Differential expression for ACE2 expression changes in treated vs control samples was performed with level 3 CMAP data of 24 hours response values using limma ( Ritchie et al , 2015 ; Methods ) . enrichment of positive / negative ACE2 expression regulators in different drug classes on their mechanism of action ( MOA ) was tested with GSEA method as implemented in R package fgsea ( Segushichev , 2016 ; Methods ) , enrichment significance ( negative log10 P values ) is shown in bar plots in ( E ) , for on 672 clinically approved drugs We further extended to a larger set of 989 clinically approved drugs tested on a total of 28 lines ( 16 cancer 12 normal ; this list is provided in Table EV2A with details including site , subtype donor demographics ) where each drug may have been tested on a different subset of ( Methods ) . performed enrichment using WHO ATC drug classification data ( World Health Organization , 2006 ) , top enriched drug classes are shown in ( F ) .

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Differential expression analysis for ACE2 expression changes in treated vs control samples was performed with the level 3 CMAP data of 24 hours response values using limma (Ritchie et al, 2015; Methods). The enrichment of positive/negative ACE2 expression regulators in different drug classes based on their mechanism of action (MOA) was tested with the GSEA method as implemented in the R package fgsea (Segushichev, 2016; Methods), and the enrichment significance (negative log10 P values) is shown in bar plots in (E), for the analysis on the 672 clinically approved drugs We further extended the analysis to a larger set of 989 clinically approved drugs tested on a total of 28 cell lines (16 cancer and 12 normal cells; this list is provided in Table EV2A with details including primary site, subtype and donor demographics) where each drug may have been tested on a different subset of cells (Methods). and performed enrichment analysis using the WHO ATC drug classification data (World Health Organization, 2006), and top enriched drug classes are shown in (F).


( A - D ) This Fig summarizes differential expression results for ACE2 upon treatment by clinically approved drugs in samples of lung kidney datasets mined from GEO database , spanning both cancer non - cancer datasets ( Methods ) . Volcano plots showing log fold - change ( X - axis ) uncorrected negative log10P value ( Y - axis ) of ACE2 expression changes are displayed in ( A ) for 74 datasets of or tissue samples from lung , involving 42 clinically approved drugs , in ( C ) for 35 kidney datasets involving 29 clinically approved drugs . Among these , we focused on top significant drugs that modulate ACE2 expression in non - cancerous or tissue samples from lung kidney , which are shown in ( B ) ( D ) , respectively , where ACE2 expression ( Y - axis ) difference between control treated groups ( X - axis ) are shown with boxplots . In title of each boxplot , GEO identifier of respective studies corresponding drug name sample type are provided . Different datasets involving experiments using same drug were analyzed presented separately , since sample types can be different across datasets . P values are computed from differential expression using limma ( Ritchie et al , 2015 ) ( Methods ) . In boxplots of panels ( B ) ( D ) , center , box edges whiskers denotes median , interquartile range of distribution in respective order , except for points that were determined to be outliers using a method that is a function of interquartile range , as in standard box plots . Abbreviations : HBEC , human bronchial epithelial .

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(A-D) This Fig summarizes the differential expression analysis results for ACE2 upon treatment by clinically approved drugs in samples of lung and kidney datasets mined from the GEO database, spanning both cancer and non-cancer datasets (Methods). Volcano plots showing the log fold-change (X-axis) and uncorrected negative log10P value (Y-axis) of ACE2 expression changes are displayed in (A) for 74 datasets of cells or tissue samples from lung, involving 42 clinically approved drugs, and in (C) for 35 kidney datasets involving 29 clinically approved drugs. Among these, we focused on the top significant drugs that modulate ACE2 expression in non-cancerous cells or tissue samples from lung and kidney, which are shown in (B) and (D), respectively, where the ACE2 expression (Y-axis) difference between control and treated groups (X-axis) are shown with boxplots. In the title of each boxplot, the GEO identifier of the respective studies and the corresponding drug name and the sample type are provided. Different datasets involving experiments using the same drug were analyzed and presented separately, since the sample types can be different across datasets. All the P values are computed from differential expression analysis using limma (Ritchie et al, 2015) (Methods). In the boxplots of panels (B) and (D), the center line, box edges and whiskers denotes the median, interquartile range and the rest of the distribution in respective order, except for points that were determined to be outliers using a method that is a function of the interquartile range, as in standard box plots. Abbreviations: HBEC, human primary bronchial epithelial cells.


( B ) In vitro degradation of UbLRad23 - GFP - 95 by purified S . cerevisiae proteasome . graph plots amount of substrate over time , estimated by monitored by plate - reader ( green ) or electronic autoradiography of SDS - PAGE gel bands ( red ) , normalized to initial substrate amount as described in Methods . ( C ) Michaelis - Menten of UbLRad23 - GFP - 95 degradation by different concentrations of purified S . cerevisiae proteasome ( green , 10 nM ; red , 40 nM ) . ( D ) UbLRad23 inhibits UbLRad23 - GFP - 95 degradation . initial degradation rates of UbLRad23 - GFP - 95 at different concentrations of purified UbLRad23 domain are plotted fitted to equation describing competitive inhibition . ( E ) UbLDsk2 domain is a competitive inhibitor of UbLRad23 - GFP - 95 degradation . Lineweaver - Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain . ( C - E ) Proteasomal degradation of UbLRad23 - GFP - 95 monitored by in a Tecan plate reader as described in Methods .

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(B) In vitro degradation of UbLRad23-GFP-95 by purified S. cerevisiae proteasome. The graph plots the amount of substrate over time, estimated by fluorescence intensity monitored by plate-reader (green) or electronic autoradiography of SDS-PAGE gel bands (red), normalized to the initial substrate amount as described in Methods. (C) Michaelis-Menten analysis of UbLRad23-GFP-95 degradation by different concentrations of purified S. cerevisiae proteasome (green, 10 nM; red, 40 nM). (D) UbLRad23 inhibits UbLRad23-GFP-95 degradation. The initial degradation rates of UbLRad23-GFP-95 at different concentrations of purified UbLRad23 domain are plotted and fitted to the equation describing competitive inhibition. (E) The UbLDsk2 domain is a competitive inhibitor of UbLRad23-GFP-95 degradation. Lineweaver-Burk plot of UbLRad23GFP95 degradation with different concentrations of purified UbLDsk2 domain. (C-E) Proteasomal degradation of UbLRad23-GFP-95 monitored by fluorescence intensity in a Tecan plate reader as described in Methods.


( A , B ) In vitro degradation of model by purified S . cerevisiae proteasome . model ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 were degraded by proteasome ( red solid circle ) can be stabilized by removing UbL domain ( no UbL , black triangles ) , by removing 95 - - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model containing UbL domain of S . cerevisiae Rad23 .

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(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23.


( A , B ) In vitro degradation of model by purified S . cerevisiae proteasome . model ( UbL - DHFR - 95 ) consisted of an N - terminal UbL domain , followed by an E . coli DHFR domain a 95 aminoacid tail derived from S . cerevisiae cytochrome b2 . UbL - DHFR - 95 were degraded by proteasome ( red solid circle ) can be stabilized by removing UbL domain ( no UbL , black triangles ) , by removing 95 - - acid tail ( no tail , blue diamonds ) or by proteasome inhibitor ( MG132 , green squares ) . ( A ) Degradation of model containing UbL domain of S . cerevisiae Rad23 . ( B ) Degradation of model containing other UbL domains or ubiquitin . graphs plot amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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

(A, B) In vitro degradation of model proteins by purified S. cerevisiae proteasome. The model proteins (UbL-DHFR-95) consisted of an N-terminal UbL domain, followed by an E. coli DHFR domain and a 95 aminoacid tail derived from S. cerevisiae cytochrome b2. UbL-DHFR-95 proteins were degraded by proteasome (red solid circle) and can be stabilized by removing the UbL domain (no UbL, black triangles), by removing the 95-amino-acid tail (no tail, blue diamonds) or by proteasome inhibitor (MG132, green squares). (A) Degradation of model proteins containing the UbL domain of S. cerevisiae Rad23. (B) Degradation of model proteins containing other UbL domains or ubiquitin. The graphs plot the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


( B ) UbL domains mediated degradation of His3 fusion with a Su9 tail , affected yeast growth under selective condition ( + 3 - AT , - his ) . Replacing UbL domains with DHFR domains rescued growth defect . expressing His3 fusion in late log phase were serially diluted stamped on selective plates . Plates were incubated at 30 ˚ C for 3 daysfor imaging .

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(B) UbL domains mediated degradation of His3 fusion proteins with a Su9 tail, and affected yeast growth under selective condition (+3-AT, -his). Replacing UbL domains with DHFR domains rescued the growth defect. Cells expressing His3 fusion proteins in late log phase were serially diluted and stamped on selective plates. Plates were incubated at 30 ˚C for 3 daysfor imaging.


( B ) profiles of expressing different substrates monitored by flow cytometry . UbLRad23 targeted YFP - Su9 for degradation . Removing Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP in .

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(B) Fluorescence profiles of cells expressing different substrates monitored by flow cytometry. UbLRad23 targeted YFP-Su9 protein for degradation. Removing the Su9 tail or replacing UbLRad23 with a DHFR domain stabilizes YFP protein in cells.


( C ) UbL domains targeted YFP substrates to degradation in presence of an initiation region . Y - axis plots abundance of UbL - YFP - tail normalized by a translation control , which is same UbL - YFP without tail . is equal to one for domains that are not recognized by proteasome , smaller than one if UbL is recognized by proteasome UbL - YFP - tail is degraded . YFP is also corrected for plasmid copy number using RFP . median of YFP / RFP for each construct was calculated from 10 , 000 collected in one flow cytometry run reflected abundance of YFP substrates in yeast as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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(C) UbL domains targeted YFP substrates to degradation in the presence of an initiation region. The Y-axis plots abundance of the UbL-YFP-tail proteins normalized by a translation control, which is the same UbL-YFP protein without the tail. The ratio is equal to one for domains that are not recognized by the proteasome, and smaller than one if the UbL is recognized by the proteasome and the UbL-YFP-tail protein is degraded. YFP fluorescence is also corrected for plasmid copy number using RFP fluorescence. The median of YFP/RFP ratio for each construct was calculated from 10,000 cells collected in one flow cytometry run and reflected the abundance of YFP substrates in yeast cells as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


( B ) In vitro degradation of Mdy2 by purified S . cerevisiae proteasome . Mdy2 ( red solid circles ) , Mdy2ΔN - 95 ( blue solid circles ) Mdy2 - 95 ( green solid circles ) were degraded . Removing N - terminal domain of Mdy2 ( Mdy2ΔN , black solid circles ) or adding its binding partner Get4 ( red open squares , dash ) or proteasome - binding competitor UbLMdy2 ( red open diamonds , dotted ) stabilized Mdy2Get4 . When Mdy2 binding buried N - terminal domain , addition of a 95 - acid tail derived from S . cerevisiae cytochrome b2 at Cterminus restored degradation of Mdy2 ( green open squares , dotted ) . graph plots amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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(B) In vitro degradation of Mdy2 proteins by purified S. cerevisiae proteasome. Mdy2 (red solid circles), Mdy2ΔN-95 (blue solid circles) and Mdy2-95 (green solid circles) were degraded. Removing the N-terminal domain of Mdy2 (Mdy2ΔN, black solid circles) or adding its binding partner Get4 (red open squares, dash line) or the proteasome-binding competitor UbLMdy2 (red open diamonds, dotted line) stabilized Mdy2Get4. When Mdy2 binding buried the N-terminal domain, addition of a 95 amino-acid tail derived from S. cerevisiae cytochrome b2 at the Cterminus restored degradation of Mdy2 (green open squares, dotted line). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


( C ) Degradation of Mdy2 in vivo . steady - state accumulation of overexpressed C - terminally HA - tagged full - Mdy2 a mutant in which N - terminal domain was deleted ( Mdy2ΔN ) in S . cerevisiae , in absence of Get4 , presence of endogenously expressed Get4 or overexpressed Get4 . Overexpressed Get4 was detected through an N - terminal Flag - tag .

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(C) Degradation of Mdy2 in vivo. The steady-state accumulation of overexpressed C-terminally HA-tagged full-length Mdy2 and a mutant in which the N-terminal domain was deleted (Mdy2ΔN) in S. cerevisiae, in the absence of Get4, the presence of endogenously expressed Get4 or overexpressed Get4. Overexpressed Get4 was detected through an N-terminal Flag-tag.


( D ) Steady - state accumulation of endogenous Mdy2 in S . cerevisiae , with deleted ( get4 ∆ ) or endogenous ( wt ) Get4 in presence or absence of proteasome inhibitor bortezomib . were determined by western blotting for HA - tag , Flag - tag , Mdy2 or Scs2 of SDS - PAGE gels of S . cerevisiae extracts . loading in each lane were estimated in lysates by western blotting for integral endoplasmic reticulum Scs2 .

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(D) Steady-state accumulation of endogenous Mdy2 in S. cerevisiae, with deleted (get4∆) or endogenous (wt) Get4 in the presence or absence of the proteasome inhibitor bortezomib. Protein levels were determined by western blotting for the HA-tag, Flag-tag, Mdy2 or Scs2 of SDS-PAGE gels of S. cerevisiae protein extracts. Protein loading levels in each lane were estimated in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.


( B ) In vitro degradation of Ubp6 by purified S . cerevisiae proteasome . Full - Ubp6 ( Ubp6 , black open circles ) a UbL deletion mutant ( Ubp6ΔN , green open circles ) escaped proteasome degradation . Attachment of a Cterminal 95 - acid tail derived from S . cerevisiae cytochrome b2 allowed proteasome to degrade Ubp6 ( Ubp6 - 95 , black solid circles Ubp6ΔN - 95 , green solid circles ) . graph plots amount of substrate estimated by electronic autoradiography in SDS - PAGE gel bands over time as normalized to initial substrate amount as described in Methods . Data points represent mean values determined from at least three repeat experiments ; error bars indicate s . e . m .

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(B) In vitro degradation of Ubp6 proteins by purified S. cerevisiae proteasome. Full-length Ubp6 (Ubp6, black open circles) and a UbL deletion mutant (Ubp6ΔN, green open circles) escaped proteasome degradation. Attachment of a Cterminal 95 amino-acid tail derived from S. cerevisiae cytochrome b2 allowed the proteasome to degrade the Ubp6 proteins (Ubp6-95, black solid circles and Ubp6ΔN-95, green solid circles). The graph plots the amount of substrate estimated by electronic autoradiography in SDS-PAGE gel bands over time as normalized to the initial substrate amount as described in Methods. Data points represent mean values determined from at least three repeat experiments; error bars indicate s.e.m.


C ) Degradation of Ubp6 proteinsin vivo . steady - state accumulation of N - terminally HA - tagged Ubp6 mutants in S . cerevisiae . Ubp6 Ubp6ΔN are stable in yeast not further stabilized by bortezomib . Attachment of a Su9 tail to Cterminus lead to degradation of Ubp6 Ubp6ΔNproteins were stabilized by bortezomib treatment .

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C) Degradation of Ubp6 proteinsin vivo. The steady-state accumulation of N-terminally HA-tagged Ubp6 and mutants in S. cerevisiae. Ubp6 and Ubp6ΔN are stable in yeast and not further stabilized by bortezomib. Attachment of a Su9 tail to the Cterminus lead to the degradation of Ubp6 and Ubp6ΔNproteins and the proteins were stabilized by bortezomib treatment.


( D ) steady - state accumulation of endogenous Ubp6 Ubp6 derivatives with short ( 19 acid ) or long ( 54 acid ) tails at their C - termini . 19 - acid tail destabilized Ubp6 54 - - acid tail lead to complete depletion of Ubp6 . were determined by western blotting for HA - tag , Ubp6 or Scs2 in SDS - PAGE gels of S . cerevisiae extracts . proteasome was inhibited by 100 µM bortezomib as indicated . loading in each lane were normalized in lysates by western blotting for integral endoplasmic reticulum Scs2 .

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(D) The steady-state accumulation of endogenous Ubp6 and Ubp6 derivatives with short (19 amino acid) or long (54 amino acid) tails at their C-termini. The 19 amino-acid tail destabilized Ubp6 and the 54-amino-acid tail lead to complete depletion of Ubp6. Protein levels were determined by western blotting for the HA-tag, Ubp6 or Scs2 in SDS-PAGE gels of S. cerevisiae protein extracts. The proteasome was inhibited by 100 µM bortezomib as indicated. Protein loading levels in each lane were normalized in all lysates by western blotting for the integral endoplasmic reticulum membrane protein Scs2.


( F ) of highlighted genes enriched in either somata or processes at transcript or level . ( Upper row : Scatterplots of transcript level ; n = 2 ; crossbar represents mean . Lower row : Boxplot of ; n = 4 ) .

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(F) Quantification of the highlighted genes enriched in either the somata or processes at the transcript or protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4).


( A ) Volcano plots representing transcript down - ( red ) or up - regulation ( yellow ) after 48 hours PTX in somatic process compartment . Representative transcripts are highlighted labelled . Linear model was used to fit transcriptome data subsequently contrasted using likelihood testing for PTX - treatment effects across compartments .

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(A) Volcano plots representing transcript down- (red) or up-regulation (yellow) after 48 hours PTX in the somatic and process compartment. Representative transcripts are highlighted and labelled. Linear model was used to fit transcriptome data and subsequently contrasted using likelihood ratio testing for PTX-treatment effects across compartments.


( A ) Real - time quantitative PCR ( RT - qPCR ) of transcripts changing differentially between compartments using compartmentalized cultures after 48 hours PTX - treatment . n = 8 independent biological replicates ( except for Plk2 ( n = 7 ) , where one measurement couldn ' t be performed due to insufficient cDNA ) ; PTX - effect was assessed by three - way ANOVA followed by Tukey ' s post - hoc multiple comparison ; * p < 0 . 05 ; * * p < 0 . 01 .

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(A) Real-time quantitative PCR (RT-qPCR) of transcripts changing differentially between compartments using the compartmentalized cultures after 48 hours PTX-treatment. n=8 independent biological replicates (except for Plk2 (n=7), where one measurement couldn't be performed due to insufficient cDNA); PTX-effect was assessed by three-way ANOVA followed by Tukey's post-hoc multiple comparison test; *p < 0.05; **p < 0.01.


( B ) Representative of single - molecule FISH ( smFISH ) in either control or 48 h PTX - treated rat hippocampal neurons ( DIV20 ) using probes specific for Add2 , Dnajc6 Sort1 ( green ) . MAP2 immunostaining ( red ) was used to visualize neuronal somata dendrites . Inserts at higher magnification illustrate PTX - dependent changes in dendritic . Scale bar = 10 μm . ( C ) of B . ( n = 3 - 4 independet biological replicates with 8 - 10 averaged per condition replicate ; two - sample Student ' s t - ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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(B) Representative images of single-molecule FISH (smFISH) in either control or 48 h PTX-treated rat hippocampal neurons (DIV20) using probes specific for Add2, Dnajc6 and Sort1 (green). MAP2 immunostaining (red) was used to visualize neuronal somata and dendrites. Inserts at higher magnification illustrate PTX-dependent changes in dendritic RNA puncta. Scale bar = 10 μm. (C) Quantification of B. (n=3-4 independet biological replicates with 8-10 cells averaged per condition and replicate; two-sample Student's t-test; *p < 0.05; **p < 0.01). B


( D ) Representative plots of genes changing after PTX at transcript level . ( Upper row : Scatterplots of transcript level ; n = 2 ; crossbar represents mean . Lower row : Boxplot of ; n = 4 ) . Boxplots : central : median ; box : 25th to 75th percentile ; whiskers : until last data point within 1 . 5x interquartile range ( IQR ) .

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(D) Representative plots of genes changing after PTX at the transcript and protein level. (Upper row: Scatterplots of transcript level; n=2; crossbar represents mean. Lower row: Boxplot of protein levels; n=4). Boxplots: central line: median; box: 25th to 75th percentile; whiskers: until last data point within 1.5x interquartile range (IQR).


( A ) Representative pictures of nascent Camk2a - peptides in mock - PTX - treated neurons . Upper Panels : Representative overview with merged channels ( grey : MAP2 - , red : Duolink - PLA signal , blue : Hoechst ) . Middle Panels : Magnifications of boxed areas marked in ( A ) . MAP2 signal was used to outline somata dendritic processes ( lines ) . Lower Panels : Negative control of puromycin - untreated with representative Duolink - signal in dendrite soma , respectively . ( Scale bars : in overview 20 μm , in inserts of higher magnification 5 μm ) . ( B ) Same as A with nascent Synapsin1 - peptides . (

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(A) Representative pictures of nascent Camk2a-peptides in mock- and PTX-treated neurons. Upper Panels: Representative overview images with merged channels (grey: MAP2-staining, red: Duolink-PLA signal, blue: Hoechst nuclear staining). Middle Panels: Magnifications of boxed areas marked in (A). MAP2 signal was used to outline somata and dendritic processes (white lines). Lower Panels: Negative control of puromycin-untreated cells with representative Duolink-signal in dendrite and soma, respectively. (Scale bars: in overview images 20 μm, in inserts of higher magnification 5 μm). (B) Same as A with nascent Synapsin1-peptides. (


( C ) Enrichment for RBP motifs in down - up - regulated genes in either compartment . Fisher ' s exact was used to perform enrichment ; FDR - corrected .

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(C) Enrichment analysis for RBP motifs in down- and up-regulated genes in either compartment. Fisher's exact test was used to perform enrichment analysis; FDR-corrected.


Immunostaining from Daam2LacZ / + with Aldh1l1 - EGFP reporter indicates that Daam2 ( LacZ + , Red ) is expressed in astrocytes ( Green ) . ( A ) Daam2 expression is observed in multiple brain regions at postnatal day 21 .

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Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (A) Daam2 expression is observed in multiple brain regions at postnatal day 21.


Immunostaining from Daam2LacZ / + with Aldh1l1 - EGFP reporter indicates that Daam2 ( LacZ + , Red ) is expressed in astrocytes ( Green ) . ( B ) Expression of Daam2 in cortex persists throughout development ( P7 to adult ) . C . of Daam2 - expressing astrocytes ( LacZ + ) with Aldh1l1 - EGFP labeled astrocytes in cortex during development . Data is presented as mean ± . Two brain sections , N = 4 for each .

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Immunostaining from Daam2LacZ/+ animals with Aldh1l1-EGFP reporter indicates that Daam2 (LacZ+, Red) is expressed in astrocytes (Green). (B) Expression of Daam2 in cortex persists throughout development (P7 to adult). C. Quantification of Daam2-expressing astrocytes (LacZ+) with Aldh1l1-EGFP labeled astrocytes in cortex during development. Data is presented as mean ± SEM. Two brain sections, N=4 for each age.


G . Representative of astrocytes labeled with Aldh1l1 - EGFP reporter in cortex OB of Daam2 cHet cKO mice at postnatal day 28 ( P28 ) . Scale bar : 50 μm . H - K . Overall complexity of astrocytes with Aldh1l1 - EGFP reporter was measured by Sholl ( Two - way ANOVA ) total process ( Student ' s t - ) . Data is presented as mean ± . n = 6 - 10 from N = 3 - 5 mice per genotype , * p < 0 . 05 , p * * * * < 0 .

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G. Representative images of astrocytes labeled with Aldh1l1-EGFP reporter in cortex and OB of Daam2 cHet and cKO mice at postnatal day 28 (P28). Scale bar: 50 μm. H-K. Overall complexity of astrocytes with Aldh1l1-EGFP reporter was measured by Sholl analysis (Two-way ANOVA) and total process length (Student's t-test). Data is presented as mean ± SEM. n= 6-10 cells from N=3-5 mice per genotype, *p<0.05, p****<0.0001


A . Representative of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex OB at P28 . Scale bar : 20 μm .

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A. Representative images of GFAP immunostaining in Daam2 cKO mice compared to controls in cortex and OB at P28. Scale bar: 20 μm.


D . Western blot showed a significant increase in amount of GFAP in Daam2 KO astrocytes . N =

D. Western blot analysis showed a significant increase in the amount of GFAP protein in Daam2 KO primary astrocytes. N=2


F . Overexpression of Daam2 via injection of AAV - GfaABC1D - myc - Daam2 in wild type P1 pups revealed reduced GFAP compared to injected with control ( AAV - GfaABC1D - GFP ) . AAV - GfaABC1D - myc - Daam2 was injected into Aldh1l1 - GFP reporter mice for morphological complexity infected ( Myc positive ) were compared with non - infected . Dotted indicates - infected . Filled triangles empty triangles indicate astrocytic branches with control reduced level of GFAP or Aldh1l1 - GFP , respectively . Scale bar : 20 μm .

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F. Overexpression of Daam2 via injection of AAV-GfaABC1D-myc-Daam2 in wild type P1 pups revealed reduced GFAP intensity compared to animals injected with control virus (AAV-GfaABC1D-GFP). AAV-GfaABC1D-myc-Daam2 was injected into Aldh1l1-GFP reporter mice for morphological complexity analysis and infected cells (Myc positive) were compared with non-infected cells. Dotted white line indicates virus-infected cells. Filled triangles and empty triangles indicate astrocytic primary branches with the control and reduced level of GFAP or Aldh1l1-GFP, respectively. Scale bar: 20 μm.


E . Schematics of astrocytic calcium imaging representative of spontaneous calcium signaling ( green ) of Daam2 cHet cKO mice injected with AAV - GfaABC1D - GCaMP6f in OB . Approximate territory of astrocytes including soma multiple microdomains is outlined in yellow . Representative traces of GCaMP6f signal in Daam2 cHet cKO astrocytes are shown in black red respectively . F - H . of amplitude ( F ) , frequency ( G ) , under curve ( AUC ) ( H ) of ∆ F / F GCaMP6f signal events in OB . Amplitude AUC of Daam2 - deficient astrocytes are significantly increased in OB . Data is presented as mean ± . Total number of is n = 16 , 15 ( F - H ) from N = 3 - 4 mice of each genotype . Student ' s t - was used for statistics , * p < 0 . 05 .

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E. Schematics of astrocytic calcium imaging and representative images of spontaneous calcium signaling (green) of Daam2 cHet and cKO mice injected with AAV-GfaABC1D-GCaMP6f virus in OB. Approximate territory of astrocytes including soma and multiple microdomains is outlined in yellow. Representative traces of GCaMP6f signal in Daam2 cHet and cKO astrocytes are shown in black and red respectively. F-H. Quantification of amplitude (F), frequency (G), and area under curve (AUC) (H) of ∆F/F GCaMP6f signal events in OB. Amplitude and AUC of Daam2-deficient astrocytes are significantly increased in OB. Data is presented as mean ± SEM. Total number of cells is n=16, 15 (F-H) from N=3-4 mice of each genotype. Student's t-test was used for statistics, *p<0.05.


A - F . ( A , D ) Schematics of whole recording in ex vivo brain slices representative traces of spontaneous EPSC IPSC in neurons in cortex from Daam2 cHet ( top , black ) cKO ( bottom , red ) mice . ( B - C , E - F ) of amplitude frequency of sEPSC sIPSC in each genotype . Data is presented as mean ± . Total number of is n = 19 , 20 ( B - C ) 17 , 13 ( E - F ) from N = 7 mice for each genotype . Student ' s t - was used for comparison , * p < 0 . 05 .

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A-F. (A, D) Schematics of whole cell recording in ex vivo brain slices and representative traces of spontaneous EPSC and IPSC in neurons in cortex from Daam2 cHet (top, black) and cKO (bottom, red) mice. (B-C, E-F) Quantification of amplitude and frequency of sEPSC and sIPSC in each genotype. Data is presented as mean ± SEM. Total number of cells is n=19, 20 (B-C) and 17, 13 (E-F) from N=7 mice for each genotype. Student's t-test was used for comparison, *p<0.05.


A . Astrocyte - specific Daam2 cKO mice show delays in buried food finding . N = 13 , 15 mice per genotype . Data is presented as mean ± . Student ' s t - was used , * p < 0 . 05 .

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A. Astrocyte-specific Daam2 cKO mice show delays in buried food finding. N=13, 15 mice per genotype. Data is presented as mean ± SEM. Student's t-test was used, *p<0.05.


E - F . qRT - PCR ( E ) immunostaining ( F ) confirm differential expression of genes found in MS profiling . qRT - PCR is repeated as triplicate is presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . Student ' s t - was used . Scale bar : 20 μm .

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E-F. qRT-PCR (E) and immunostaining (F) confirm differential expression of genes found in MS profiling. qRT-PCR is repeated as triplicate and is presented as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001. Student's t-test was used. Scale bar: 20 μm.


F - J . Representative of GFAP immunostaining ( F ) ( G - J ) in controls , Daam2 icKO , Slc4a4 icKO , double icKO 6 after tamoxifen injection . Scale bar : 20 μm . Data is presented as mean ± . n = 10 - 24 , N = 3 - 4 mice per genotype . Scale bar : 20 μm . Two - way ANOVA is used for statistics . [ * ] [ # ] indicates comparison with control double icKO , respectively . * * p < 0 . 01 , p * * * < 0 . 001 , # # p < 0 . 01 .

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F-J. Representative images of GFAP immunostaining (F) and quantification (G-J) in controls, Daam2 icKO, Slc4a4 icKO, and double icKO 6 weeks after tamoxifen injection. Scale bar: 20 μm. Data is presented as mean ± SEM. n=10-24 images, N=3-4 mice per genotype. Scale bar: 20 μm. Two-way ANOVA is used for statistics. [*] and [#] indicates comparison with control and double icKO, respectively. **p<0.01, p***<0.001, ##p<0.01.


( A ) Chromosome 7 of S . bayanus is incompatible with S . cerevisiae genome . chromosome 7 replacement stain ( Sc + Sb - chr7 ) exhibited serious growth defects when grown on non - fermentable carbon source ( glycerol ) . respiration defects could be partially rescued when a whole set of S . bayanus chromosomes were provided ( Sc + Sb - chr7 x Sb - ρ⁰ ) , or fully rescued with addition of S . bayanus mtDNA ( Sc + Sb - chr7 - ρ⁰ x Sb ) . cultures were serially - diluted spotted onto YPD ( glucose ) or YPG ( glycerol ) plates . plates were then incubated at 28 ° C until colonies were easily observed . Sc , S . cerevisiae . Sb , S . bayanus . ρ⁰ , mtDNA - less strains .

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

(A) Chromosome 7 of S. bayanus is incompatible with the S. cerevisiae genome. The chromosome 7 replacement stain (Sc+Sb-chr7) exhibited serious growth defects when grown on the non-fermentable carbon source (glycerol). The respiration defects could be partially rescued when a whole set of S. bayanus chromosomes were provided (Sc+Sb-chr7 x Sb-ρ⁰), or fully rescued with the addition of S. bayanus mtDNA (Sc+Sb-chr7-ρ⁰ x Sb). Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed. Sc, S. cerevisiae. Sb, S. bayanus. ρ⁰, mtDNA-less strains.


( B ) Ectopically - expressing Sc - CCM1 rescues respiration defect of Sc + Sb - chr7 . Spot assays for wild - type S . cerevisiae ( Sc ) chromosome 7 replacement stain ( Sc + Sb - chr7 ) carrying an empty plasmid ( + vector ) or plasmid with S . cerevisiae CCM1 gene ( + Sc - CCM1 ) .

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(B) Ectopically-expressing Sc-CCM1 rescues the respiration defect of Sc+Sb-chr7. Spot assays for the wild-type S. cerevisiae (Sc) and the chromosome 7 replacement stain (Sc+Sb-chr7) carrying an empty plasmid (+ vector) or the plasmid with the S. cerevisiae CCM1 gene (+ Sc-CCM1).


( C ) Sb - CCM1 incompatibility could only be rescued in presence of S . bayanus mtDNA . native CCM1 ORF in S . cerevisiae was replaced by either Sb - CCM1 ( Sc + Sb - CCM1 ) or Sc - CCM1 ( Sc + Sc - CCM1 , as a control ) coding regions . Crossing Sc + Sb - CCM1 with mtDNA - less Sb - ρ⁰ ( Sc + Sb - CCM1 x Sb - ρ⁰ ) could not rescue growth defect .

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(C) Sb-CCM1 incompatibility could only be rescued in the presence of S. bayanus mtDNA. The native CCM1 ORF in S. cerevisiae was replaced by either Sb-CCM1 (Sc+Sb-CCM1) or Sc-CCM1 (Sc+Sc-CCM1, as a control) coding regions. Crossing the Sc+Sb-CCM1 strain with the mtDNA-less Sb-ρ⁰ strain (Sc+Sb-CCM1 x Sb-ρ⁰) could not rescue the growth defect.


( D ) CCM1 incompatibility is symmetric between S . cerevisiae S . bayanus . CCM1 ORF in S . bayanus was replaced by either Sc - CCM1 ( Sb + Sc - CCM1 ) or Sb - CCM1 ( Sb + Sb - CCM1 , as a control ) coding regions . Only Sb + Sc - CCM1 exhibited respiration defects defect could not be rescued even when a whole set of S . cerevisiae chromosomes were provided ( Sb + Sc - CCM1 x Sc - ρ⁰ ) .

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(D) CCM1 incompatibility is symmetric between S. cerevisiae and S. bayanus. The CCM1 ORF in S. bayanus was replaced by either Sc-CCM1 (Sb+Sc-CCM1) or Sb-CCM1 (Sb+Sb-CCM1, as a control) coding regions. Only the Sb+Sc-CCM1 strain exhibited respiration defects and the defect could not be rescued even when a whole set of S. cerevisiae chromosomes were provided (Sb+Sc-CCM1 x Sc-ρ⁰).


( E ) CCM1 incompatibility probably occurred during divergence between S . bayanus common ancestor of S . cerevisiae , S . paradoxus , S . mikatae S . kudriavzevii . endogenous CCM1 in S . cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts . Only carrying S . bayanus CCM1 displayed growth defects on glycerol - containing plates .

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(E) CCM1 incompatibility probably occurred during the divergence between S. bayanus and the common ancestor of S. cerevisiae, S. paradoxus, S. mikatae and S. kudriavzevii. The endogenous CCM1 in S. cerevisiae was replaced with its orthologous alleles from other Saccharomyces sensu stricto yeasts. Only the strain carrying S. bayanus CCM1 displayed growth defects on glycerol-containing plates.


( A ) Sb - Ccm1 is transported efficiently to S . cerevisiae mitochondria . Total extracts ( total ) , fractions ( cyto ) or mitochondrial fractions ( mito ) were hybridized with different antibodies to detect . G6PDH ( glucose - 6 - phosphate dehydrogenase ) Cox2 served as mitochondrial markers , respectively . Both Sc - Ccm1 Sb - Ccm1 were fused with c - Myc at C - terminus expressed from a single - copy plasmid . Myc - tagged Ccm1 consistently appeared as double bands , which were probably due to some unknown modifications .

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(A) Sb-Ccm1 protein is transported efficiently to S. cerevisiae mitochondria. Total cell extracts (total), cytosolic fractions (cyto) or mitochondrial fractions (mito) were hybridized with different antibodies to detect the proteins. G6PDH (glucose-6-phosphate dehydrogenase) and Cox2 served as cytosolic and mitochondrial markers, respectively. Both Sc-Ccm1 and Sb-Ccm1 were fused with c-Myc at the C-terminus and expressed from a single-copy plasmid. The Myc-tagged Ccm1 proteins consistently appeared as double bands, which were probably due to some unknown protein modifications.


( B ) level of 15S rRNA is reduced in Sc + Sb - CCM1 . isolated from Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) was hybridized with gene - specific probes using Northern blots .

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(B) The level of 15S rRNA is reduced in Sc+Sb-CCM1 cells. RNA isolated from Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells was hybridized with gene-specific probes using Northern blots.


( C ) Sb - Ccm1 interacts weakly with S . cerevisiae 15S rRNA . level of Ccm1 - bound 15S rRNA was measured using quantitative PCR following mitochondrial immunoprecipitation of S . cerevisiae expressing Sc - CCM1 - 13Myc or Sb - CCM1 - 13Myc . Data was normalized to control group without Myc tag to obtain relative fold enrichment . Graph was plotted using data from three independent repeats for each p value was calculated by unpaired , two - sided student t - . Error bars indicate S . D . * * * , p value < 0 . 001 .

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(C) Sb-Ccm1 interacts weakly with S. cerevisiae 15S rRNA. The level of Ccm1-bound 15S rRNA was measured using quantitative PCR analysis following mitochondrial RNA immunoprecipitation of S. cerevisiae cells expressing Sc-CCM1-13Myc or Sb-CCM1-13Myc. Data was normalized to the control group without the Myc tag to obtain the relative fold enrichment. Graph was plotted using data from three independent repeats for each strain and p value was calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001.


( D ) Overexpressing Sb - CCM1 rescues respiration defect of Sc + Sb - CCM1 . cultures were serially - diluted spotted onto YPD ( glucose ) or YPG ( glycerol ) plates . plates were then incubated at 28 ° C until colonies were easily observed .

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(D) Overexpressing Sb-CCM1 rescues the respiration defect of Sc+Sb-CCM1 cells. Cell cultures were serially-diluted and spotted onto YPD (glucose) or YPG (glycerol) plates. The plates were then incubated at 28°C until colonies were easily observed.


( E ) steady - state level of mtDNA - encoded is reduced in Sc + Sb - CCM1 . Immunoblotting for mitochondrial in Sc + Sb - CCM1 ( labeled as Sb ) or Sc + Sc - CCM1 ( labeled as Sc ) . Cox1 , Cox2 Cox3 are mtDNA - encoded IV subunits . Tom20 is a nucleus - encoded mitochondrial .

(E) The steady-state level of mtDNA-encoded proteins is reduced in Sc+Sb-CCM1 cells. Immunoblotting for the mitochondrial proteins in Sc+Sb-CCM1 (labeled as Sb) or Sc+Sc-CCM1 (labeled as Sc) cells. Cox1, Cox2 and Cox3 are mtDNA-encoded complex IV subunits. Tom20 is a nucleus-encoded mitochondrial protein.


( A ) Tetrad reveals that respiration defect of Sc + Sb - CCM1 can be rescued by mutations in mitochondrial or genomes . suppressor clones were crossed with Sc + Sb - CCM1 - ρ⁰ tetrads from cross were analyzed . A non - Mendelian 4 : 0 segregation pattern for improved respiration is expected if suppressor mutation is in mtDNA ( left panel ) . Otherwise , a 2 : 2 segregation pattern would be observed if rescue effect involved a single mutation ( right panel ) .

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(A) Tetrad analysis reveals that the respiration defect of Sc+Sb-CCM1 cells can be rescued by mutations in mitochondrial or nuclear genomes. The suppressor clones were crossed with the Sc+Sb-CCM1-ρ⁰ strain and the tetrads from the cross were analyzed. A non-Mendelian 4:0 segregation pattern for improved respiration is expected if the suppressor mutation is in mtDNA (left panel). Otherwise, a 2:2 segregation pattern would be observed if the rescue effect involved a single nuclear mutation (right panel).


( A ) Reconstituted Sb - CCM1 mutants can rescue incompatibility of Sb - CCM1 . Among 94 suppressor clones , 20 of them carry mutations in Sb - CCM1 gene . These mutants were reconstructed in Sc + Sb - CCM1 to their function . Superscripts next to Sb - CCM1 indicate respective acid substitutions numbers in parentheses indicate number of independent clones in our suppressor screen . Sc , wild - type S . cerevisiae .

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(A) Reconstituted Sb-CCM1 mutants can rescue the incompatibility of Sb-CCM1. Among 94 suppressor clones, 20 of them carry mutations in the Sb-CCM1 gene. These mutants were reconstructed in Sc+Sb-CCM1 cells to test their function. Superscripts next to Sb-CCM1 indicate the respective amino acid substitutions and numbers in parentheses indicate the number of independent clones in our suppressor screen. Sc, wild-type S. cerevisiae.


( C ) 400th residue plays a determinant role in S . cerevisiae - S . bayanus incompatibility . Both Sb - CCM1E375Q Sb - CCM1D400N mutants relieved incompatibility in S . cerevisiae , but only Sb - CCM1D400N caused respiration defects in S . bayanus . Sb - CCM1E375Q mutant was selected as a control in this experiment since it was a charged - to - polar substitution similar to Sb - CCM1D400N also appeared three times in our screen . Sb , wild - type S . bayanus .

(C) The 400th residue plays a determinant role in the S. cerevisiae-S. bayanus incompatibility. Both Sb-CCM1E375Q and Sb-CCM1D400N mutants relieved the incompatibility in S. cerevisiae, but only Sb-CCM1D400N caused respiration defects in S. bayanus. The Sb-CCM1E375Q mutant was selected as a control in this experiment since it was a charged-to-polar substitution similar to Sb-CCM1D400N and also appeared three times in our screen. Sb, wild-type S. bayanus.


( D ) structural shows that intramolecular suppressor mutations were located on predicted PPR motifs . predicted structure of Ccm1 is shown at left . Blue green colors indicate two α - helices in each predicted PPR motif color indicates additional α - helices in N - terminus middle region . Red color indicates positions of suppressor mutations . Predicted PPR motifs occupy around 52 % of whole ( 453 of 871 residues ) . box at right displays a magnified version of some PPR motifs where substitutions are located .

(D) Protein structural analysis shows that all intramolecular suppressor mutations were located on the predicted PPR motifs. The predicted protein structure of Ccm1 is shown at left. Blue and green colors indicate the two α-helices in each predicted PPR motif and white color indicates additional α-helices in the N-terminus and the middle region. Red color indicates the positions of the suppressor mutations. Predicted PPR motifs occupy around 52% of the whole protein (453 of 871 residues). The box at right displays a magnified version of some PPR motifs where the substitutions are located.


( A ) 15S rRNA are increased in suppressor clones . Total was isolated from replacement strains ( Sc + Sc - CCM1 Sc + Sb - CCM1 ) , two suppressor strains ( Sc + Sb - CCM1D400N Sc + Sb - CCM1E375Q ) one mitochondrial suppressor then subjected to quantitative PCR analyses . Relative mRNA abundance was calculated by normalizing 15S rRNA level to ATP6 mRNA level . Graph was plotted using data from five independent repeats for each p values were calculated by unpaired , two - sided student t - . Error bars indicate S . D . * * * , p value < 0 . 001 . * * , p value < 0 . 01 . Asterisks in black , comparing Sc + Sc - CCM1 with others . Asterisks in gray , comparing Sc + Sb - CCM1 with others .

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(A) 15S rRNA levels are increased in the suppressor clones. Total RNA was isolated from the replacement strains (Sc+Sc-CCM1 and Sc+Sb-CCM1), two nuclear suppressor strains (Sc+Sb-CCM1D400N and Sc+Sb-CCM1E375Q) and one mitochondrial suppressor strain and then subjected to quantitative PCR analyses. Relative mRNA abundance was calculated by normalizing the 15S rRNA level to the ATP6 mRNA level. Graph was plotted using data from five independent repeats for each strain and p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. ***, p value < 0.001. **, p value < 0.01. Asterisks in black, comparing Sc+Sc-CCM1 with others. Asterisks in gray, comparing Sc+Sb-CCM1 with others.


( B ) steady - state level of mtDNA - encoded is increased in suppressor clones . extracts from replacement suppressor strains were hybridized with antibodies against G6PDH , Cox1 , Cox2 or Cox3 .

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(B) The steady-state level of mtDNA-encoded proteins is increased in the suppressor clones. Protein extracts from the replacement and suppressor strains were hybridized with antibodies against G6PDH, Cox1, Cox2 or Cox3.


( B ) Seven of 12 tested PPR genes exhibit incompatibility between S . cerevisiae at least one other Saccharomyces sensu stricto species . doubling time of S . cerevisiae carrying endogenous PPR genes ( Sc ) or orthologs of other yeast species ( Sp , Sm , Sk Sb ) were measured in YPD ( glucose ) or YPG ( glycerol ) media . measurements were then normalized to doubling time of Sc to obtain relative doubling time . Graphs were plotted using data from at least three independent repeats for each . Sc , S . cerevisiae . Sp , S . paradoxus . Sm , S . mikatae . Sk , S . kudriavzevii . Sb , S . bayanus . n . d . , growth not detected . p values were calculated by unpaired , two - sided student t - . Error bars indicate S . D . * , p value < 0 . 05 . * * , p value < 0 . 01 . * * * , p value < 0 . 001 .

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(B) Seven of 12 tested PPR genes exhibit incompatibility between S. cerevisiae and at least one other Saccharomyces sensu stricto species. The doubling time of S. cerevisiae cells carrying the endogenous PPR genes (Sc) or orthologs of other yeast species (Sp, Sm, Sk and Sb) were measured in YPD (glucose) or YPG (glycerol) media. All the measurements were then normalized to the doubling time of Sc to obtain the relative doubling time. Graphs were plotted using data from at least three independent repeats for each strain. Sc, S. cerevisiae. Sp, S. paradoxus. Sm, S. mikatae. Sk, S. kudriavzevii. Sb, S. bayanus. n.d., growth not detected. p values were calculated by unpaired, two-sided student t-test. Error bars indicate S.D. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001.


( A ) expression of AR was determined using Western blots ( overexpressing AR in J82 knocking down AR in UMUC3 ) .

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(A) The expression of AR was determined using Western blots (overexpressing AR in J82 cells and knocking down AR in UMUC3 cells).


( B ) Relative expression of top 20 up - regulated circRNAs from GEO dataset GSE92675 ( BCa tissues compared to para - tissues ) in response to AR modulation in J82 UMUC3 . # indicates an with a relatively low expression ( qRT - PCR cycle number > 35 ) , thus circRNA was excluded for further .

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(B) Relative expression of the top 20 up-regulated circRNAs from the GEO dataset GSE92675 (BCa tumor tissues compared to para-tumor tissues) in response to AR modulation in J82 and UMUC3 cells. The # indicates an RNA with a relatively low expression (qRT-PCR cycle number > 35), thus the circRNA was excluded for further analysis.


( E ) Real - time quantitative PCR ( qRT - PCR ) results show knock down efficiency for these 4 circRNAs in J82 oe - AR ( J82 - AR ) .

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(E) Real-time quantitative PCR (qRT-PCR) results show the knock down efficiency for these 4 circRNAs in J82 oe-AR (J82-AR) cells.


( F ) Transwell invasion assays showed that knock down of hsa _ circ _ 0084171 ( circ _ oo84171 ) , but not other circRNAs , can partly reverse oeAR - increased invasion in J82 . Scale bar , 100 μm .

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(F) Transwell invasion assays showed that knock down of hsa_circ_0084171 (circ_oo84171), but not the other circRNAs, can partly reverse oeAR-increased invasion in J82 cells. Scale bar, 100 μm.


( G ) MTT assays showed that shcirc _ 0084171 can partly reverse oeAR - increased cisplatin chemo - resistance ( 2 μg / ml ) in J82 . To calculate mean , value of 0 µg / ml cisplatin groups were set as 1 .

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(G) MTT assays showed that shcirc_0084171 can partly reverse oeAR-increased cisplatin chemo-resistance (2 μg/ml) in J82 cells. To calculate the mean, the value of 0 µg/ml cisplatin groups were set as 1.


( H ) qRT - PCR results showed that overexpression ( oe ) efficacy for hsa _ circ _ 0084171 ( oecirc _ 0084171 ) in UMUC3 - shAR .

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(H) The qRT-PCR results showed that the overexpression (oe) efficacy for hsa_circ_0084171 (oecirc_0084171) in UMUC3-shAR cells.


( I ) Transwell invasion assays showed that oecirc _ 0084171 can partly rescue shAR induced invasion deduction in UMUC3 . Scale bar , 100 μm .

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(I) Transwell invasion assays showed that oecirc_0084171 can partly rescue shAR induced invasion deduction in UMUC3 cells. Scale bar, 100 μm.


( J ) MTT assays showed that oecirc _ 0084171 can partly overcome shAR effect for cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 .

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(J) MTT assays showed that oecirc_0084171 can partly overcome shAR effect for cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


B ) relative expression of circFNTA was elevated in multiple BCa lines ( T24 , J82 , UMUC3 , 5637 ) compared to normal bladder epithelial SVHUC as shown by qRT - PCR .

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B) The relative expression of circFNTA was elevated in multiple BCa cell lines (T24, J82, UMUC3, and 5637) compared to normal bladder epithelial cell line SVHUC as shown by qRT-PCR.


C ) expression of circFNTA GAPDH mRNA in J82 UMUC3 treated with or without RNase R was detected by qRT - PCR .

C) The expression of circFNTA and GAPDH mRNA in J82 and UMUC3 cells treated with or without RNase R was detected by qRT-PCR.


D ) in situ hybridization ( FISH ) demonstrated that circFNTA was predominantly localized to cytoplasm of J82 UMUC3 . DAPI was mainly localized to cytoplasm . Scale bar , 20 μm .

D) RNA Fluorescence in situ hybridization (FISH) demonstrated that circFNTA was predominantly localized to the cytoplasm of J82 and UMUC3 cells. DAPI was mainly localized to the cytoplasm. Scale bar, 20 μm.


E ) Two shcircFNTA plasmids using PLKO . 1 vector were transfected into UMUC3 . knock down efficacy was measured by qRT - PCR .

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E) Two shcircFNTA plasmids using PLKO.1 vector were transfected into UMUC3 cells. The knock down efficacy was measured by qRT-PCR.


G ) qRT - PCR results show oecircFNTA efficacy in J82 compared with PWPI vector ( left panel ) oecircFNTA or oelinearFNTA in J82 ( right panel ) .

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G) The qRT-PCR results show oecircFNTA efficacy in J82 cells compared with PWPI vector (left panel) and oecircFNTA or oelinearFNTA in J82 cells (right panel).


Transwell invasion assays showed that shcircFNTA can decrease UMUC3 invasion ( H ) more profoundly than oelinearFNTA as compared with oevector group . Scale bar , 100 μm .

Transwell invasion assays showed that shcircFNTA can decrease UMUC3 cell invasion (H) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.


Transwell invasion assays showed that shcircFNTA can increase J82 invasion ( I ) more profoundly than oelinearFNTA as compared with oevector group . Scale bar , 100 μm .

Transwell invasion assays showed that shcircFNTA can increase J82 cells invasion (I) more profoundly than oelinearFNTA as compared with the oevector group. Scale bar, 100 μm.


J ) MTT assays showed that oecircFNTA enhances J82 chemo - resistance to cisplatin ( 0 , 1 , 2 , 5 , 10 μg / ml were tested ) . This effect was more obvious in oecircFNTA group as in oelinearFNTA group ( upper panel ) . MTT assays showed that shcircFNTA increases UMUC3 chemo - sensitivity to cisplatin ( lower panel ) .

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J) MTT assays showed that oecircFNTA enhances J82 cells chemo-resistance to cisplatin (0, 1, 2, 5, 10 μg/ml were tested). This effect was more obvious in the oecircFNTA group as in the oelinearFNTA group (upper panel). MTT assays showed that shcircFNTA increases UMUC3 cells chemo-sensitivity to cisplatin (lower panel).


K ) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with adjacent para - tissues ( N = 41 ) representative are shown . Scale bar , 250 μm .

K) FISH analyses revealed that circFNTA was significantly upregulated in BCa tissues as compared with the adjacent para-tumor tissues (N=41 patients) and representative images are shown. Scale bar, 250 μm.


L ) qRT - PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para - tissues ( N = 23 ) .

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L) The qRT-PCR assays also demonstrated that circFNTA is significantly elevated in fresh BCa tissues compared to para-tumor tissues (N=23 patients).


( A ) expression of AR FNTA ( circFNTA host gene ) were determined using Western blot showed that shAR can decrease FNTA expression in UMUC3 oeAR can increase FNTA expression in J82 .

(A) The protein expression of AR and FNTA (circFNTA host gene) were determined using Western blot and showed that shAR can decrease FNTA protein expression in UMUC3 cells and oeAR can increase FNTA protein expression in J82 cells.


( B ) qRT - PCR results showed that mRNA of FNTA are not changed dramatically in UMUC3 with shAR J82 with oeAR .

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(B) qRT-PCR results showed that mRNA levels of FNTA are not changed dramatically in UMUC3 cells with shAR and J82 cells with oeAR.


( C ) mRNA of 4 editing genes ( ADAR , ADAR2 , DHX9 , QKI ) were tested with manipulated AR ( UMUC3 in left panel J82 in right panel ) . Results showed ADAR2 mRNA is negatively correlated with AR expression .

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(C) The mRNA levels of 4 RNA editing genes (ADAR, ADAR2, DHX9, and QKI) were tested with manipulated AR (UMUC3 cells in left panel and J82 cells in right panel). Results showed ADAR2 mRNA is negatively correlated with AR expression.


( D ) Western blot also confirmed that ADAR2 can be negatively regulated by AR at level .

(D) Western blot analysis also confirmed that ADAR2 can be negatively regulated by AR at the protein level.


( E ) Knock down efficiency of AR ADAR2 ( shADAR2 # 1 shADAR # 2 ) in UMUC3 , as determined by qRT - PCR .

(E) Knock down efficiency of AR and ADAR2 (shADAR2#1 and shADAR#2) in UMUC3 cells, as determined by qRT-PCR.


( F ) shADAR2 # 1 shADAR # 2 could both reverse shAR - dependent circFNTA reduction in UMUC3 .

(F) shADAR2#1 and shADAR#2 could both reverse shAR-dependent circFNTA reduction in UMUC3 cells.


( G ) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR - dependent UMUC3 invasion reduction . Scale bar , 100 μm .

(G) Transwell invasion assays confirmed that both shADAR2 constructs can partly reverse shAR-dependent UMUC3 cells invasion reduction. Scale bar, 100 μm.


( H ) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR - dependent effects on cisplatin chemo - resistance ( 2 μg / ml ) in UMUC3 .

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(H) MTT assays confirmed that both shADAR2 constructs can partly overcome shAR-dependent effects on cisplatin chemo-resistance (2 μg/ml) in UMUC3 cells.


( J ) ChIP assays confirmed that AR can directly bind to ADAR2 AREI / II ( - 572nt to - 555nt ) .

(J) ChIP assays confirmed that AR can directly bind to ADAR2 AREI/II (-572nt to -555nt).


( K ) ChIP pull - down products were amplified by qRT - PCR reaction . IgG antibody was used as a negative control .

(K) ChIP pull-down DNA products were amplified by qRT-PCR reaction. The IgG antibody was used as a negative control.


( M ) Co - transfection of ARE Wild type ( WT ) or Mutant ( Mut ) ADAR2 promoter pGL3 - Luciferase constructs into UMUC3 with / without shAR ( left panel ) , J82 with / without oeAR ( right panel ) . luciferase was used to detect promoter activity .

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(M) Co-transfection of ARE Wild type (WT) or Mutant (Mut) ADAR2 promoter pGL3-Luciferase constructs into UMUC3 cells with/without shAR (left panel), and J82 cells with/without oeAR (right panel). The luciferase assay was used to detect promoter activity.


( A ) Western blot showed that shcircFNTA can decrease FNTA expression in UMUC3 ( upper panel ) oecircFNTA can increase FNTA expression , that this was more profound than using oelinearFNTA in J82 ( lower panel ) .

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(A) Western blot analysis showed that shcircFNTA can decrease FNTA expression in UMUC3 cells (upper panel) and oecircFNTA can increase FNTA expression, and that this was more profound than using oelinearFNTA in J82 cells (lower panel).


( B ) A pull - down was used to binding of circFNTA to 11 miRNAs ( predicted by online database ) in UMUC3 . results showed that miR - 328 - 5p , miR - 370 - 3p miR - 920 can be pulled - down by circFNTA .

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(B) A RNA pull-down assay was used to test the binding of circFNTA to 11 miRNAs (predicted by online database) in UMUC3 cells. The results showed that miR-328-5p, miR-370-3p and miR-920 can be pulled-down by circFNTA.


( C ) Relative miRNA expressions were quantified by qRT - PCR to miRNA overexpression efficacy .

(C) Relative miRNA expressions were quantified by qRT-PCR to test miRNA overexpression efficacy.


Transwell invasion ( D ) confirmed that only oemiR - 370 - 3p can reverse oecircFNTA induced invasion in J82 ,

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Transwell invasion assay (D) confirmed that only oemiR-370-3p can reverse oecircFNTA induced invasion in J82 cells,


MTT assays ( E ) confirmed that oemiR - 370 - 3p can reverse oecircFNTA induced cisplatin chemo - resistance ( 2 μg / ml ) in J82 . Scale bar , 100 μm .

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MTT assays (E) confirmed that oemiR-370-3p can reverse oecircFNTA induced cisplatin chemo-resistance (2 μg/ml) in J82 cells. Scale bar, 100 μm.


Transducing miR - 370 - 3p inhibitor into UMUC3 can reverse FNTA reduction by shcircFNTA shown by Western blot ( F )

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Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Western blot (F)


Transducing miR - 370 - 3p inhibitor into UMUC3 can reverse FNTA reduction by shcircFNTA shown by Transwell ( G ) .

Transducing the miR-370-3p inhibitor into UMUC3 cells can reverse FNTA protein reduction by shcircFNTA shown by Transwell assay (G).


( H ) MTT assays also confirmed that transducing with a miR - 370 - 3p inhibitor in UMUC3 can partly reverse cisplatin chemo - resistance ( 2 μg / ml ) reduction by shcircFNTA .

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(H) MTT assays also confirmed that transducing with a miR-370-3p inhibitor in UMUC3 cells can partly reverse cisplatin chemo-resistance (2 μg/ml) reduction by shcircFNTA.


( J ) transfection efficacy of oecircFNTA WT oecircFNTA Mut was tested by qRT - PCR .

(J) The transfection efficacy of oecircFNTA WT and oecircFNTA Mut was tested by qRT-PCR.


( K ) Transfection with oecircFNTA WT could increase J82 invasion , while oecircFNTA Mut cannot . Scale bar , 100 μm .

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(K) Transfection with oecircFNTA WT could increase J82 cells invasion, while oecircFNTA Mut cannot. Scale bar, 100 μm.


( L ) Tramsfection with oecircFNTA WT could increase J82 cisplatin chemo - resistance ( 2 μg / ml ) , while oecircFNTA Mut did not have this effect .

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(L) Tramsfection with oecircFNTA WT could increase J82 cells cisplatin chemo-resistance (2 μg/ml), while oecircFNTA Mut did not have this effect.


( N ) Luciferase reporter activity after transfection of WT Mut FNTA 3 ' UTR reporter constructs into UMUC3 ( left panel ) , comparing transduced miR - 370 - 3p inhibitor vs Ctrl J82 ( right panel ) , comparing oemiR - 370 - 3p vs control PLV .

(N) Luciferase reporter activity after transfection of WT and Mut FNTA 3'UTR reporter constructs into UMUC3 cells (left panel), comparing transduced miR-370-3p inhibitor vs Ctrl and J82 cells (right panel), and comparing oemiR-370-3p vs control PLV.


( A ) Western blots show knock - down efficiency of shFNTA in J82 UMUC3 .

(A) Western blots show the knock-down efficiency of shFNTA in J82 and UMUC3 cells.


( B ) Transwell assays indicated that shFNTA can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Quantifications are shown on right . Scale bar , 100 μm .

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(B) Transwell assays indicated that shFNTA can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Quantifications are shown on the right. Scale bar, 100 μm.


( C ) MTT assays also confirmed that shFNTA can reverse oecircFNTA - increased chemo - resistance to cisplatin ( 2 μg / ml ) in J82 UMUC3 .

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(C) MTT assays also confirmed that shFNTA can reverse oecircFNTA-increased chemo-resistance to cisplatin (2 μg/ml) in J82 and UMUC3 cells.


Transwell assays ( D , with quantitations at right ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Scale bar , 100 μm .

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Transwell assays (D, with quantitations at the right) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


MTT assays ( E ) indicated that treatment with Tipifarnib ( farnesyltransferase inhibitor ) can partly reverse oecircFNTA - increased invasion in J82 UMUC3 . Scale bar , 100 μm .

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MTT assays (E) indicated that treatment with Tipifarnib (farnesyltransferase inhibitor) can partly reverse oecircFNTA-increased invasion in J82 and UMUC3 cells. Scale bar, 100 μm.


( F ) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 did not change KRAS level significantly .

(F) Western blots showed that shcircFNTA in UMUC3 or oecircFNTA in J82 cells did not change KRAS protein level significantly.


( G ) Raf - 1 RBD GST - pull - down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 ( upper panel ) , oecircFNTA can increase KRAS activity in J82 ( lower panel ) .

(G) Raf-1 RBD GST-pull-down assays confirmed that shcircFNTA can decease KRAS activity in UMUC3 cells (upper panel), and oecircFNTA can increase KRAS activity in J82 cells (lower panel).


( H ) Western blots showed that shcircFNTA can decrease ERK1 / 2 MEK1 / 2 phosphorylation in UMUC3 ( left panel ) , oecircFNTA can increase their phosphorylation in J82 ( right panel ) .

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(H) Western blots showed that shcircFNTA can decrease ERK1/2 and MEK1/2 phosphorylation in UMUC3 cells (left panel), and oecircFNTA can increase their phosphorylation in J82 cells (right panel).


( A ) UMUC3 - PLKO UMUC3 - shcircFNTA were used to establish a subcutaneous nude mouse xenograft model , cisplatin ( 1 mg / kg ) was intraperitoneally administered . of tumors are shown after mice were sacrificed ( N = 6 ) . ( B ) Compared with vector ( PLKO ) group , growth rate was significantly inhibited in shcircFNTA nude mice . ( C ) weights also decreased in shcircFNTA group .

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(A) UMUC3-PLKO and UMUC3-shcircFNTA cells were used to establish a subcutaneous nude mouse xenograft model, and cisplatin (1 mg/kg) was intraperitoneally administered. Images of tumors are shown after mice were sacrificed (N= 6). (B) Compared with the vector (PLKO) group, the tumor growth rate was significantly inhibited in shcircFNTA nude mice. (C) Tumor weights also decreased in the shcircFNTA group.


( D - E ) qRT - PCR results using mouse tissues revealed that circFNTA expression was significantly reduced ( D ) , expression of its host gene FNTA was correspondently decreased in shcircFNTA group as compared to PLKO group ( E ) .

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(D-E) The qRT-PCR results using mouse tumor tissues revealed that circFNTA expression was significantly reduced (D), and the expression of its host gene FNTA was correspondently decreased in the shcircFNTA group as compared to the PLKO group (E).


( F ) Representative immunohistochemistry ( ) detecting FNTA , p - ERK1 / 2 p - MEK1 / 2 in PLKO tumurs as compared to shcircFNTA xenografted tumors . Scale bars : 100 μm ( upper panels ) 25 μm ( lower panels ) . ( G ) of relative revealed that targeting circFNTA led to decreased expression of FNTA , p - ERK1 / 2 p - MEK1 / 2 .

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(F) Representative immunohistochemistry (IHC) images detecting FNTA, p-ERK1/2 and p-MEK1/2 in PLKO tumurs as compared to shcircFNTA xenografted tumors. Scale bars: 100 μm (upper panels) and 25 μm (lower panels). (G) Quantification of relative IHC staining intensity revealed that targeting circFNTA led to decreased expression of FNTA, p-ERK1/2 and p-MEK1/2.


( H ) tail vein injection model was established to observe lung metastasis ( N = 6 ) . Representative of lung metastasis foci in gross microscopic HE examination . Scale bars : 400 μm ( upper panels ) 100 μm ( lower panels ) . ( I ) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to PLKO group . ( J ) Targeting circFNTA significantly reduced lung metastatic foci as compared to PLKO group .

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(H) The tumor tail vein injection model was established to observe lung metastasis (N=6). Representative images of lung metastasis foci in gross and microscopic HE examination. Scale bars: 400 μm (upper panels) and 100 μm (lower panels). (I) Knocking down circFNTA significantly decreased metastatic foci numbers as compared to the PLKO group. (J) Targeting circFNTA significantly reduced lung metastatic foci as compared to the PLKO group.


( A ) Autophagic markers from mixed midbrain gba + / + , gba + / − , gba − / − cultures were analyzed via western blotting using antibodies as indicated .

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(A) Autophagic markers from mixed midbrain gba+/+, gba+/−, and gba−/− cultures were analyzed via western blotting using antibodies as indicated.


( B ) Autophagic flux was analyzed in gba + / + gba − / − midbrain neurons . Neurons were treated with 100 nM bafilomycin A1 1 μM rapamycin flux assayed by comparing LC3I LC3II via western blotting . β - actin was used as a loading control . ( C ) Densitometry analyzes of autophagy of ( B ) expressed as a of LC3II / LC3I . Error bars , ± . * p < 0 . 05 .

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(B) Autophagic flux was analyzed in gba+/+ and gba−/− midbrain neurons. Neurons were treated with 100 nM bafilomycin A1 and 1 μM rapamycin and flux assayed by comparing LC3I and LC3II levels via western blotting. β-actin was used as a loading control.(C) Densitometry analyzes of autophagy of (B) expressed as a ratio of LC3II/LC3I. Error bars, ± SEM. *p < 0.05.


( D ) Accumulation of p62 / SQSTM1 was analyzed via immunoblotting . β - actin was used as a loading control .

(D) Accumulation of p62/SQSTM1 was analyzed via immunoblotting. β-actin was used as a loading control.


( E ) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO . were extracted analyzed via western blotting stained with anti - ubiquitin antibodies .

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(E) Midbrain neurons were treated with either 10 μM MG132 or vehicle control DMSO. Proteins were extracted and analyzed via western blotting and stained with anti-ubiquitin antibodies.


( F ) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated . β - actin was used as a loading control .

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(F) Ubiquitin linkage of isolated midbrain analyzed using western blotting with antibodies as indicated. β-actin was used as a loading control.


( G ) Proteasomal activity ( chymotrypsin activity ) was measured using Proteasome Glo where activity is proportional to released luciferase . RFU , relative luciferase units . Data represent mean ± ( n = 3 , each experiment contained triplicates for each genotype ) . * p < 0 . 05 .

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(G) Proteasomal activity (chymotrypsin activity) was measured using the Proteasome Glo assay where activity is proportional to the released luciferase. RFU, relative luciferase units. Data represent the mean ± SEM (n = 3, each experiment contained triplicates for each genotype). *p < 0.05.


( A ) of soluble insoluble α - synuclein from midbrain of P1 gba + / + gba − / − mice . TX - 100 soluble insoluble ( SDS / Urea ) factions were isolated analyzed via western blotting with α - synuclein antibodies . β - actin was used as a loading control .

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(A) Levels of soluble and insoluble α-synuclein from midbrain of P1 gba+/+ and gba−/− mice. TX-100 soluble and insoluble (SDS/Urea) factions were isolated and analyzed via western blotting with α-synuclein antibodies. β-actin was used as a loading control.


( B ) Sagittal brain sections from gba + / + , gba + / − , gba − / − mice were stained with α - synuclein antibodies . Scale bar , 1 , 000 μm .

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(B) Sagittal brain stem sections from gba+/+, gba+/−, and gba−/− mice were stained with α-synuclein antibodies. Scale bar, 1,000 μm.


( A ) Neurons astrocytes from gba + / + , gba + / − , gba − / − mice were stained with TMRM . mean florescence in mitochondria was analyzed via confocal microscopy ( n = 3 , > 32 analyzed / experiment ) .

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(A) Neurons and astrocytes from gba+/+, gba+/−, and gba−/− mice were stained with TMRM. The mean florescence intensity in mitochondria was analyzed via confocal microscopy (n = 3, >32 cells analyzed/experiment).


( B ) Neurons from gba + / + gba − / − bathed in TMRM containing recording solution analyzed via confocal microscopy . After 1 min , 1 μM oligomycin was added followed by 1 μM FCCP ( n = 5 , three analyzed / experiment ) .

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(B) Neurons from gba+/+ and gba−/− bathed in TMRM containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 1 min, 1 μM oligomycin was added followed by 1 μM FCCP (n = 5, three cells analyzed/experiment).


( C ) Neurons from gba + / + gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . bathed in 25 nM TMRM 5 mM methyl pyruvate containing recording solution analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three analyzed / experiment ) .

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(C) Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).


( D ) As in ( C ) , except were bathed in 10 mM methyl succinate , ( n = 3 , four / experiment ) . data in this figure represent mean ± , * p < 0 . 05 . Neurons from gba + / + gba − / − incubated with 5 mM methyl pyruvate 5 min prior to imaging . bathed in 25 nM TMRM 5 mM methyl pyruvate containing recording solution analyzed via confocal microscopy . After 90 s , 1 μM oligomycin was added , followed by 1 μM FCCP ( n = 3 , three analyzed / experiment ) .

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(D) As in (C), except cells were bathed in 10 mM methyl succinate, (n = 3, four cells/experiment). All data in this figure represent the mean ± SEM, *p < 0.05. Neurons from gba+/+ and gba−/− incubated with 5 mM methyl pyruvate 5 min prior to imaging. Cells bathed in 25 nM TMRM and 5 mM methyl pyruvate containing recording solution and fluorescence intensity analyzed via confocal microscopy. After 90 s, 1 μM oligomycin was added, followed by 1 μM FCCP (n = 3, three cells analyzed/experiment).


( A ) Oxygen consumption rates of gba + / + , gba + / − , gba − / − mixed midbrain cultures . Basal oxygen consumption was measured over 3 min . maximal ( uncoupled ) rate was measured via addition of FCCP nonmitochondrial oxygen consumption analyzed via addition of antimycin A ( n = 3 , three runs / experiment ) .

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(A) Oxygen consumption rates of gba+/+, gba+/−, and gba−/− mixed midbrain cultures. Basal oxygen consumption was measured over 3 min. The maximal (uncoupled) rate was measured via the addition of FCCP and the nonmitochondrial oxygen consumption analyzed via the addition of antimycin A (n = 3, three runs/experiment).


( B ) I - NADH : Ubiquinone reductase activity from gba + / + , gba + / − , gba − / − brains expressed as a to citrate synthase ( n = 3 ) .

(B) Complex I-NADH: Ubiquinone reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( C ) II - III : Succinate dehydeogenase , cytochome c reductase activity from gba + / + , gba + / − , gba − / − brains expressed as a to citrate synthase ( n = 3 ) .

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(C) Complex II-III: Succinate dehydeogenase, cytochome c reductase activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( D ) IV activity from gba + / + , gba + / − , gba − / − brains expressed as a to citrate synthase ( n = 3 ) .

(D) Complex IV activity from gba+/+, gba+/−, and gba−/− brains expressed as a ratio to citrate synthase (n = 3).


( E ) Neurons were treated with 3 nM decylTPP MitoQ10 for 48 hr stained with TMRM analyzed via confocal microscopy ( n = 3 , > 16 / experiment ) .

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(E) Neurons were treated with 3 nM decylTPP and MitoQ10 for 48 hr and stained with TMRM and fluorescence intensity analyzed via confocal microscopy (n = 3, >16 cells/experiment).


( F ) gba + / + gba − / − treated with 3 nM decylTPP MitoQ10 for 48 hr were subjected to immunoblotting using indicted antibodies . data in this figure represent mean ± , * p < 0 . 05 .

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(F) gba+/+and gba−/− cells treated with 3 nM decylTPP and MitoQ10 for 48 hr were subjected to immunoblotting using the indicted antibodies. All data in this figure represent the mean ± SEM, *p < 0.05.


( A ) Neurons astrocytes were immunostained for cytochrome c stained with Hoechst . Far left panels were additionally immunostained for GFAP MAP2 to mark neurons astrocytes . " A " in nucleus denotes astrocytes . Scale bar , 20 μm . ( B ) Mitochondrial was blind counted from in ( A ) with neurons scored as MAP2 positive astrocytes as GFAP positive . A " normal " is classified as a mixture of fused fragmented mitochondria that comprise a wild - type network . ( n = 3 ; 100 counted / experiment ) .

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(A) Neurons and astrocytes were immunostained for cytochrome c and stained with Hoechst. Far left panels were additionally immunostained for GFAP and MAP2 to mark neurons and astrocytes. "A" in nucleus denotes astrocytes. Scale bar, 20 μm.(B) Mitochondrial morphology was blind counted from cells in (A) with neurons scored as MAP2 positive and astrocytes as GFAP positive. A "normal" morphology is classified as a mixture of fused and fragmented mitochondria that comprise a wild-type network. (n = 3; 100 cells counted/experiment).


( C ) of from gba + / + , gba + / − , gba − / − isolated mitochondria were analyzed via immunoblotting with indicated antibodies .

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(C) Levels of morphology proteins from gba+/+, gba+/−, and gba−/− isolated mitochondria were analyzed via immunoblotting with the indicated antibodies.


( D ) Midbrain gba + / + , gba + / − , gba − / − astrocytes expressing GFP - DRP1K38A were immunostained for cytochrome c mitochondrial morphologies analyzed ; concurrently were stained with TMRM analyzed via confocal microscopy . Scale bar , 20 μm ( n = 3 , > 15 / experiment ) . data in this figure represent mean ± .

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(D) Midbrain gba+/+, gba+/−, and gba−/− astrocytes expressing GFP-DRP1K38A were immunostained for cytochrome c and mitochondrial morphologies analyzed; concurrently cells were stained with TMRM and fluorescence intensity analyzed via confocal microscopy. Scale bar, 20 μm (n = 3, >15 cells/experiment). All data in this figure represent the mean ± SEM.


( A ) Midbrain astrocytes were transfected with GFP - LC3 , treated with 100 nM bafilomycin A1 , stained with MitoTracker Red imaged using confocal microscopy .

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(A) Midbrain astrocytes were transfected with GFP-LC3, treated with 100 nM bafilomycin A1, and stained with MitoTracker Red and imaged using confocal microscopy.


( B ) Midbrain gba + / + gba − / − neurons astrocytes were transfected with YFP - Parkin . Half transfected were treated for 1 hr with 10 μM FCCP bathed in TMRM containing recording solution imaged using confocal microscopy . Scale bar , 20 μm . ( C ) TMRM of from ( B ) was analyzed via confocal microscopy . Data represent mean ± , ( n = 3 , > 4 analyzed per experiment ) .

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(B) Midbrain gba+/+ and gba−/− neurons and astrocytes were transfected with YFP-Parkin. Half the transfected cells were treated for 1 hr with 10 μM FCCP and all bathed in TMRM containing recording solution and imaged using confocal microscopy. Scale bar, 20 μm.(C) TMRM fluorescence intensity of cells from (B) was analyzed via confocal microscopy. Data represent the mean ± SEM, (n = 3, >4 cells analyzed per experiment).


( D ) Mitochondria within midbrain neurons were uncoupled by addition of 10 μM FCCP for 1 6 hr . PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba + / + gba − / − neurons . Mitochondrial CI subunit NDUFS4 was used as a loading control .

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(D) Mitochondria within midbrain neurons were uncoupled by the addition of 10 μM FCCP for 1 and 6 hr. PINK1 expression analyzed via immunoblotting on isolated mitochondria from gba+/+ and gba−/− neurons. Mitochondrial CI subunit NDUFS4 was used as a loading control.


( A ) Representative of immunofluorescence ( with anti - PlxnB2 antibodies ) of phenotype of COS7 transfected with wild - type or mutated PlxnB2 , compared to collapsed phenotype induced by cognate ligand Sema4C ( 1 μg / ml ) in expressing WT receptor . ( B ) stacked bar graph shows fraction of collapsed PlxnB2 - expressing , identified as having a surface minor of 2000 μm2 ( measured by ImageJ software ) , out of 50 - 100 counted per each condition , from different fields n > 3 experiments ) . statistical was performed comparing WT untreated with other groups , by Fisher ' s Exact , which indicated a * * * * * p value < 0 . 00001 for Sema4C - treated for G842C - PlxnB2 - expressing ( confirmed by ANOVA on values , shown in Appendix Fig . S2 ) . By Fisher ' s , only other mutants significantly different from WT were R820H ( p < 0 . 001 ) R843Q ( p < 0 . 01 ) , although ANOVA on values shown in Appendix Fig . S2 didn ' t confirm such significance for other PlxnB2 variants .

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(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.


( A ) graphs show of viability in shPlxnB2 - tranduced agnospheres , compared to their respective scramble - transfected controls , after 24 , 48 , 72 , 96 hours of growth in culture . Values are mean ± SD of n ≥ 3 independent experiments ( 6 technical replicates for each ) . statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons ( Graph Pad Prism 8 . 0 . 0 ) : * * p = 0 . 0053 ; * * * * p < 0 . 0001 .

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(A) The graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001.


( C ) After transplantation in immunodeficient mice of CUP described above , volume of ADX43 - ( D ) ADX901 - shPlxnB2 tumors was assessed , in comparison with respective controls . Plotted values indicate mean ± SD ( n = 8 each group ) . statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons : * * p = 0 . 0060 ; * * * * p < 0 . 0001 .

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(C) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.