determined by hypergeometric test. (B) Metaplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Histone H4K5 acetylation levels are represented by normalized reads that obtained in the H4K5Ac ChIP-seq analysis. TSS represents transcription start site and TTS represents transcription termination site. (C) Boxplot of histone H4K5 acetylation levels of TEs in wild type, arid2/3/4, and epcr1/2 plants. Asterisks indicate that H4K5Ac levels is significantly higher in the arid2/3/4 (P=2.4210-13) and epcr1/2 (P=1.5210-8) mutants than in the wild type as determined by Welch's Two-Sample t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( D ) The nuclei with condensed , intermediate , or decondensed heterochromatin status , as marked by DAPI staining and H3K27me signals . ( E ) Percentages of nuclei with condensed , intermediate , or decondensed heterochromatin signals in the wild type and the arid2 / 3 / 4 mutant . n = 113 .

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(D) The nuclei with condensed, intermediate, or decondensed heterochromatin status, as marked by DAPI staining and H3K27me signals. (E) Percentages of nuclei with condensed, intermediate, or decondensed heterochromatin signals in the wild type and the arid2/3/4 mutant. n=113.


( F ) Abundance of Pol IV - dependent siRNAs in the epcr1 / 2 , arid2 / 3 / 4 , nrpd1 , nrpe1 , and hda6 mutants relative to the wild type [ ( Mut - WT ) / ( Mut + WT ) ] from chromosome 3 of Arabidopsis .

(F) Abundance of Pol IV-dependent siRNAs in the epcr1/2, arid2/3/4, nrpd1, nrpe1, and hda6 mutants relative to the wild type [(Mut-WT)/(Mut+WT)] from chromosome 3 of Arabidopsis.


( H ) Boxplot showing levels of Pol IV - dependent siRNAs that are up - regulated in the hda6 mutant . Asterisks indicate that siRNA levels in the mutants are significantly ( P → 0 ) up - regulated compared to the wild type as determined by paired student t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range .

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

(H) Boxplot showing levels of Pol IV-dependent siRNAs that are up-regulated in the hda6 mutant. Asterisks indicate that siRNA levels in the mutants are significantly (P→0) up-regulated compared to the wild type as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( A ) Metaplot of CG , CHG , and CHH methylation of genes and TEs in the genomes of wild type , epcr1 / 2 , and arid2 / 3 / 4 plants . H indicates C , A , or T .

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

(A) Metaplot of CG, CHG, and CHH methylation of genes and TEs in the genomes of wild type, epcr1/2, and arid2/3/4 plants. H indicates C, A, or T.


( B ) Metaplot of CG , CHG , and CHH methylation on chromosome 3 of Arabidopsis in wild type , epcr1 / 2 , and arid2 / 3 / 4 plants .

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(B) Metaplot of CG, CHG, and CHH methylation on chromosome 3 of Arabidopsis in wild type, epcr1/2, and arid2/3/4 plants.


( D ) Boxplot of DNA methylation in the wild type , nrpe1 , epcr1 / 2 , and arid2 / 3 / 4 mutants . The hypo - DMRs identified in the nrpe1 mutant was defined as RdDM target loci . Asterisks indicate that DNA methylation is significantly ( P < 0 . 001 ) decreased in the mutants as determined by paired student t - test . Horizontal lines represent the median , and the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range .

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

(D) Boxplot of DNA methylation in the wild type, nrpe1, epcr1/2, and arid2/3/4 mutants. The hypo-DMRs identified in the nrpe1 mutant was defined as RdDM target loci. Asterisks indicate that DNA methylation is significantly (P<0.001) decreased in the mutants as determined by paired student t-test. Horizontal lines represent the median, and the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( E ) Heat maps of CG , CHG , and CHH methylation at RdDM target loci . The hypo - DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type , nrpe1 , eprc1 / 2 , and arid2 / 3 / 4 mutants . Black and light yellow indicate low methylation and high methylation , respectively .

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

(E) Heat maps of CG, CHG, and CHH methylation at RdDM target loci. The hypo-DMRs identified in the nrpe1 mutant were defined as RdDM target loci and analyzed in the wild type, nrpe1, eprc1/2, and arid2/3/4 mutants. Black and light yellow indicate low methylation and high methylation, respectively.


( A ) Scatter plots showing the effect of the epcr1 / 2 and arid2 / 3 / 4 mutations on CG , CHG , and CHH methylation at the co - upregulated TEs and genes in the epcr1 / 2 and arid2 / 3 / 4 mutants . TE methylation and gene DNA methylation refer , respectively , to methylation of the TE body and methylation of the 1 - kb - gene - promoter region .

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

(A) Scatter plots showing the effect of the epcr1/2 and arid2/3/4 mutations on CG, CHG, and CHH methylation at the co-upregulated TEs and genes in the epcr1/2 and arid2/3/4 mutants. TE methylation and gene DNA methylation refer, respectively, to methylation of the TE body and methylation of the 1-kb-gene-promoter region.


( B ) Box plots of CG , CHG , and CHH methylation in the wild type , arid2 / 3 / 4 , and epcr1 / 2 mutants at the co - upregulated TEs and genes in the arid2 / 3 / 4 and epcr1 / 2 mutants . Asterisks indicate statistical significance as determined by paired student t test . CHG methylation of co - upregulated TEs is significantly increased in arid2 / 3 / 4 ( P = 1 . 2610 - 4 ) and epcr1 / 2 ( P = 6 . 310 - 9 ) ; CHH methylation of co - upregulated genes is significantly decreased in arid2 / 3 / 4 ( P = 3 . 510 - 6 ) and epcr1 / 2 ( P = 6 . 410 - 7 ) . Horizontal lines represent the median , the bottom and top of the box represent the 25th and 75th percentile . The whiskers represent data range within 1 . 5 × of the interquantile range .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Box plots of CG, CHG, and CHH methylation in the wild type, arid2/3/4, and epcr1/2 mutants at the co-upregulated TEs and genes in the arid2/3/4 and epcr1/2 mutants. Asterisks indicate statistical significance as determined by paired student t test. CHG methylation of co-upregulated TEs is significantly increased in arid2/3/4 (P=1.2610-4) and epcr1/2 (P=6.310-9); CHH methylation of co-upregulated genes is significantly decreased in arid2/3/4 (P=3.510-6) and epcr1/2 (P=6.410-7). Horizontal lines represent the median, the bottom and top of the box represent the 25th and 75th percentile. The whiskers represent data range within 1.5× of the interquantile range.


( C ) Overexpression of DRD1 fails to restore the silencing of EPCR1 / 2 target loci in the epcr1 / 2 mutant background .

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(C) Overexpression of DRD1 fails to restore the silencing of EPCR1/2 target loci in the epcr1/2 mutant background.


( F - G ) Thermal shift assay traces of human Teneurin4WT ( F ) and human Teneurin4Mut ( G ) in the presence of physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentration ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) .

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

(F-G) Thermal shift assay traces of human Teneurin4WT (F) and human Teneurin4Mut (G) in the presence of physiological calcium concentrations (+ Ca2+; black), low calcium concentration (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey).


( A ) Dimensionless Kratky plots for Teneurin4WT ( left ) and Teneurin4Mut ( right ) in physiological calcium concentrations ( + Ca2 + ; black ) , low calcium concentrations ( + / - Ca2 + ; blue and red , respectively ) and no calcium ( - Ca2 + ; grey ) . I ( 0 ) and Rg were obtained from Guinier analysis ( Fig EV5A , B ) . Crosshairs indicate the maximum position for a fully folded globular protein .

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

(A) Dimensionless Kratky plots for Teneurin4WT (left) and Teneurin4Mut (right) in physiological calcium concentrations (+ Ca2+; black), low calcium concentrations (+/- Ca2+; blue and red, respectively) and no calcium (- Ca2+; grey). I(0) and Rg were obtained from Guinier analysis (Fig EV5A, B). Crosshairs indicate the maximum position for a fully folded globular protein.


( B ) Pair - distance distribution plots for Teneurin4 proteins colored as in A .

(B) Pair-distance distribution plots for Teneurin4 proteins colored as in A.


( A ) Representative images of HEK293 - T cells transfected with GFP ( top ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; bottom ) for 24h and stained with Alexa 568 Phalloidin ( magenta ) and Dapi ( blue ) . Images are maximum intensity projections of 14 - 16 stacks . Scale bar , 10 µm . ( B ) Zoom shows the regions indicated in A . White arrows indicate the tip of the filopodium from the transfected cell . Scale bar , 2 µm . (

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

(A) Representative images of HEK293-T cells transfected with GFP (top), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP- Teneurin4Mut (GFP-Ten4Mut; bottom) for 24h and stained with Alexa 568 Phalloidin (magenta) and Dapi (blue). Images are maximum intensity projections of 14-16 stacks. Scale bar, 10 µm. (B) Zoom shows the regions indicated in A. White arrows indicate the tip of the filopodium from the transfected cell. Scale bar, 2 µm. (


( C ) Average number of filopodia per cell in cells transfected with GFP , GFP - Teneurin4WT ( Ten4 WT ; red ) and GFP - Teneurin4Mut ( Ten4Mut ; blue ) . ( D ) Fraction of filopodia enriched with GFP per cell in GFP - , GFP - Teneurin4WT ( Ten4WT ; blue ) - and GFP - Teneurin4Mut ( Ten4Mut ; red ) - transfected cells . (

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

(C) Average number of filopodia per cell in cells transfected with GFP, GFP-Teneurin4WT (Ten4 WT; red) and GFP- Teneurin4Mut (Ten4Mut; blue). (D) Fraction of filopodia enriched with GFP per cell in GFP-, GFP-Teneurin4WT (Ten4WT; blue)- and GFP-Teneurin4Mut (Ten4Mut; red)-transfected cells. (


( A ) Representative images of non - adherent K562 cells electroporated with GFP ( left ) , GFP - Teneurin4WT ( GFP - Ten4WT ; middle ) or GFP - Teneurin4Mut ( GFP - Ten4Mut ; right ) . Zoomed images show the formation of cellular clusters in cells expressing GFP - Ten4WT and GFP - Ten4Mut , but not in GFP - expressing cells . Scale bar top panel , 50 µm . Scale bar bottom panel , 20 µm .

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

(A) Representative images of non-adherent K562 cells electroporated with GFP (left), GFP-Teneurin4WT (GFP-Ten4WT; middle) or GFP-Teneurin4Mut (GFP-Ten4Mut; right). Zoomed images show the formation of cellular clusters in cells expressing GFP-Ten4WT and GFP-Ten4Mut, but not in GFP-expressing cells. Scale bar top panel, 50 µm. Scale bar bottom panel, 20 µm.


( C ) Fraction of cell clusters detected per image in GFP - ( black ) , GFP - Teneurin4WT ( blue ) - and GFP - Teneurin4Mut ( red ) - electroporated cells .

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

(C) Fraction of cell clusters detected per image in GFP- (black), GFP-Teneurin4WT (blue)- and GFP-Teneurin4Mut (red)-electroporated cells.


( a ) TbMCU - GFP co - localization with MitoTracker ( MT ) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP ( Pearson ' s correlation coefficient ( PCC ) : 0 . 912 ) . ( b , c ) TbMCU co - localization with MitoTracker in the mitochondria of PCF ( b ) or BSF ( c ) trypanosomes , as detected by immunofluorescence analyses with antibodies against TbMCU . PCC : 0 . 930 and 0 . 920 , respectively . Merge images show the co - localization in yellow . DIC , differential interference contrast microscopy . Scale bar , 10 μm ( a - c ) .

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

(a) TbMCU-GFP co-localization with MitoTracker (MT) in mitochondria of PCF trypanosomes as detected by immunofluorescence analyses with antibodies against GFP (Pearson's correlation coefficient (PCC): 0.912). (b,c) TbMCU co-localization with MitoTracker in the mitochondria of PCF (b) or BSF (c) trypanosomes, as detected by immunofluorescence analyses with antibodies against TbMCU. PCC: 0.930 and 0.920, respectively. Merge images show the co-localization in yellow. DIC, differential interference contrast microscopy. Scale bar, 10 μm (a-c).


( d , e ) Growth of BSF ( d ) and PCF ( e ) trypanosomes in the absence ( black lines , − Tet ) or presence ( red lines , + Tet ) of 1 μg ml − 1 tetracycline for the indicated number of days . Values are mean ± s . d . ( n = 3 ) .

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

(d,e) Growth of BSF (d) and PCF (e) trypanosomes in the absence (black lines, −Tet) or presence (red lines, +Tet) of 1 μg ml−1 tetracycline for the indicated number of days. Values are mean±s.d. (n=3).


( f , g ) Northern blot analyses of TbMCU RNAi of BSF ( f ) and PCF ( g ) trypanosomes grown in the absence ( 0 ) and presence ( 2 - 8 ; 2 - 10 ) of tetracycline ( top ) . Tubulin is shown as a loading control ( bottom ) . Markers are shown on the right . The transcript of TbMCU , including the 5 ′ - and 3 ′ - UTR was ~ 1 . 5 kb in length .

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

(f,g) Northern blot analyses of TbMCU RNAi of BSF (f) and PCF (g) trypanosomes grown in the absence (0) and presence (2-8; 2-10) of tetracycline (top). Tubulin is shown as a loading control (bottom). Markers are shown on the right. The transcript of TbMCU, including the 5′- and 3′-UTR was ~1.5 kb in length.


( h , i ) Western blot analyses of TbMCU RNAi of BSF ( h ) and PCF ( i ) trypanosomes grown in the absence ( 0 ) or presence ( 2 - 8 ; 2 - 10 ) of tetracycline . Total lysates ( 30 μg ) were subjected to 10 % SDS - polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU ( top ) . One band of ~ 30 kDa was detected in T . brucei homogenates . Membranes were stripped and re - incubated with antibody against the voltage - dependent anion channel ( TbVDAC ) as a loading control ( bottom ) .

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

(h,i) Western blot analyses of TbMCU RNAi of BSF (h) and PCF (i) trypanosomes grown in the absence (0) or presence (2-8; 2-10) of tetracycline. Total lysates (30 μg) were subjected to 10% SDS-polyacrylamide gel electrophoresis before transfer to a nitrocellulose membrane and then stained with antibodies against TbMCU (top). One band of ~30 kDa was detected in T. brucei homogenates. Membranes were stripped and re-incubated with antibody against the voltage-dependent anion channel (TbVDAC) as a loading control (bottom).


( a ) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5 - N . After several pulses of Ca2 + ( 8 μM final concentration ) cells ( 5 × 107 trypanosomes grown in the absence ( black tracing ) or presence ( green tracing ) of tetracycline ) were added to the reaction medium ( 2 . 45 ml ) and the reaction was started adding 50 μM digitonin . Ruthenium red ( RR , 40 μM , yellow and red tracing ) , and FCCP ( 5 μM ) were added where indicated . A decrease in fluorescence indicates decreasing medium Ca2 + or increasing vesicular Ca2 + . ( b ) Relative Ca2 + uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1 . 0 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(a) The reaction buffer contained 2 mM succinate and 1 μM Calcium Green 5-N. After several pulses of Ca2+ (8 μM final concentration) cells (5 × 107 trypanosomes grown in the absence (black tracing) or presence (green tracing) of tetracycline) were added to the reaction medium (2.45 ml) and the reaction was started adding 50 μM digitonin. Ruthenium red (RR, 40 μM, yellow and red tracing), and FCCP (5 μM) were added where indicated. A decrease in fluorescence indicates decreasing medium Ca2+ or increasing vesicular Ca2+. (b) Relative Ca2+ uptake at 700 s as compared with that of control trypanosomes grown in the absence of tetracycline considered as 1.0 (−Tet) (means±s.d., n=3, **P0.001, Student's t-test).


( c ) Similar conditions as in a except that further CaCl2 additions ( 8 μM each time ) were added to − Tet trypanosomes to show the high mitochondrial capacity to take up Ca2 + . EGTA ( 8 μM ) and CaCl2 ( Ca2 + ) were added where indicated to show the presence of medium Ca2 + . ( d ) Relative initial rate of Ca2 + uptake by + Tet as compared with − Tet trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(c) Similar conditions as in a except that further CaCl2 additions (8 μM each time) were added to −Tet trypanosomes to show the high mitochondrial capacity to take up Ca2+. EGTA (8 μM) and CaCl2 (Ca2+) were added where indicated to show the presence of medium Ca2+. (d) Relative initial rate of Ca2+ uptake by +Tet as compared with −Tet trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( e ) Representative traces of Ca2 + uptake kinetics in digitonin - permeabilized TbMCU BSF trypanosomes cultured in the absence ( − Tet ) or presence ( + Tet ) of tetracycline . The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate . Ca2 + uptake was monitored over time using 1 μM Calcium Green - 5N . Ruthenium red ( RR , 40 μM , yellow and red tracings ) , oligomycin ( Oligo , 2 . 5 μg ml − 1 ) , EGTA ( 0 . 4 μM ) and CaCl2 ( pulses of 0 . 4 μM ) were added where indicated . ( f ) Relative Ca2 + uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * ( + Tet ± RR ) , * * ( − Tet ± RR ) , * * * ( − Tet / + Tet ) , P0 . 001 , Student ' s t - test ) .

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

(e) Representative traces of Ca2+ uptake kinetics in digitonin-permeabilized TbMCU BSF trypanosomes cultured in the absence (−Tet) or presence (+Tet) of tetracycline. The reactions were started adding 40 μM digitonin in the presence of 1 mM ATP and 500 μM sodium orthovanadate. Ca2+ uptake was monitored over time using 1 μM Calcium Green-5N. Ruthenium red (RR, 40 μM, yellow and red tracings), oligomycin (Oligo, 2.5 μg ml−1), EGTA (0.4 μM) and CaCl2 (pulses of 0.4 μM) were added where indicated. (f) Relative Ca2+ uptake at 200 s as compared with that of control BSF trypanosomes grown in the absence of tetracycline considered as 1 (−Tet) (means±s.d., n=3, *(+Tet±RR), **(−Tet±RR), ***(−Tet/+Tet) , P0.001, Student's t-test).


( a ) PCFtrypanosomes ( 5 × 107 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing 2 mM succinate , and 5 μM safranine , and the reaction was started with digitonin ( 50 μM ) . ADP ( 10 μM ) , oligomycin ( Oligo , 2 μg ml − 1 ) , CaCl2 ( 40 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( b ) Changes in safranine fluorescence after addition of ADP or Ca2 + to − Tet and + Tet PCFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

(a) PCFtrypanosomes (5 × 107 cells) were added to the reaction buffer (2.45 ml) containing 2 mM succinate, and 5 μM safranine, and the reaction was started with digitonin (50 μM). ADP (10 μM), oligomycin (Oligo, 2 μg ml−1), CaCl2 (40 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (b) Changes in safranine fluorescence after addition of ADP or Ca2+ to −Tet and +Tet PCFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


( c ) BSFtrypanosomes ( 2 × 108 cells ) were added to the reaction buffer ( 2 . 45 ml ) containing EGTA ( 20 μM ) , ATP ( 1 mM ) , sodium orthovanadate ( 500 μM ) and 12 . 5 μM safranine . The reaction was started with digitonin ( 40 μM ) . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated . ( d , e ) Changes in safranine fluorescence after addition of Ca2 + ( d ) or FCCP ( e ) to − Tet and + Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

(c) BSFtrypanosomes (2 × 108 cells) were added to the reaction buffer (2.45 ml) containing EGTA (20 μM), ATP (1 mM), sodium orthovanadate (500 μM) and 12.5 μM safranine. The reaction was started with digitonin (40 μM). CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated. (d,e) Changes in safranine fluorescence after addition of Ca2+ (d) or FCCP (e) to −Tet and +Tet BSFtrypanosomes (means±s.d., n=3, **P0.01, Student's t-test).


( a ) Comparison of AMP / ATP ratios between PCF trypanosomes grown in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi , and with or without glucose ( Gl ) , expressed as fold increase ( means ± s . d . , n = 3 ; * P0 . 05 ; * * P0 . 01 , Student ' s t - test ) .

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

(a) Comparison of AMP/ATP ratios between PCF trypanosomes grown in the absence (−Tet) or presence (+Tet) of tetracycline to induce RNAi, and with or without glucose (Gl), expressed as fold increase (means±s.d., n=3; *P0.05; **P0.01, Student's t-test).


( b ) Comparison of AMP / ATP ratios between ( − Tet ) and ( + Tet ) PCF trypanosomes grown in a glucose - depleted medium ( SDM - 80 ) containing 5 . 2 mM L - proline for 2 days after 2 - day growth in SDM - 79 ± tetracycline , expressed as fold increase ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

(b) Comparison of AMP/ATP ratios between (−Tet) and (+Tet) PCF trypanosomes grown in a glucose-depleted medium (SDM-80) containing 5.2 mM L-proline for 2 days after 2-day growth in SDM-79±tetracycline, expressed as fold increase (means±s.d., n=3, **P0.01, Student's t-test).


( c ) Fluorescence microscopic images of TbATG8 . 2 in PCF trypanosomes cultured in SDM - 79 in the absence ( WT ) or presence ( RNAi ) of tetracycline to induce RNAi ; scale bars , 10 μm . ( d ) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM - 79 or SDM - 80 medium in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . Over 500 cells from three experiments with 20 random fields / experiment were analysed ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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(c) Fluorescence microscopic images of TbATG8.2 in PCF trypanosomes cultured in SDM-79 in the absence (WT) or presence (RNAi) of tetracycline to induce RNAi; scale bars, 10 μm. (d) Quantification of autophagosome formation in RNAi PCF trypanosomes grown in SDM-79 or SDM-80 medium in the presence (+Tet) or absence (−Tet) of tetracycline. Over 500 cells from three experiments with 20 random fields/experiment were analysed (means±s.d., n=3, **P0.001, Student's t-test).


( e ) Western blot analysis of ATG8 . 2 or tubulin in PCFtrypanosomes cultured in SDM - 79 or SDM - 80 in the absence ( − Tet ) or presence ( + Tet ) of tetracycline to induce RNAi . Cell lysates ( 30 μg ) were fractionated in 12 % urea - SDS - PAGE gel . Immunoblots were labelled with anti - ATG8 . 2 or anti - tubulin antibody . ( f ) Quantification of ATG8 . 2 - II / ( ATG8 . 2 - II + ATG8 . 2 - I ) presented as mean ratios ± s . d . ( n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

(e) Western blot analysis of ATG8.2 or tubulin in PCFtrypanosomes cultured in SDM-79 or SDM-80 in the absence (−Tet) or presence (+ Tet) of tetracycline to induce RNAi. Cell lysates (30 μg) were fractionated in 12% urea-SDS-PAGE gel. Immunoblots were labelled with anti-ATG8.2 or anti-tubulin antibody. (f) Quantification of ATG8.2-II/(ATG8.2-II+ATG8.2-I) presented as mean ratios±s.d. (n=3, **P0.01, Student's t-test).


( g ) Groups of five mice were infected with WT T . brucei ( black line ) or trypanosomes transfected with the construct for RNAi of TbMCU ( red line ) . Doxycycline ( green line ; 200 μg ml − 1 ) was given in the drinking water throughout a 30 - day period .

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(g) Groups of five mice were infected with WT T. brucei (black line) or trypanosomes transfected with the construct for RNAi of TbMCU (red line). Doxycycline (green line; 200 μg ml−1) was given in the drinking water throughout a 30-day period.


( h ) Parasitemia levels in surviving mice were monitored at 2 - day intervals after infection . The results are means ± s . d . ( n = 2 ) .

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(h) Parasitemia levels in surviving mice were monitored at 2-day intervals after infection. The results are means±s.d. (n=2).


( a ) Growth of TbMCU - KO BSF in the absence ( black line ) or presence ( red line ) of 1 μg ml − 1 tetracycline for the indicated number of days . Addition of 10 mM threonine to the medium ( green line ) partially rescued the mutant BSF trypanosomes but had no effect on + Tet cells ( yellow line ; means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(a) Growth of TbMCU-KO BSF in the absence (black line) or presence (red line) of 1 μg ml−1 tetracycline for the indicated number of days. Addition of 10 mM threonine to the medium (green line) partially rescued the mutant BSF trypanosomes but had no effect on +Tet cells (yellow line; means±s.d., n=3, **P0.001, Student's t-test).


( b ) Southern blot analysis of the BSF conditional KOs . Genomic DNA from the T . brucei SM parental strain ( + / + ) , single - ( + / − ) or double - allele ( − / − ) KO cells .

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(b) Southern blot analysis of the BSF conditional KOs. Genomic DNA from the T. brucei SM parental strain (+/+), single- (+/−) or double-allele (−/−) KO cells.


( c ) Northern blot analysis of wild - type ( WT ) and TbMCU - KO BSF cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . Tubulin is shown as a loading control ( bottom panel ) .

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(c) Northern blot analysis of wild-type (WT) and TbMCU-KO BSF cultured in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. Tubulin is shown as a loading control (bottom panel).


( d ) Western blot analysis of total cell lysates . Proteins from BSF trypanosomes cultured in the presence ( + Tet ) or absence ( − Tet ) of tetracycline were detected using anti - HA antibody . TbVDAC was used as a loading control .

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

(d) Western blot analysis of total cell lysates. Proteins from BSF trypanosomes cultured in the presence (+Tet) or absence (−Tet) of tetracycline were detected using anti-HA antibody. TbVDAC was used as a loading control.


( e ) Immunofluorescence analysis of control ( upper panels ) and TbMCU - KO ( lower panels ) trypanosomes . TbMCU co - localized with MitoTracker ( MT ) in the control parasites ( + Tet ; PCC = 0 . 808 ) . Scale bar , 10 μm .

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

(e) Immunofluorescence analysis of control (upper panels) and TbMCU-KO (lower panels) trypanosomes. TbMCU co-localized with MitoTracker (MT) in the control parasites (+Tet; PCC=0.808). Scale bar, 10 μm.


( f ) Conditional TbMCU - KO BSF trypanosomes were grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline for 2 days . BSF trypanosomes ( 2 × 108 cells ) were incubated as in Fig . 2e . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 20 μM . A representative trace ( red or black ) of Ca2 + uptake from one of three independent experiments is shown .

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

(f) Conditional TbMCU-KO BSF trypanosomes were grown in the presence (+Tet) or absence (−Tet) of tetracycline for 2 days. BSF trypanosomes (2 × 108 cells) were incubated as in Fig. 2e. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 20 μM. A representative trace (red or black) of Ca2+ uptake from one of three independent experiments is shown.


( g ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSF trypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 ( 50 μM ) , EGTA ( 200 μM ) and FCCP ( 10 μM ) were added where indicated .

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

(g) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSF trypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 (50 μM), EGTA (200 μM) and FCCP (10 μM) were added where indicated.


( h , i ) Representative traces of mitochondrial membrane potential of digitonin - permeabilized BSFtrypanosomes grown in the presence ( + Tet ) or absence ( − Tet ) of tetracycline . The reactions were incubated as in Fig . 3c . CaCl2 25 , 50 and 100 μM CaCl2 ) or FCCP ( i ) to + Tet and − Tet BSFtrypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(h,i) Representative traces of mitochondrial membrane potential of digitonin-permeabilized BSFtrypanosomes grown in the presence (+Tet) or absence (−Tet) of tetracycline. The reactions were incubated as in Fig. 3c. CaCl2 25, 50 and 100 μM CaCl2) or FCCP (i) to +Tet and −Tet BSFtrypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( a ) Rhod - 2 fluorescence in control or overexpressing TbMCU ( TbMCU - OE ) PCF . Scale bar , 10 μm . ( b , c ) Rhod - 2 ( mitochondrial ) ( b ) and Fluo - 4 ( cytosolic ) ( c ) fluorescence in control ( − Tet ) and overexpressing TbMCU ( + Tet ) PCF ( means ± s . d . , n = 3 , * * P0 . 05 , Student ' s t - test ) ; NS , not significant .

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

(a) Rhod-2 fluorescence in control or overexpressing TbMCU (TbMCU-OE) PCF. Scale bar, 10 μm. (b,c) Rhod-2 (mitochondrial) (b) and Fluo-4 (cytosolic) (c) fluorescence in control (−Tet) and overexpressing TbMCU (+Tet) PCF (means±s.d., n=3, **P0.05, Student's t-test); NS, not significant.


( d ) Ca2 + uptake by digitonin - permeabilized PCF overexpressing TbMCU . Trypanosomes were incubated as in Fig . 2c . Multiple pulses of CaCl2 ( arrowheads ) were added to a final concentration of 8 μM . ( e ) Ca2 + uptake in TbMCU - OE PCF ( + Tet ) 200 s after addition of Ca2 + , as compared with uninduced trypanosomes considered as 1 ( − Tet ) ( means ± s . d . , n = 3 , * * P0 . 01 , Student ' s t - test ) .

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

(d) Ca2+ uptake by digitonin-permeabilized PCF overexpressing TbMCU. Trypanosomes were incubated as in Fig. 2c. Multiple pulses of CaCl2 (arrowheads) were added to a final concentration of 8 μM. (e) Ca2+ uptake in TbMCU-OE PCF (+Tet) 200 s after addition of Ca2+, as compared with uninduced trypanosomes considered as 1 (−Tet) (means±s.d., n=3, **P0.01, Student's t-test).


( f ) Mitochondrial membrane potential of digitonin - permeabilized control ( − Tet ) and TbMCU - OE ( + Tet ) PCF . The reactions were incubated as in Fig . 3a .

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

(f) Mitochondrial membrane potential of digitonin-permeabilized control (−Tet) and TbMCU-OE (+Tet) PCF. The reactions were incubated as in Fig. 3a.


( g ) Changes in safranine fluorescence after addition of ADP , Ca2 + or FCCP to - Tet and + Tet PCF trypanosomes ( means ± s . d . , n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(g) Changes in safranine fluorescence after addition of ADP, Ca2+ or FCCP to -Tet and +Tet PCF trypanosomes (means±s.d., n=3, **P0.001, Student's t-test).


( h ) Mitochondrial oxidative stress as measured with MitoSOX Red in control ( C ) and TbMCU - overexpressing ( OE ) PCF permeabilized with 50 μM digitonin in the presence of 0 . 4 mM Ca2 + . Controls were treated with 2 μM antimycin A ( C + AA ) , or were trypanosomes exposed to 0 . 4 mM Ca2 + in the absence of digitonin ( − DIG + Ca2 + ) , and trypanosomes permeabilized with digitonin in the absence of Ca2 + ( C , yellow line ) . ( i ) Quantification of change at 900 s . Results are expressed as mean fluorescence ± s . d . ( n = 3 , * * P0 . 001 , Student ' s t - test ) .

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

(h) Mitochondrial oxidative stress as measured with MitoSOX Red in control (C) and TbMCU-overexpressing (OE) PCF permeabilized with 50 μM digitonin in the presence of 0.4 mM Ca2+. Controls were treated with 2 μM antimycin A (C+AA), or were trypanosomes exposed to 0.4 mM Ca2+ in the absence of digitonin (−DIG+Ca2+), and trypanosomes permeabilized with digitonin in the absence of Ca2+ (C, yellow line). (i) Quantification of change at 900 s. Results are expressed as mean fluorescence±s.d. (n=3, **P0.001, Student's t-test).


( j ) Growth of trypanosomes in the absence ( control , black lines ) or presence ( TbMCU - OE , red lines ) of 1 μg ml − 1 tetracycline ( means ± s . d . , n = 3 ) . ( k ) Dead cells in representative fields of control ( − Tet ) and TbMCU - OE ( + Tet ) PCF are indicated ( white arrows ) . Scale bar , 10 μm .

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

(j) Growth of trypanosomes in the absence (control, black lines) or presence (TbMCU-OE, red lines) of 1 μg ml−1 tetracycline (means±s.d., n=3). (k) Dead cells in representative fields of control (−Tet) and TbMCU-OE (+Tet) PCF are indicated (white arrows). Scale bar, 10 μm.


( l ) Cell viability on apoptotic challenge of control ( − Tet ) or TbMCU - OE ( + Tet ) PCF trypanosomes with H2O2 and C2 - ceramide . The percentage of cell death was obtained from means ± s . d . by analysing > 60 fields ( including > 400 cells ) ( n = 3 ) . * * P0 . 01 , Student ' s t - test .

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

(l) Cell viability on apoptotic challenge of control (−Tet) or TbMCU-OE (+Tet) PCF trypanosomes with H2O2 and C2-ceramide. The percentage of cell death was obtained from means±s.d. by analysing >60 fields (including >400 cells) (n=3). **P0.01, Student's t-test.


( A ) Confocal images of representative eye cryosections immunostained with anti - Lamp1 antibody from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on , at 10 AM ( diurnal condition ) . Enlarged boxes highlight Lamp1 - positive structures ( white arrowheads ) in the RPE . Autofluorescence from lipofuscin granules are visible in all cryosections of miR - 211 - / - compared to control littermates . Nuclei were counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer .

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

(A) Confocal images of representative eye cryosections immunostained with anti-Lamp1 antibody from 2-month-old miR-211-/- and control mice sacrificed 3h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1-positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules are visible in all cryosections of miR-211-/- compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.


( B ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative Western blot analysis of the Lamp1 , Cln5 , Trpml1 , Sqstm1 / p62 , Cathepsin D ( CtsD ) and LC3 proteins from WT and miR - 211 - / - mice . Note a decrease of both proCtsD and its maturation CtsD heavy chain ( hc ) . The plots show the quantification of the indicated proteins normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 , * * p ≤ 0 . 01 . Data information : ( RPE ) Retinal Pigment

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD) and LC3 proteins from WT and miR-211-/- mice. Note a decrease of both proCtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05, **p ≤ 0.01. Data information: (RPE) Retinal Pigment Epithelium


( C ) Cathepsin B ( CtsB ) activity in RPE lysates from 2 - month - old miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . CtsB was reduced in miR - 211 - / - compared to control mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * p ≤ 0 . 05 . Data information : ( RPE ) Retinal Pigment

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

(C) Cathepsin B (CtsB) activity in RPE lysates from 2-month-old miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). CtsB was reduced in miR-211-/- compared to control mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05. Data information: (RPE) Retinal Pigment Epithelium


( D ) Representative images of conventional TEM analysis of RPE of both 2 - month - old and 3 - month - old WT and miR - 211 - / - mice . miR - 211 - / - mice show accumulation of phagolysosomes ( red arrows ) in the RPE . Scale bar 2 μm . Enlarged boxes highlight phagolysosome - like structures containing poorly processed POS . Scale bar 1 . 5 μm . Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin - like granules ( blue arrows ) in 3 - month - old miR - 211 - / - compared to control WT mice . Melanosomes are indicated by white asterisks . Scale bar 500 nm ( n = 3 mice ) . ( E ) Graphs showing number of lipofuscin - like granules ( n / 100 μm2 ) from the RPE of 3 - month - old miR - 211 - / - mice as in ( D ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) * * * p ≤ 0 . 005 ( n = 6 mice ) . Data information : ( RPE ) Retinal Pigment

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

(D) Representative images of conventional TEM analysis of RPE of both 2-month-old and 3-month-old WT and miR-211-/- mice. miR-211-/- mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome-like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin-like granules (blue arrows) in 3-month-old miR-211-/- compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm (n=3 mice). (E) Graphs showing number of lipofuscin-like granules (n/100 μm2) from the RPE of 3-month-old miR-211-/- mice as in (D). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT) ***p ≤ 0.005 (n=6 mice). Data information: (RPE) Retinal Pigment Epithelium


( A ) Representative confocal images of eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old WT mice sacrificed 3h after light on at 10 AM ( diurnal condition - LIGHT ) and 3h after light off at 10 PM ( nocturnal condition - DARK ) , nuclei were counterstained with DAPI ( blue ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

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

(A) Representative confocal images of eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old WT mice sacrificed 3h after light on at 10 AM (diurnal condition - LIGHT) and 3h after light off at 10 PM (nocturnal condition - DARK), nuclei were counterstained with DAPI (blue). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( B ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated on the X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . Mann and Whitney test * p ≤ 0 . 05 ( n = 3 mice for each time point ) . Values from 12h dark - adapted mice ( T0 ) were set to one .

(B) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated on the X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. Mann and Whitney test *p ≤ 0.05 (n=3 mice for each time point). Values from 12h dark-adapted mice (T0) were set to one.


( C ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) and anti - ZO - 1 ( red ) antibodies from 2 - month - old miR - 211 - / - and WT control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Enlarged boxes highlight Ezrin staining in both basal infoldings ( BI ) and apical extension ( AE ) of the RPE . At least n = 6 mice per group . Scale bar 10 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( BI ) RPE basal infoldings ( AE ) RPE apical extensions .

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

(C) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) and anti-ZO-1 (red) antibodies from 2-month-old miR-211-/- and WT control mice sacrificed 3h after light on at 10 AM (diurnal condition). Enlarged boxes highlight Ezrin staining in both basal infoldings (BI) and apical extension (AE) of the RPE. At least n = 6 mice per group. Scale bar 10 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer; (BI) RPE basal infoldings (AE) RPE apical extensions.


( D ) qRT - PCR assay for Ezrin from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after light on at 10 AM . The graph shows the expression level of Ezrin normalized to Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - vs WT ) , * * * p ≤ 0 . 005 ( n = 6 mice ) .

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

(D) qRT-PCR assay for Ezrin from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after light on at 10 AM. The graph shows the expression level of Ezrin normalized to Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT), ***p ≤ 0.005 (n=6 mice).


( E ) RPE were isolated at 10 AM ( diurnal condition ) from 2 - month - old WT and miR - 211 - / - mice . Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR - 211 - / - mice . The graph shows the quantification of Ezrin normalized to the Gapdh loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 5 mice ) Mann and Whitney test ( miR - 211 - / - vs WT ) , * * p ≤ 0 . 01 .

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

(E) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative image at low exposure of Western blot analysis of the Ezrin protein from WT and miR-211-/- mice. The graph shows the quantification of Ezrin normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=5 mice) Mann and Whitney test (miR-211-/- vs WT), **p ≤ 0.01.


( F ) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2 - month - old miR - 211 - / - mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m . ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one . ( G ) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2 - month - old WT mice during dark / light and light / dark transitions . Mice were sacrificed at determined time points as indicated in X axis . Scheme at the top describes light / dark adaptation regime . Values normalized to Hprt , represent means ± s . e . m ( n = 3 mice for each time point ) . Values from 12h dark adapted mice ( T0 ) were set to one .

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

(F) Graph representing the kinetic expression pattern of Ezrin levels in the RPE from 2-month-old miR-211-/- mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m. (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one. (G) Graph representing the kinetic expression pattern of Trpm1 levels in the RPE from 2-month-old WT mice during dark/light and light/dark transitions. Mice were sacrificed at determined time points as indicated in X axis. Scheme at the top describes light/dark adaptation regime. Values normalized to Hprt, represent means ± s.e.m (n=3 mice for each time point). Values from 12h dark adapted mice (T0) were set to one.


( H ) qRT - PCR assay for Mtmr10 and Klf13 from RPE 2 - month - old WT and miR - 211 - / - mice sacrificed 3h after on at 10 AM . The graph shows the expression level of these genes normalized to the Hprt . Bar graphs represent mean values ± s . e . m . ( n = 3 mice )

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

(H) qRT-PCR assay for Mtmr10 and Klf13 from RPE 2-month-old WT and miR-211-/- mice sacrificed 3h after on at 10 AM. The graph shows the expression level of these genes normalized to the Hprt. Bar graphs represent mean values ± s.e.m. (n=3 mice)


( A ) Representative images from ARPE - 19 cells treated with DMSO and NSC668394 . All cells were fixed and stained with anti - LC3 ( green ) and anti - LAMP1 ( red ) antibodies and nuclei were counterstained with DAPI ( blue ) . Scale bar 5 μm . ( B ) The plot shows the quantification of the numbers of LC3 / LAMP1 - positive vesicles per cell ( n ≥ 100 ) . Values represent means ± s . e . m from 3 independent experiments . Student ' s t - test ( NSC668394 vs DMSO ) * * * p ≤ 0 . 005 .

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

(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm. (B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005.


( C ) Representative Western blots of LAMP1 , EZRIN , EZRIN - pT567 , CLN5 , TRPML1 , SQSTM1 / p62 , CTSD and LC3 proteins from DMSO or NSC668394 treated cells . Note an increase of both proCTSD and its maturation CTSD heavy chain ( hc ) . The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control . Bar graphs represent mean values ± s . e . m . of at least n = 6 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.


( D ) Representative images of RFP - GFP - LC3 assay in ARPE - 19 cells transiently transfected with RFP - GFP - LC3 and treated with DMSO or NSC668394 . Nuclei were counterstained with DAPI ( blue ) . Scale bars : 5 μm . ( E ) Box plots showing quantitative analysis of RFP + GFP + puncta ( Autophagosome ) and RFP + GFP - puncta ( Autolysosome ) ( n ≥ 100 cells ) from 3 independent experiments . Box limits represent 25th percentile and 75th percentile ; horizontal lines represent medians ; whiskers display min . to max . values . Student ' s t - test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm. (E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome ) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.


( F ) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B ( CTSB ) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis . Scale bar 10 μm . ( G ) Graph showing the mean values of intensity of cresyl violet fluorescence ± s . e . m . of representative data ( n ≥ 100 cells ) from 3 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( H ) Cathepsin B ( CTSB ) activity in lysates from NSC668394 - treated and DMSO - control ARPE - 19 cells . Graph showing the percentage of values ± s . e . m . of CTSB activity from 4 independent experiments . Mann and Whitney test ( NSC668394 vs DMSO ) * p ≤ 0 . 05 .

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

(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.


( A ) Representative images from HC assay of HeLaTFEB - GFP cells transfected with control ( siCTRL ) or siPPP3CB , and subjected to the indicated conditions . Analysis of TFEB nuclear translocation in HeLaTFEB - GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394 , silenced for EZRIN ( siEZR ) or serum - starved ( stv ) . Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB - GFP cells . Starvation ( stv ) was used as control . Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB . Scale bar 5 μm .

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

(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.


( B ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared to control ; n = 3 independent experiments were performed . Mann and Whitney test ( NSC668394 and stv vs DMSO ; siEZR vs siCTRL ) * p ≤ 0 . 05 .

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(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.


( C ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells and subjected to the silencing of PPP3CB . At least 3 independent experiments were performed . Mann and Whitney test ( siPPP3CB vs siCTRL ) * p ≤ 0 . 05 .

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

(C) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells and subjected to the silencing of PPP3CB. At least 3 independent experiments were performed. Mann and Whitney test (siPPP3CB vs siCTRL) *p ≤ 0.05.


( D ) qRT - PCR assay for PPP3CB from HeLaTFEB - GFP cells transfected with siPPP3CB and siCTRL . The graph shows the reduction in PPP3CB expression level normalized to the Hprt . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( siPPP3CB vs siCTRL ) , * * * p ≤ 0 . 005 ( n = 6 independent experiments ) .

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

(D) qRT-PCR assay for PPP3CB from HeLaTFEB-GFP cells transfected with siPPP3CB and siCTRL. The graph shows the reduction in PPP3CB expression level normalized to the Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (siPPP3CB vs siCTRL), ***p ≤ 0.005 (n=6 independent experiments).


( E ) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis . Torin treatment was used as control .

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(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.


( F ) Representative traces of ML1 - GCaMP3 normalized fluorescence recorded in transiently transfected ARPE - 19 cells . During time - lapse recording , cells were stimulated with ML - SA1 ( 20 µM ) after addition ( arrowhead ) of DMSO or NSC668394 . Where indicated , the specific ML1 inhibitor ML - SI3 ( 10 µM ) was added . Bar graph reports the mean values ± s . e . m . of the time required by fluorescence to decay to half of the ML - SA1 - induced peak . n = 50 cells from 3 independent experiments . * * * p < 0 . 005 by Mann and Whitney test .

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(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.


( G ) Nuclear translocation of TFEB in stable HeLaTFEB - GFP cells subjected to the indicated conditions is reduced after Ca2 + chelator BAPTA treatment . Scale bar 5 μm .

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(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.


H ) The graph shows the mean ± s . e . m . of the percentage of nuclear TFEB translocation in Ezrin - inhibited cells compared with DMSO under Ca2 + chelator BAPTA treatment . At least 3 independent experiments were performed . Mann and Whitney test ( stv , stv + BAPTA , BAPTA , NSC668394 , NSC668394 + BAPTA vs DMSO ) * p ≤ 0 . 05 .

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H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.


( I ) qRT - PCR analysis for TFEB target genes ( TRPML1 , BECLIN , MAPLC3B and LAMP1 ) was performed on ARPE - 19 cells treated with DMSO or NSC668394 . Bar graphs represent mean values ± s . e . m . of at least 3 independent experiments . Mann and Whitney * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 005 .

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

(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.


( A ) Confocal images of representative eye cryosections immunostained with anti - Ezrin ( green ) / anti - ZO - 1 ( red ) , anti - Ezrin - pT567 ( green ) / anti - ZO - 1 ( red ) , anti - Lamp1 ( green ) and anti - Sqstm1 / p62 ( green ) antibodies from 6 - month - old miR - 211 - / - sacrificed five months after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . Data information : ( RPE ) Retinal Pigment Epithelium ; ( OS ) outer segment ; ( ONL ) outer nuclear layer .

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

(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.


( B ) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6 - month - old miR - 211 - / - mice . Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1 , Ezrin , Ezrin - pT567 , Sqstm1 / p62 and LC3 . The plots show the quantification of the indicated proteins normalized to the β - Actin loading control . Bar graphs represent mean values ± s . e . m . of independent experiments ( n = 6 mice ) . Mann and Whitney test ( DMSO vs WT ; NSC668394 vs DMSO ) , * p ≤ 0 . 05 , * * * p ≤ 0 . 005 .

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

(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.


( C ) Cathepsin B ( CtsB ) activity in RPE lysates from miR - 211 - / - and WT mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . RPE was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . CtsB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of CtsB activity ± s . e . m . of independent experiments ( n = 3 mice ) Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) , * p ≤ 0 . 05 . ( D ) GUSB activity from retina from miR - 211 - / - and control mice sacrificed 3h after light on at 10 AM ( diurnal condition ) . Retina was isolated 1 week after DMSO or NSC668394 treatment from 2 - month - old miR - 211 - / - mice . GUSB was rescued in NSC668394 - treated miR - 211 - / - compared to DMSO - control miR - 211 - / - mice . Bar graphs represent percentage of GUSB activity ± s . e . m . of independent experiments ( n = 3 mice ) . Mann and Whitney test ( DMSO - miR - 211 - / - vs WT ; NSC668394 - miR - 211 - / - vs DMSO - miR - 211 - / - ) .

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

(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05. (D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).


( A ) Representative images of conventional TEM analysis of RPE of WT , DMSO - treated miR - 211 - / - mice and NSC668394 - treated miR - 211 - / - three - month - old mice . NSC668394 - treated miR - 211 - / - mice show rescue of accumulation of double - membrane phagolysosome - like structures containing poorly processed POS ( red arrows ) compared to DMSO - treated miR - 211 - / - mice . Scale bar 1 mm . Data information : ( OS ) outer segment ( RPE ) Retinal Pigment Epithelium .

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

(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm. Data information: (OS) outer segment (RPE) Retinal Pigment Epithelium.


( B ) Confocal images of representative eye cryosections immunostained with anti - Rhodopsin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment , which are sacrificed 3h after light on at 10 AM ( diurnal condition ) . Nuclei are counterstained with DAPI ( blue ) . At least n = 6 mice per group . Scale bar 100 μm . The enlarged box highlights rhodopsin accumulation in the RPE ( green spots ) . Scale bar 5 μm . Autofluorescence from lipofuscin granules from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Representative images of retina cryosections immunostained with anti - Cone Arrestin ( green ) antibody from WT and miR - 211 - / - mice at three months of age after DMSO or NSC668394 treatment . Nuclei are counterstained with DAPI ( blue ) . N = at least 6 mice per group . Scale bar 100 μm . Data information : ( OS ) outer segment ; ( ONL ) outer nuclear layer ; ( RPE ) Retinal Pigment Epithelium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm. Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.


( C ) Graphs showing number of lipofuscin granules from the RPE of mice as in ( B ) . Bar graphs represent mean values ± s . e . m . Mann and Whitney test ( miR - 211 - / - DMSO vs WT and miR - 211 - / - NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 . * p ≤ 0 . 05 ( n = 6 mice ) .

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(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).


( D ) Graphs show cone percentage ( cones / area ) from the retina of mice treated as in ( B ) . Error bars represent s . e . m . Mann and Whitney test ( DMSO vs WT and NSC668394 vs DMSO treated mice ) * * * p ≤ 0 . 005 ( n = 6 mice ) .

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(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).