( A ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701 ( green ) , PRC11 - 168 ( pink ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of PRC11 - 168 ( pink ) , MBP - CENP - E2605 - 2701 ( green ) and MBP - CENP - E2605 - 2701 / PRC11 - 168 ( orange ) . A shift in the elution volume was only seen in the presence of both CENP - E and PRC1 . ( B ) Top . Size - exclusion chromatography elution profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of MBP - CENP - E2605 - 2701FDN ( green ) and MBP - CENP - E2605 - 2701 FDN / PRC11 - 168 ( orange ) . No shift in the elution profile was observed .

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(A) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701 (green), PRC11-168 (pink) and MBP-CENP-E2605-2701/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of PRC11-168 (pink), MBP-CENP-E2605-2701 (green) and MBP-CENP-E2605-2701/PRC11-168 (orange). A shift in the elution volume was only seen in the presence of both CENP-E and PRC1. (B) Top. Size-exclusion chromatography elution profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of MBP-CENP-E2605-2701FDN (green) and MBP-CENP-E2605-2701 FDN/PRC11-168 (orange). No shift in the elution profile was observed.


( B ) Representative images of live HeLa cells in mitosis transiently transfected with either GFP - Kif4A1133 - 1165 or GFP - Kif4A1133 - 1232 incubated with SiR - Tubulin . Scale bar , 10μm . Quantification of cells with GFP - Kif4A1133 - 1165 ( n = 12 ) or GFP - Kif4A1133 - 1232 ( n = 13 ) localization to the central spindle . Data represented from 2 independent experiments .

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(B) Representative images of live HeLa cells in mitosis transiently transfected with either GFP-Kif4A1133-1165 or GFP-Kif4A1133-1232 incubated with SiR-Tubulin. Scale bar, 10μm. Quantification of cells with GFP-Kif4A1133-1165 (n=12) or GFP-Kif4A1133-1232 (n=13) localization to the central spindle. Data represented from 2 independent experiments.


( C ) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1 . Size - exclusion chromatography elution profile of GST - Kif4A1133 - 1232 ( green ) and PRC11 - 168 ( pink ) , or together ( orange ) . Bottom , Coomassie - stained gel showing the size - exclusion chromatography profile of GST - Kif4A1133 - 1232 ( green ) , PRC11 - 168 ( pink ) and GST - Kif4A1133 - 1232 / PRC11 - 168 ( orange ) .

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(C) A shift in the elution volume was only seen in the presence of both Kif4A and PRC1. Size-exclusion chromatography elution profile of GST-Kif4A1133-1232 (green) and PRC11-168 (pink), or together (orange). Bottom, Coomassie-stained gel showing the size-exclusion chromatography profile of GST-Kif4A1133-1232 (green), PRC11-168(pink) and GST-Kif4A1133-1232/PRC11-168 (orange).


( A ) Representative images of GFP - PRC1 ( magenta ) mixed with rhodamine - microtubules . Fire blue - green intensity LUT used to show tubulin intensity and microtubule overlaps . ( B ) Representative images of 647GST - CENP - E2639 - 2671 ( yellow ) mixed with rhodamine - microtubules .

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(A) Representative images of GFP-PRC1 (magenta) mixed with rhodamine-microtubules. Fire blue-green intensity LUT used to show tubulin intensity and microtubule overlaps. (B) Representative images of 647GST-CENP-E2639-2671 (yellow) mixed with rhodamine-microtubules.


( D ) Representative immunofluorescence images of HeLa cells stained for DNA , microtubules , PRC1 and with Alexa647 - labelled GST - CENP - E2639 - 2671 showing it also recognizes PRC1 in cells . Experiments were repeated > 3 times .

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(D) Representative immunofluorescence images of HeLa cells stained for DNA, microtubules, PRC1 and with Alexa647-labelled GST-CENP-E2639-2671 showing it also recognizes PRC1 in cells. Experiments were repeated >3 times.


( F ) Representative kymograph showing microtubule - microtubule sliding in the presence of 2 . 5 nM PRC1 and 50 nM full - length CENP - E . ( G ) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall .

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(F) Representative kymograph showing microtubule-microtubule sliding in the presence of 2.5 nM PRC1 and 50 nM full-length CENP-E. (G) Example kymograph showing a free microtubule sliding until reaching the end of the immobilized microtubule where it slows down to a stall.


( D ) Live - cell imaging of metaphase spindles in HeLa cells transiently transfected wild - type , phosphomimetic and non - phosphorylatable mutants of GFP - CENP - E2605 - 2701 ( monomeric ) and GFP - GST - CENP - E2605 - 2701 ( dimeric ) and stained for tubulin using SiR - Tubulin . The fraction of cells localizing to the overlapping microtubules is represented as a percentage . Scalebar , 10 μm .

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(D) Live-cell imaging of metaphase spindles in HeLa cells transiently transfected wild-type, phosphomimetic and non-phosphorylatable mutants of GFP-CENP-E2605-2701 (monomeric) and GFP-GST-CENP-E2605-2701 (dimeric) and stained for tubulin using SiR-Tubulin. The fraction of cells localizing to the overlapping microtubules is represented as a percentage. Scalebar, 10 μm.


( G ) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP - GST - CENP - E2605 - 2701 and mutants at 9 . 7μm , quantified in F . Asterisks indicate ordinary One - way Anova test significance value . * * * * P < 0 . 0001 .

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(G) Bar graph showing mean and standard error for GFP fluorescence intensity at peak fluorescence for GFP-GST-CENP-E2605-2701 and mutants at 9.7μm, quantified in F. Asterisks indicate ordinary One-way Anova test significance value. ****P<0.0001.


( B , C ) Elution profile from a size - exclusion chromatography ( SEC , black line , left y - axis ) run with subsequent multi - angle light scattering ( MALS , right y - axis ) analysis for PRC11 - 168 and PRC11 - 168 MEE . Outcome of the MALS analysis for the peak is presented in pink ( molecular weight , right y - axis ) .

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(B, C) Elution profile from a size-exclusion chromatography (SEC, black line, left y-axis) run with subsequent multi-angle light scattering (MALS, right y-axis) analysis for PRC11-168 and PRC11-168 MEE. Outcome of the MALS analysis for the peak is presented in pink (molecular weight, right y-axis).


( D ) Fire purple - yellow intensity LUT used to show tubulin intensity and microtubule overlaps . Microtubules are shown alone or incubated with 2 . 5 nM full - length PRC1 WT or PRC1 MEE . Scalebar , 10 μm . Experiment was replicated 2 times .

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(D) Fire purple-yellow intensity LUT used to show tubulin intensity and microtubule overlaps. Microtubules are shown alone or incubated with 2.5 nM full-length PRC1 WT or PRC1 MEE. Scalebar, 10 μm. Experiment was replicated 2 times.


( A ) Representative immunofluorescence images of HeLa cells in late anaphase and telophase , expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules , GFP and CENP - E are in white , green and red respectively . DNA is in blue . ( B ) Graph showing the percentage of cells with a PRC1 - localized at the site of abscission for cells treated in ( A ) . Mean and standard deviation are presented , n = 156 and 141 for cells expressing PRC1 - WT and - MEE respectively . Data from 4 biological replicates and 1 technical replicate . Asterisks indicate a T - test significance value . * * * * P < 0 . 0001 . ( C ) Graph showing the percentage of cells with CENP - E localized at the abscission site for cells treated in ( A ) , mean and standard deviation are presented , n = 58 and 66 for cells expressing PRC1 - WT and - MEE respectively . Data from 2 biological replicates . p - value ( p = 0 . 11 ) calculated for an unpaired T - test .

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(A) Representative immunofluorescence images of HeLa cells in late anaphase and telophase, expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules, GFP and CENP-E are in white, green and red respectively. DNA is in blue. (B) Graph showing the percentage of cells with a PRC1-localized at the site of abscission for cells treated in (A). Mean and standard deviation are presented, n= 156 and 141 for cells expressing PRC1-WT and -MEE respectively. Data from 4 biological replicates and 1 technical replicate. Asterisks indicate a T-test significance value. ****P<0.0001. (C) Graph showing the percentage of cells with CENP-E localized at the abscission site for cells treated in (A), mean and standard deviation are presented, n= 58 and 66 for cells expressing PRC1-WT and -MEE respectively. Data from 2 biological replicates. p-value (p=0.11) calculated for an unpaired T-test.


( G ) Representative live - cell images of HeLa cells expressing GFP - PRC1 - WT or - MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline - induced Cas 9 expression . Microtubules ( SiR - Tubulin ) and DNA ( SPY650 ) are shown in red . The cell membrane ( CellMask ) is highlighted in white and GFP - PRC1 is in green . Scalebar , 10 μm .

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(G) Representative live-cell images of HeLa cells expressing GFP-PRC1-WT or -MEE and depleted for endogenous PRC1 using a PRC1 siRNA and a PRC1 sgRNA after doxycycline-induced Cas 9 expression. Microtubules (SiR-Tubulin) and DNA (SPY650) are shown in red. The cell membrane (CellMask) is highlighted in white and GFP-PRC1 is in green. Scalebar, 10 μm.


( J ) Representative immunofluorescence images of cells expressing GFP - PRC1 - WT or - MEE after 72 hours siRNA depletion and doxycycline - induced knockout of endogenous PRC1 . DNA , microtubules and GFP - PRC1 / GFP - PRC1 - MEE are in blue , red and green respectively . Scalebar , 10μm .

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(J) Representative immunofluorescence images of cells expressing GFP-PRC1-WT or -MEE after 72 hours siRNA depletion and doxycycline-induced knockout of endogenous PRC1. DNA, microtubules and GFP-PRC1/GFP-PRC1-MEE are in blue, red and green respectively. Scalebar, 10μm.


G . Endogenous MITOL enhances the K48 - polyubiquitin chains of Parkin . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with CCCP ( 10 µM ) for indicated time . MG132 ( 30 μM ) was added 3 h after CCCP treatment . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IP - IB assay with the indicated antibodies . K48 - polyubiquitin chains were immunoblotted with K48 - specific ubiquitin antibody .

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G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.


E . MITOL regulates mitophagy . MITOL WT HeLa cells , MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0 . 5µg ( + ) and 5µg ( + + ) stably expressing HA ‐ Parkin and mt ‐ Keima were treated with CCCP ( 10µM ) for 8h . Then , mKeima was measured at 488 ( pH 7 ) and 561 ( pH 4 ) nm lasers using Flow Cytometer . Percentages of mitophagy were calculated from 30 , 000 cells in each independent experiment . Data represent the mean ± SD of three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


C . Endogenous MITOL translocates to the ER in later phase of mitophagy . EGFP - MITOL knock in HeLa cells were transfected with HA - Parkin and treated with DMSO or CCCP ( 10 µM ) as indicated times . Cells were fixed , permeabilized and subjected to immunofluorescence analysis with the indicated antibodies . Colocalization was quantified by Manders ' s coefficient . Means ± SEM of more than 10 cells obtained from three independent experiments . For statistical analysis , a one - way ANOVA with Tukey ' s multiple comparisons test were performed , * * * * P < 0 . 0001 . Scale bar represents 1 μm .

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C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.


E . ER - localized MITOL is transported from the mitochondria , and is not newly synthesized protein . HeLa cells stably expressing HA - Parkin were co - transfected with vectors for MITOL - KikGR and FLAG - FKBP38 . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel . Right panel is schematic experimental model for KikGR in this study .

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E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.


C . Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL . WT or MITOL KO HeLa cells stably expressing HA - Parkin were treated with DMSO or CCCP ( 10 µM ) for 48 h . Lysates of cells were fractionated into cytosolic , mitochondrial and ER fractions , and then subjected to an IB assay with the indicated antibodies . Phospho - Ser65 - Parkin is detected by using specific antibody . Right panels are the quantification of IB about Phospho - Ser65 - Parkin protein levels of the ER fraction . The data represent the mean ± SD for three independent experiments . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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

C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


D . Parkin is detected at the ER in the absence of MITOL . HeLa cells were transfected with vectors for KikGR - Parkin . After ultraviolet light ( 365 nm ) exposure to the whole plate , cells were incubated with or without CCCP ( 10 µM ) for indicated time , and monitored for red and green KikGR fluorescence . Scale bar , 10 μm . Higher magnification images of the boxed regions are shown in the small panel .

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D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.


D . MITOL KO increases degradation of ER targeting FKBP38 . HeLa cells stably expressing HA - Parkin were transfected with mitochondrial targeting FLAG - FKBP38 ( FKBP38ActA ) and ER targeting FLAG - FKBP38 ( FKBP38IYFFT ) , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies . Another cell lysates were subsequently separated on a Phos - tag gel , followed by IB with anti - Parkin antibodies ( Phos - tag ) .

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D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).


E - G . MITOL partly prevents CCCP - induced cell death by reducing Parkin - mediated degradation of FKBP38 . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were transfected with the indicated vectors , and treated with DMSO or CCCP ( 10 µM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting the percentage of dead cells using fluorescence microscopy . Data represent the mean ± SD of five independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . KR , FKBP38 K271 / 273R .

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E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.


A . Mislocalization of Bcl - 2 is observed in MITOL KO cells in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin were transfected with FLAG - Bcl2 and treated with DMSO or CCCP ( 10 µM ) for the indicated times . Cells were immunostained with indicated antibody . Scale bar , 1 μm .

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A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.


B . MITOL prevents caspase - dependent cell death in mitophagy . WT or MITOL KO HeLa cells stably expressing HA - Parkin and HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without Z - VAD - FMK ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 . C . Cell death in mitophagy caused by MITOL deficiency depends on calcium leak . WT and MITOL KO HeLa cells stably expressing HA - Parkin or HeLa cells were treated with DMSO or CCCP ( 10 µM ) with or without BAPTA - AM ( 10 μM ) for 24 h . Cells were stained with propidium iodide ( PI ) for counting fluorescence microscopy of the percentage of dead cells . Data represent the mean ± SD of three independent experiments ( > 100 individual cells were counted ) . For statistical analysis , a one - way ANOVA with Tukey post - test was performed , * * P < 0 . 01 .

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B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.


( E ) Northern blot analysis of circBtnl1 in different tissues of two - month - old mice , 18S RNA served as a loading control .

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(E) Northern blot analysis of circBtnl1 in different tissues of two-month-old mice, 18S RNA served as a loading control.


( F ， G ) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues ( F ) and organoid ( G ) . Green region shows the distribution of circBtnl1 using antisense probe , gray region shows the nuclei staining by DAPI . For ( F ) ， scale bars ， 5um ; for ( G ) ， scale bars ， 20 um .

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(F， G) RNA fluorescence in situ hybridization of circBtnl1 in small intestine tissues (F) and organoid (G). Green region shows the distribution of circBtnl1 using antisense probe, gray region shows the nuclei staining by DAPI. For (F)，scale bars，5um; for (G)，scale bars，20 um.


( C ) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1 + / + and circBtnl1 - / - mice . For all representative images , at least three independent experiments were performed with similar results . For every microscope field , average Ki67 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(C) Representative images of Ki67 staining of in small intestinal tissues from circBtnl1+/+ and circBtnl1-/- mice. For all representative images, at least three independent experiments were performed with similar results. For every microscope field, average Ki67+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( E ) H & E staining of small intestine tissues from indicated mice at different time points ( D0 - D7 ) after 8 Gy radiation damage . Number of crypts were calculated as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . n = 5 fields were calculated for each group . Scale bar , 50 μm .

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(E) H&E staining of small intestine tissues from indicated mice at different time points (D0 - D7) after 8 Gy radiation damage. Number of crypts were calculated as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel. n = 5 fields were calculated for each group. Scale bar, 50 μm.


( G ) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm . Three independent experiments were performed with similar results , and representative experiments are shown . For every microscope field , average Olmf4 + ISC numbers per crypt are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test in right panel in right panel .

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(G) Confocal microscopy of ISCs makers Olmf4 in organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm. Three independent experiments were performed with similar results, and representative experiments are shown. For every microscope field, average Olmf4+ ISC numbers per crypt are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test in right panel in right panel.


( B , C ) CircBtnl1 was co - localized with Ddx3y protein in small intestine tissue ( B ) and organoid ( C ) by RNA FISH , followed by immunofluorescence staining . Scale bars , 5 μm .

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(B, C) CircBtnl1 was co-localized with Ddx3y protein in small intestine tissue (B) and organoid (C) by RNA FISH, followed by immunofluorescence staining. Scale bars, 5 μm.


( G ) Electrophoretic mobility shift assay ( EMSA ) using biotin - labeled circBtnl1 and recombinant Ddx3y .

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(G) Electrophoretic mobility shift assay (EMSA) using biotin-labeled circBtnl1 and recombinant Ddx3y.


( H ) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates , followed by RNA pulldown assay and Western blot .

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(H) Truncated fragments of biotinylated circBtnl1 were incubated with small intestine crypts lysates, followed by RNA pulldown assay and Western blot.


( B ) Small intestine crypt cells were isolated from circBtnl1 + / + and circBtnl1 - / - two - month - old mice , followed by RNA extraction and q - PCR analysis for top ten upregulated transcription factors . n = 3 independent samples . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 by two - tailed Student ' s t - test .

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(B) Small intestine crypt cells were isolated from circBtnl1+/+ and circBtnl1-/- two-month-old mice, followed by RNA extraction and q-PCR analysis for top ten upregulated transcription factors. n = 3 independent samples. Results of relative fold changes are shown as means ± SD. **P < 0.01 by two-tailed Student's t-test.


( D ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 sense , antisense and control probes in mice ISCs . ( E ) Immunoblotting analysis of Ddx3y in RNA pull - down samples by Atf4 and control probes in circBtnl1 + / + and circBtnl1 - / - mice ISCs .

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(D) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 sense, antisense and control probes in mice ISCs. (E) Immunoblotting analysis of Ddx3y in RNA pull-down samples by Atf4 and control probes in circBtnl1+/+ and circBtnl1-/- mice ISCs.


( J ) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 20 μm .

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(J) Confocal microscopy of ATF4 in Ddx3y knockdown and overexpression organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 20 μm.


( K , L ) Expression levels of Ddx3y and Atf4 in circBtnl1 + / + and circBtnl1 - / - ISCs were examined by real - time PCR ( K ) and Western blot ( L ) , three independent experiments were performed . Results of relative fold changes are shown as means ± SD . * * P < 0 . 01 , NS , not significant , by two - tailed Student ' s t - test .

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(K, L) Expression levels of Ddx3y and Atf4 in circBtnl1+/+ and circBtnl1-/- ISCs were examined by real-time PCR (K) and Western blot (L), three independent experiments were performed. Results of relative fold changes are shown as means ± SD. ** P < 0.01, NS, not significant, by two-tailed Student's t-test.


( D , E ) ChIP assay was performed using anti - H3K4me3 ( D ) or anti - H3K27me3 ( E ) with circBtnl1 + / + and circBtnl1 - / - ISCs lysates . * * P < 0 . 01 by two - tailed Student ' s t - test . Data are representative of at least three independent experiments .

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(D, E) ChIP assay was performed using anti-H3K4me3 (D) or anti-H3K27me3 (E) with circBtnl1+/+ and circBtnl1-/- ISCs lysates. **P < 0.01 by two-tailed Student's t-test. Data are representative of at least three independent experiments.


( H ) Confocal microscopy of Sox9 in Atf4 knockdown ( Atf4 KD ) organoids from circBtnl1 + / + and circBtnl1 - / - mice . Scale bars , 30 μm .

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(H) Confocal microscopy of Sox9 in Atf4 knockdown (Atf4 KD) organoids from circBtnl1+/+ and circBtnl1-/- mice. Scale bars, 30 μm.


( D ) H & E staining of small intestinal tissues from indicated mice . Two - month - old mice were used and n = 3 fields were taken for each group . Scale bar , 100 μm . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel .

(D) H&E staining of small intestinal tissues from indicated mice. Two-month-old mice were used and n = 3 fields were taken for each group. Scale bar, 100 μm. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel.


( E ) H & E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage . Two - month - old mice were used and n = 3 fields were taken for each group . Number of crypts were calculated as means ± SD . * P < 0 . 05 , * * P < 0 . 01 by two - tailed Student ' s t - test in right panel . Scale bar , 50 μm .

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(E) H&E staining of intestinal tissues from indicated mice at different time points after 8 Gy of radiation damage. Two-month-old mice were used and n = 3 fields were taken for each group. Number of crypts were calculated as means ± SD. *P < 0.05, **P < 0.01 by two-tailed Student's t-test in right panel. Scale bar, 50 μm.


( F ) Representative organoid images of circBtnl1 + / + ; sgSox9 and circBtnl1 - / - ; sgSox9 mice , sgCtrl served as control . Organoid formation ratios ( n = 3 ) are shown . Organoid formation ratios are shown as mean ± s . d . * P < 0 . 05 by two - tailed Student ' s t - test . Scale bars , 200 μm .

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(F) Representative organoid images of circBtnl1+/+; sgSox9 and circBtnl1-/-; sgSox9 mice, sgCtrl served as control. Organoid formation ratios (n = 3) are shown. Organoid formation ratios are shown as mean ± s. d. *P < 0.05 by two-tailed Student's t-test. Scale bars, 200 μm.


( a ) Kaplan - Meier survival curves of combined male and female WT and Atg5Tg mice no . 25 ( P0 . 001 ; n = 65 for WT mice and n = 70 for Atg5Tg mice ) . ( b , c ) Lifespan extension in male Atg5Tg mice no . 25 ( P0 . 001 ; n = 32 for WT mice and n = 36 for Atg5Tg mice no . 25 ) ( b ) and female Atg5Tg mice no . 25 ( P0 . 001 ; n = 33 for WT mice and n = 34 for Atg5Tg mice no . 25 ) ( c ) . ( d - f ) Kaplan - Meier survival curves of combined male and female WT and Atg5Tg mice no . 43 ( d ) , no . 47 ( e ) and no . 471 ( f ) ( P0 . 001 ; n = 25 for WT mice and n = 30 for Atg5Tg mice ) . P - values were calculated using the log - rank test .

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(a) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 25 (P0.001; n=65 for WT mice and n=70 for Atg5Tg mice). (b,c) Lifespan extension in male Atg5Tg mice no. 25 (P0.001; n=32 for WT mice and n=36 for Atg5Tg mice no. 25) (b) and female Atg5Tg mice no. 25 (P0.001; n=33 for WT mice and n=34 for Atg5Tg mice no. 25) (c). (d-f) Kaplan-Meier survival curves of combined male and female WT and Atg5Tg mice no. 43 (d), no. 47 (e) and no. 471 (f) (P0.001; n=25 for WT mice and n=30 for Atg5Tg mice). P-values were calculated using the log-rank test.


( g ) Expression levels of Atg5 and Atg12 - Atg5 conjugate , the conversion of LC3 I into LC3 II , and p62 level in the hearts of Atg5 Tg mice . Whole - tissue extracts were prepared from 3 - month - old Atg5 Tg mice and WT littermates , and analysed by western blotting using the indicated antibodies . β - Actin served as a control . ( h ) Densitometric analysis of the signals on the blots shown in ( g ) . The bars represent the mean ± s . d . ( n = 3 ) .

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(g) Expression levels of Atg5 and Atg12-Atg5 conjugate, the conversion of LC3 I into LC3 II, and p62 level in the hearts of Atg5 Tg mice. Whole-tissue extracts were prepared from 3-month-old Atg5 Tg mice and WT littermates, and analysed by western blotting using the indicated antibodies. β-Actin served as a control. (h) Densitometric analysis of the signals on the blots shown in (g). The bars represent the mean±s.d. (n=3).


( a ) Western blot analysis showing enhanced levels of Atg5 , Atg12 - Atg5 conjugate and LC3 conversion , and reduced p62 levels in the indicated tissues of 18 - month - old Atg5 Tg mice no . 25 ( T ) and WT mice ( W ) . ( b ) Densitometric analysis of the signals on the blots shown in ( a ) .

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(a) Western blot analysis showing enhanced levels of Atg5, Atg12-Atg5 conjugate and LC3 conversion, and reduced p62 levels in the indicated tissues of 18-month-old Atg5 Tg mice no. 25 (T) and WT mice (W). (b) Densitometric analysis of the signals on the blots shown in (a).


( c ) Electron microscopy images of autophagosomes ( arrows ) and late - autophagic compartments ( arrowheads ) in the liver tissues of 18 - month - old Atg5 Tg mice no . 25 and WT mice . Scale bars are indicated . ( d ) The numbers of autophagosomes and autolysosomes per cell ( n = 50 ) in the electron microscopic images shown in ( c ) were counted . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(c) Electron microscopy images of autophagosomes (arrows) and late-autophagic compartments (arrowheads) in the liver tissues of 18-month-old Atg5 Tg mice no. 25 and WT mice. Scale bars are indicated. (d) The numbers of autophagosomes and autolysosomes per cell (n=50) in the electron microscopic images shown in (c) were counted. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( e ) Fluorogenic comparison of basal autophagy activity between GFP - LC3 Tg mice and Atg5 / GFP - LC3 double Tg mice . Whole bodies of 8 - week - old male ( left ) and female ( right ) GFP - LC3 Tg ( LC3 ) , and LC3 / LC3 - LC3Tg mice ( Atg5 / LC3 ) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC ( Kodak Molecular Imaging Software , Version 4 . 0 ) .

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(e) Fluorogenic comparison of basal autophagy activity between GFP-LC3 Tg mice and Atg5/GFP-LC3 double Tg mice. Whole bodies of 8-week-old male (left) and female (right) GFP-LC3 Tg (LC3), and LC3/LC3-LC3Tg mice (Atg5/LC3) were visualized using a Kodak Image Station 4000MM equipped with a filter set for FITC (Kodak Molecular Imaging Software, Version 4.0).


( f ) Western blot analysis showing autophagy markers in the liver of GFP - LC3 mice . GFP - LC3 mice were injected for 5 days with siRNA - Atg5 or Atg5 - HA cDNA through the tail vein . Whole - liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies . β - Actin served as a control .

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(f) Western blot analysis showing autophagy markers in the liver of GFP-LC3 mice. GFP-LC3 mice were injected for 5 days with siRNA-Atg5 or Atg5-HA cDNA through the tail vein. Whole-liver tissue lysates were prepared and subjected to immunoblotting using the indicated antibodies. β-Actin served as a control.


( a ) Representative physical pictures of 18 - month - old WT and Atg5 Tg mice fed with a regular diet . ( b ) Body weight chart of ageing - induced obesity in WT and Atg5 Tg mice . The mice were fed with a regular diet for 24 months ( n = 180 - - 250 ) and their body weights were monitored monthly . The bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(a) Representative physical pictures of 18-month-old WT and Atg5 Tg mice fed with a regular diet. (b) Body weight chart of ageing-induced obesity in WT and Atg5 Tg mice. The mice were fed with a regular diet for 24 months (n=180--250) and their body weights were monitored monthly. The bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


. ( c ) Average daily oral food intake normalized to body weight , measured on regular diet . The food intake of mice ( n = 18 - 24 ) was measured for 2 weeks at 3 - , 6 - , 12 - and 18 - month - old age .

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. (c) Average daily oral food intake normalized to body weight, measured on regular diet. The food intake of mice (n=18-24) was measured for 2 weeks at 3-, 6-, 12- and 18-month-old age.


d ) Quantification of gonadal fat pads from 18 - month - old mice ( n = 11 ) . * * * P0 . 0001 versus control ; Student ' s t - test . ( e , f ) Quantification of visceral ( e ) and subcutaneous ( f ) fat pads of 18 - month - old mice ( n = 6 ) . * * * P0 . 0001 versus control ; Student ' s t - test .

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d) Quantification of gonadal fat pads from 18-month-old mice (n=11). ***P0.0001 versus control; Student's t-test. (e,f) Quantification of visceral (e) and subcutaneous (f) fat pads of 18-month-old mice (n=6). ***P0.0001 versus control; Student's t-test.


( g ) Enhanced glucose tolerance of Atg5 Tg mice . WT and Atg5 Tg mice were starved overnight and then given with an i . p injection of glucose ( 1 mg g − 1 body weight ) . The results are the mean ± s . d . of 12 - week - old WT and Atg5 Tg mice ( n = 12 ) . * P0 . 01 , * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(g) Enhanced glucose tolerance of Atg5 Tg mice. WT and Atg5 Tg mice were starved overnight and then given with an i.p injection of glucose (1 mg g−1 body weight). The results are the mean±s.d. of 12-week-old WT and Atg5 Tg mice (n=12). *P0.01, **P0.001, ***P0.0001 versus control; Student's t-test.


( h ) Enhanced insulin sensitivity of Atg5 Tg mice . WT and Atg5 Tg mice were starved for 6 h and then i . p injected with 0 . 75 IU soluble insulin . * P0 . 01 , * * P0 . 001 versus control ; Student ' s t - test .

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(h) Enhanced insulin sensitivity of Atg5 Tg mice. WT and Atg5 Tg mice were starved for 6 h and then i.p injected with 0.75 IU soluble insulin. *P0.01, **P0.001 versus control; Student's t-test.


( i ) Plasma levels of triglycerol ( TG ) in WT and Atg5 Tg mice . The mice were fasted for 3 h before measurement . Young mice ( 4 - week old , n = 8 ) ; old mice ( 18 - month old , n = 8 ) . * * * P0 . 0001 versus control ; Student ' s t -

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(i) Plasma levels of triglycerol (TG) in WT and Atg5 Tg mice. The mice were fasted for 3 h before measurement. Young mice (4-week old, n=8); old mice (18-month old, n=8). ***P0.0001 versus control; Student's t-test


( j ) Plasma leptin levels in WT and Atg5 Tg mice ( n = 8 - 12 ) . * * * P0 . 0001 versus control ; Student ' s t - test . (

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(j) Plasma leptin levels in WT and Atg5 Tg mice (n=8-12). ***P0.0001 versus control; Student's t-test. (


( k ) GSH / GSSG ratio in the liver ( n = 4 males , three times trial ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(k) GSH/GSSG ratio in the liver (n=4 males, three times trial). **P0.001, ***P0.0001 versus control; Student's t-test.


( l ) Respiration rates of mitochondria in WT and Atg5 Tg MEFs ( n = 2 ) .

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(l) Respiration rates of mitochondria in WT and Atg5 Tg MEFs (n=2).


( m ) Wire - hang endurance test of Atg5 Tg and WT mice ( n = 10 ) . All the bars represent the mean ± s . d . * * * P0 . 0001 versus control ; Student ' s t - test .

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(m) Wire-hang endurance test of Atg5 Tg and WT mice (n=10). All the bars represent the mean±s.d. ***P0.0001 versus control; Student's t-test.


( a ) Comparison of Atg12 - Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis . MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13 , after which cell extracts were examined by western blot analysis using anti - Atg5 and anti - β - actin antibodies .

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(a) Comparison of Atg12-Atg5 conjugate levels in WT and Atg5 Tg MEFs by western blot analysis. MEFs from WT and Atg5 Tg mice were cultured at embryonic day 13, after which cell extracts were examined by western blot analysis using anti-Atg5 and anti-β-actin antibodies.


( b ) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no . 6 by rapamycin and / or Baf . A1 . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf . A1 , and cell extracts were then analysed by western blotting . The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β - actin .

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(b) Increased conversion of LC3 and reduced expression of p62 in Atg5 Tg MEFs no. 6 by rapamycin and/or Baf.A1. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 in passage number 3 were incubated for 48 h with 10 μM rapamycin in the absence or presence of Baf.A1, and cell extracts were then analysed by western blotting. The signals on the blot were quantified by densitometric analysis and represented as the ratio of p62 to β-actin.


( c , d ) Increased resistance of Atg5 Tg MEFs to oxidative stress . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated for 24 h with 50 , 100 , 200 and 300 μM H2O2 ( c ) or 300 μM H2O2 in the absence or presence of 5 mM 3 - MA or 20 nM Baf . A1 ( Baf . A1 ) ( d ) . Cell viability was assessed by propidium iodide staining . The values are the mean ± s . e . m . ( n = 3 ) . * * P0 . 001 , * * * P0 . 0001 versus control ; Student ' s t - test .

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(c,d) Increased resistance of Atg5 Tg MEFs to oxidative stress. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated for 24 h with 50, 100, 200 and 300 μM H2O2 (c) or 300 μM H2O2 in the absence or presence of 5 mM 3-MA or 20 nM Baf.A1 (Baf.A1) (d). Cell viability was assessed by propidium iodide staining. The values are the mean±s.e.m. (n=3). **P0.001, ***P0.0001 versus control; Student's t-test.


( e ) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress . Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope . Scale bars , 50 μm .

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(e) Representative photographs showing the resistance of Atg5 Tg MEFs to oxidative stress. Primary cultured WT and Atg5 Tg MEFs were treated with 300 μM H2O2 for 24 h and then observed under a light microscope. Scale bars, 50 μm.


( f ) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs . WT MEFs no . 1 and Atg5 Tg MEFs no . 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf . A1 , and cell extracts were analysed by western blotting . About 10 and 30 μg of proteins were used for LC3 ( upper panel ) and p62 ( bottom panel ) detection , respectively . β - Actin served as a control .

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(f) Effect of oxidative stress on the autophagy activity in Atg5 Tg MEFs. WT MEFs no. 1 and Atg5 Tg MEFs no. 6 were treated with 100 μM H2O2 for 24 h in the presence or absence of 20 nM Baf.A1, and cell extracts were analysed by western blotting. About 10 and 30 μg of proteins were used for LC3 (upper panel) and p62 (bottom panel) detection, respectively. β-Actin served as a control.


Representative images for the TIF analysis shown in ( C ) . Shown are POT1Y223C / - hESCs and an unedited wild - type hESC clone ( WT / WT ) isolated in parallel , as well as a POT1 WT / Q623H hESC and a contemporaneously isolated hECS clones ( WT / WT ) . Scale bars = 10µm . Box and whisker plot ( showing min , max and average ) quantifying telomere - dysfunction induced foci ( TIF ) in two POT1 Y223C / - hESC lines , one POT1 Q623H / Q623H , and one POT1WT / Q623H hESC lines compared to wild - type unedited control hESCs . Cells were analyzed 3 months post targeting . Cells were stained for TRF1 and γ - H2Ax . POT1Y223C / - cells have increased TIFs compared to an unedited wild - type cell clone , isolated and cultured in parallel ( P < 0 . 01 , two - tailed Mann - Whitney test , one biological replicate ) .

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Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).


Images of telomere and centromere FISH on metaphase spreads of cell lines as in ( B ) . Cy3 Tel - C PNA probes were used . Arrows indicate chromosomal abnormalities . E ) Quantification of chromosomal abnormalities as determined by FISH analysis in ( D ) .

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Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).


B ) Quantification of telomere length data from two STELA experiments using the same genomic DNA , shown in ( A ) and ( Appendix Figure 2C ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


B ) TIF analysis of wild - type , POT1WT / Y223C hESCs , and the indicated controls using antibodies against TRF1 and γ - H2AX . Scale bars = 10µm . Lentiviral expression of TPP1 ∆ PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response . TPP1 ∆ PBD infected cells were analyzed 72h post infection and without selection .

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B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.


E ) Quantification of two STELA experiments , including that shown in ( D ) . Indicated in red is the mean telomere length and the error bars indicate the standard deviation .

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E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


D ) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

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D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


C ) Representative genotyping outcomes of hCD34 + cells nucleofected sgRNA - 1 , xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS . Data information : The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


B ) Telomere restriction fragment assay for the five subjects shown in ( A ) . Mean telomere length is indicated .

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B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


( A ) Representative micrographs of human skin sections stained with hematoxylin - eosin ( H & E ) ( left ) and anti - IL - 38 antibody ( right ) from normal patients ( n = 11 ) and tumors of cSCC patients ( n = 13 ) . Scale bars represent 100 μm . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . ( B ) Relative expression of IL - 38 in human normal tissues ( n = 9 ) and cSCC ( n = 18 ) were analyzed using Geo Datasets ( GSE98767 ) . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(A) Representative micrographs of human skin sections stained with hematoxylin-eosin (H&E) (left) and anti-IL-38 antibody (right) from normal patients (n = 11) and tumors of cSCC patients (n = 13). Scale bars represent 100 μm. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. (B) Relative expression of IL-38 in human normal tissues (n = 9) and cSCC (n = 18) were analyzed using Geo Datasets (GSE98767). Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( C and D ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 32 weeks to induce skin tumors . ( C ) Representative micrographs of mouse normal skin ( n = 6 ) and tumor ( n = 6 ) sections stained with anti - IL - 38 antibody . The graph shows the quantification of mean IL - 38 expression per high - powered field in tissues . Scale bars represent 100 μm . ( D ) Relative expression levels of Il - 38 in normal skin ( n = 5 ) and tumors ( n = 5 ) of mice were quantified using qPCR . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(C and D) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 32 weeks to induce skin tumors. (C) Representative micrographs of mouse normal skin (n = 6) and tumor (n = 6) sections stained with anti-IL-38 antibody. The graph shows the quantification of mean IL-38 expression per high-powered field in tissues. Scale bars represent 100 μm. (D) Relative expression levels of Il-38 in normal skin (n = 5) and tumors (n = 5) of mice were quantified using qPCR. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( F ) Representative western blot bands indicating IL - 38 in mouse normal skin ( n = 3 ) and DMBA / TPA - induced tumors ( n = 3 ) . The graph shows the quantification of mean IL - 38 expression in tissues . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(F) Representative western blot bands indicating IL-38 in mouse normal skin (n = 3) and DMBA/TPA-induced tumors (n = 3). The graph shows the quantification of mean IL-38 expression in tissues. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( B ) Representative photographs of Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA for 32 weeks . White triangles indicate the tumors on the back of mice . ( C Average tumor number ( C ) per mouse from Il - 38f / f ( n = 12 ) and K14Cre / + - Il - 38f / f mice ( n = 8 ) treated with DMBA / TPA . . Error bars represent the mean ± SD ( C ) All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test (

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(B) Representative photographs of Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA for 32 weeks. White triangles indicate the tumors on the back of mice. (C Average tumor number (C) per mouse from Il-38f/f (n = 12) and K14Cre/+-Il-38f/f mice (n = 8) treated with DMBA/TPA.. Error bars represent the mean ± SD (C) All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test (C


( A ) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f ( n = 5 ) mice after treated with DMBA for 24h . Red triangles indicate the γH2AX + positive cells . Scale bars represent 100 μm . The graph shows the number of γH2AX + cells per high - powered field . Data information : Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test .

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(A) Representative immunohistochemical staining micrographs of γH2AX in the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f (n = 5) mice after treated with DMBA for 24h. Red triangles indicate the γH2AX+ positive cells. Scale bars represent 100 μm. The graph shows the number of γH2AX+ cells per high-powered field. Data information: Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test.


( A The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( A ) Representative micrograph sections stained with hematoxylin - eosin ( H & E ) from the skin of Il - 38f / f ( n = 5 ) and K14Cre / + - Il - 38f / f mice ( n = 5 ) Scale bars represent 100 μm . The graph shows average epidermal thickness .

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(A The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (A) Representative micrograph sections stained with hematoxylin-eosin (H&E) from the skin of Il-38f/f (n = 5) and K14Cre/+-Il-38f/f mice (n = 5) Scale bars represent 100 μm. The graph shows average epidermal thickness.


B ) The dorsal hair of normal C57 / BL6 mice were shaved and treated with DMBA / TPA twice a week for 3 weeks to induce the skin inflammation . ( B ) Representative micrograph sections stained with anti - Ki67 antibody from the skin of Il - 38f / f ( n = 6 ) and K14Cre / + - Il - 38f / f mice ( n = 6 ) . Scale bars represent 100 μm . The graph shows average numbers of Ki67 + cells per high - powered field .

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B) The dorsal hair of normal C57/BL6 mice were shaved and treated with DMBA/TPA twice a week for 3 weeks to induce the skin inflammation. (B) Representative micrograph sections stained with anti-Ki67 antibody from the skin of Il-38f/f (n = 6) and K14Cre/+-Il-38f/f mice (n = 6). Scale bars represent 100 μm. The graph shows average numbers of Ki67+ cells per high-powered field.


( G and H ) Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) after overexpression ( G ) or knockdown ( H ) of IL - 38 . Error bars represent the mean ± SD . All data are biological replicates . * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using Student ' s t test . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

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(G and H) Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) after overexpression (G) or knockdown (H) of IL-38. Error bars represent the mean ± SD. All data are biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test. NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( I and J ) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL - 38 overexpression ( I ) or knockdown ( J ) . NC , Negative control ; LV , Lentivirus ; Oe , Overexpression .

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(I and J) Representative western blot bands indicating cyclin E and cyclin A in A431 cells after IL-38 overexpression (I) or knockdown (J). NC, Negative control; LV, Lentivirus; Oe, Overexpression.


( A and B ) Pull - down assay of IL - 38 ( 1 - 152 aa ) ( A ) or IL - 38 ( 20 - 152 aa ) ( B ) with IL - 1Rrp2 - Fc , IL - 1RAPL1 - Fc , IL1RAcp - Fc , and IgG - Fc fusion proteins .

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(A and B) Pull-down assay of IL-38 (1-152 aa) (A) or IL-38 (20-152 aa) (B) with IL-1Rrp2-Fc, IL-1RAPL1-Fc, IL1RAcp-Fc, and IgG-Fc fusion proteins.


( C ) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector ( left ) or an expression vector encoding IL - 1Rrp2 ( right ) were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) or IL - 36β ( 200 ng / ml ) at different times .

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(C) Cell extracts prepared from 293T cells transfected with a mock mammalian expression vector (left) or an expression vector encoding IL-1Rrp2 (right) were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) or IL-36β (200 ng/ml) at different times.


( D ) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 at different concentrations .

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

(D) Cell extracts prepared from 293T cells transfected with an expression vector encoding IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 at different concentrations.


( F ) Cell extracts prepared from 293T cells transfected with HA - tagged full - length or deletion mutants of IL - 1Rrp2 , were probed using anti - HA or anti - β - actin antibodies . Oe , overexpression .

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

(F) Cell extracts prepared from 293T cells transfected with HA-tagged full-length or deletion mutants of IL-1Rrp2, were probed using anti-HA or anti-β-actin antibodies. Oe, overexpression.


( G ) Cell extracts prepared from 293T cells transfected with full - length or deletion mutants of IL - 1Rrp2 were probed using anti - phospho - JNK , anti - JNK , or anti - β - actin antibodies after stimulation with IL - 38 ( 200 ng / ml ) at different times .

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 B-MEASURED_VAR O O O B-MEASURED_VAR 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

(G) Cell extracts prepared from 293T cells transfected with full-length or deletion mutants of IL-1Rrp2 were probed using anti-phospho-JNK, anti-JNK, or anti-β-actin antibodies after stimulation with IL-38 (200 ng/ml) at different times.


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after IL - 1Rrp2 interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

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

Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after IL-1Rrp2 interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Representative images of the scratch assay ( left ) and wound closure rate ( right ) of A431 cells ( n = 5 ) treated with recombinant IL - 38 ( 200 ng / mL ) ( F ) after JNK interference . Error bars represent the mean ± SD . All data are biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; p values were calculated using one - way ANOVA ,

O O O 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 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

Representative images of the scratch assay (left) and wound closure rate (right) of A431 cells (n = 5) treated with recombinant IL-38 (200 ng/mL) (F) after JNK interference. Error bars represent the mean ± SD. All data are biological replicates. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using one-way ANOVA , F


Expression of Tardbp , total Sort1 and exon 17b Sort1 mRNAs quantified by Q - PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses . Values were first normalized to Gapdh mRNA levels , then plotted as average ± SEM relative to the value of scrambled shRNA ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Expression of Tardbp, total Sort1 and exon 17b Sort1 mRNAs quantified by Q-PCR in primary hippocampal neurons infected with either scrambled or Tardbp shRNA lentiviruses. Values were first normalized to Gapdh mRNA levels, then plotted as average ± SEM relative to the value of scrambled shRNA (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of cell lysates and culture supernatants showing TDP - 43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Uninfected cultures were used as an additional control . β - actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants , respectively . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Western blots of cell lysates and culture supernatants showing TDP-43 and Sortilin expression in primary hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Uninfected cultures were used as an additional control. β-actin immunoblotting and Coomasise Blue staining were used to control equal loading of lysates and supernatants, respectively. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses , immunolabled for BDNF ( green ) , SCG2 ( red ) and counterstained with DAPI ( blue ) in orthogonal views ( dashed lines ) showing colocalization of BDNF and SCG2 in the soma ( white arrows ) .

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Representative photomicrographs of cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses, immunolabled for BDNF (green), SCG2 (red) and counterstained with DAPI (blue) in orthogonal views (dashed lines) showing colocalization of BDNF and SCG2 in the soma (white arrows).


Colocalization between BDNF and SCG2 - positive vesicles expressed as percentage of total BDNF immunoreactivity ± SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Colocalization between BDNF and SCG2-positive vesicles expressed as percentage of total BDNF immunoreactivity ±SEM in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Colocalization between BDNF and SCG2 - positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF / SCG2 colocalization expressed as percentage of total BDNF immunoreactivity ( N = 3 independent experiments , n > 50 individual cells per condition in each experiment ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Colocalization between BDNF and SCG2-positive vesicles in hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses together with either EGFP or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF/SCG2 colocalization expressed as percentage of total BDNF immunoreactivity (N=3 independent experiments, n>50 individual cells per condition in each experiment). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion induced by KCl treatment assessed by in - situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * , p < 0 . 05 ; * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

BDNF secretion induced by KCl treatment assessed by in-situ BDNF ELISA in cultured hippocampal neurons infected with scrambled or Tardbp shRNA lentiviruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). *, p<0.05; ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


BDNF secretion in cultured hippocampal neurons infected with scrambled ( left ) or Tardbp ( right ) shRNA lentiviruses together with either EGFP , Sort1 shRNA or Sortilin rescue viruses . Histogram bars show average ± SEM BDNF levels normalized to a standard curve ( N = 3 independent experiments ; n = 3 wells per condition in each experiment ) . * * * , p < 0 . 001 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

BDNF secretion in cultured hippocampal neurons infected with scrambled (left) or Tardbp (right) shRNA lentiviruses together with either EGFP, Sort1 shRNA or Sortilin rescue viruses. Histogram bars show average ± SEM BDNF levels normalized to a standard curve (N=3 independent experiments; n=3 wells per condition in each experiment). ***, p<0.001. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrographs of TDP - 43 ( green ) and NeuN ( red ) immunohistochemistry in hippocampal CA1 of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Arrows denote areas of depleted TDP - 43 immunoreactivity in the mutant .

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Representative photomicrographs of TDP-43 (green) and NeuN (red) immunohistochemistry in hippocampal CA1 of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Arrows denote areas of depleted TDP-43 immunoreactivity in the mutant.


Percentage of TDP - 43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus ( HC [ - CA1 ] ) of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Percentage of TDP-43 positive cells relative to NeuN cells in CA1 and the remaining hippocampus (HC [-CA1]) of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


TDP - 43 intensity in TDP - 43 / NeuN double - positive cells in CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition ; 5 sections per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

TDP-43 intensity in TDP-43/NeuN double-positive cells in CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition; 5 sections per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of Tardbp mRNA in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SEM relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

O O B-MEASURED_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 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

Expression of Tardbp mRNA in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SEM relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Western blots of micro - dissected CA1 and the remaining hippocampus showing expression of TDP - 43 , full - length and truncated ( soluble ) Sortilin , and GAPDH in Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Bands corresponding to soluble Sortilin are indicated ( black arrows ) . GAPDH immunoblotting was used to control for equal loading . Molecular weights are indicated in kDa . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Western blots of micro-dissected CA1 and the remaining hippocampus showing expression of TDP-43, full-length and truncated (soluble) Sortilin, and GAPDH in Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Bands corresponding to soluble Sortilin are indicated (black arrows). GAPDH immunoblotting was used to control for equal loading. Molecular weights are indicated in kDa. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


TDP - 43 expression in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TDP-43 expression in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of exon 17b Sort1 and total Sort1 mRNAs in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice quantified by Q - PCR . Results are presented as average ± SD relative to levels in Tardbpfx / fx mice , after normalization to Gapdh mRNA . ( N = 3 mice per condition ; * * , p < 0 . 01 Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Expression of exon 17b Sort1 and total Sort1 mRNAs in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice quantified by Q-PCR. Results are presented as average ± SD relative to levels in Tardbpfx/fx mice, after normalization to Gapdh mRNA. (N=3 mice per condition; **, p<0.01 Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Expression of full - length ( left ) and truncated ( soluble , right ) Sortilin protein in micro - dissected CA1 and the remaining hippocampus of Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SD of densitometry relative to GAPDH ( N = 3 mice per condition ; * * , p < 0 . 01 ) . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Expression of full-length (left) and truncated (soluble, right) Sortilin protein in micro-dissected CA1 and the remaining hippocampus of Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SD of densitometry relative to GAPDH (N=3 mice per condition; **, p<0.01). Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx / fx mouse .

Representative photomicrograph of Golgi staining in hippocampus of a Tardbpfx/fx mouse.


Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx / fx and CamKIIaCRE ; Tardbpfx / fx mice . Results are presented as average ± SEM ( N = 3 mice per condition , 20 neurons per mouse ) . * , p < 0 . 05 ; * * , p < 0 . 01 . Data information : Results are presented as average ± standard error of the mean ( SEM ) . All statistical tests were performed using 2 - way ANOVA with Sidak post hoc analysis .

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

Sholl analysis of apical and basal dendritic arbors of CA1 pyramidal neurons from Tardbpfx/fx and CamKIIaCRE;Tardbpfx/fx mice. Results are presented as average ± SEM (N=3 mice per condition, 20 neurons per mouse). *, p<0.05; **, p<0.01. Data information: Results are presented as average ± standard error of the mean (SEM). All statistical tests were performed using 2-way ANOVA with Sidak post hoc analysis.


High - magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx / fx mouse . Black arrows denote selected dendritic spines .

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High-magnification representative photomicrograph of Golgi staining of the dendritic arbour of a CA1 pyramidal neuron in a Tardbpfx/fx mouse. Black arrows denote selected dendritic spines.