( F ) Induction of autophagy by TBD . HeLa stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or constructs encoding indicated BCN1 variants . After 24 h , Flag ‐ tagged were detected by immunoblotting frequency of with > 5 GFP - LC3 + ( GFP - LC3VAC ) was assessed ( mean values ± s . d . , n = 3 ; * P0 . 01 , * * P0 . 001 versus pcDNA3 . 1 ‐ transfected ) .

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(F) Induction of autophagy by the TBD. HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or constructs encoding the indicated BCN1 variants. After 24 h, Flag‐tagged proteins were detected by immunoblotting and the frequency of cells with >5 GFP-LC3+cytosolic puncta (GFP-LC3VAC cells) was assessed (mean values±s.d., n=3; *P0.01, **P0.001 versus pcDNA3.1‐transfected cells).


( G , H ) Mechanisms of autophagy induction by TBD . ( G ) U2OS stably expressing RFP - FYVE were transfected pcDNA3 . 1 or plasmids for expression of TBD , BCN1 or BCN1ΔTBD in combination with a control siRNA ( siUNR ) or siRNAs that effectively deplete VPS4 , BCN1 TAK1 . Forty ‐ eight hours later , of exhibiting FYVE - RFP + dots ( FYVE - RFP + ) was assessed . ( G ) . Alternatively , HeLa stably expressing GFP - LC3 were transfected with pcDNA3 . 1 or with a TBD ‐ encoding plasmids plus siUNR or siRNAs specific indicated . Forty ‐ eight hours later , of GFP - LC3VAC was determined ( H ) . Results are mean values ± s . d . ( n = 3 , * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected ) .

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(G, H) Mechanisms of autophagy induction by the TBD. (G) U2OS cells stably expressing RFP-FYVE were transfected pcDNA3.1 or plasmids for the expression of TBD, BCN1 or BCN1ΔTBD in combination with a control siRNA (siUNR) or siRNAs that effectively deplete VPS4, BCN1 and TAK1. Forty‐eight hours later, the percentage of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+) was assessed. (G). Alternatively, HeLa cells stably expressing GFP-LC3 were transfected with pcDNA3.1 or with a TBD‐encoding plasmids plus siUNR or siRNAs specific the indicated proteins. Forty‐eight hours later, the percentage of GFP-LC3VAC cells was determined (H). Results are mean values±s.d. (n=3, *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( I ) Inhibition of TBD ‐ induced autophagy by dominant ‐ negative ( DN ) TAK1 . were co ‐ transfected pcDNA3 . 1 or vectors encoding WT TAK1 ( TAK1WT ) or a DN TAK1 variant ( TAK1K63W ) alone or together with a TBD ‐ encoding plasmid for 24 h , followed by of GFP - LC3VAC ( mean values ± s . d . , n = 3 ; * P0 . 01 versus pcDNA3 . 1 ‐ transfected ) .

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(I) Inhibition of TBD‐induced autophagy by dominant‐negative (DN) TAK1. Cells were co‐transfected pcDNA3.1 or vectors encoding WT TAK1 (TAK1WT) or a DN TAK1 variant (TAK1K63W) alone or together with a TBD‐encoding plasmid for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus pcDNA3.1‐transfected cells).


Comparison of SWATH - MS - to DDA - MS - strategies ( MS1 XIC spectral counting ) with regard to consistency ( as judged upon - level SEC chromatogram robustness towards increasing requirements on number of consecutive detections ) precision ( judged on correlation between sibling peptide SEC chromatograms ) of overall performance in error - controlled - centric query of CORUM complexes in respective level chromatogram sets ( also see Appendix Figure S3C ) . TP , true positive assignments according to error model , P , positives .

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Comparison of SWATH-MS-based quantification to DDA-MS-based strategies (MS1 XIC and spectral counting) with regard to consistency (as judged based upon protein-level SEC chromatogram robustness towards increasing requirements on the number of consecutive detections) and precision (judged based on the correlation between sibling peptide SEC chromatograms) of quantification and overall performance in error-controlled complex-centric query of CORUM complexes in the respective protein level chromatogram sets (also see Appendix Figure S3C). TP, true positive assignments according to error model, P, all positives.


Peptide - level SEC chromatograms are grouped by UniprotKB identifier to detect co - elution signals indicative of elution ranges / peaks . on external calibration of apparent analyte molecular per SEC fraction , signals can be attributed to likely assembled or monomeric state ( also see Appendix Figure S4 ) . For TCPE , three distinct elution signals , numbered 1 - 3 , are detected , two in assembled one in monomer elution range .

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Peptide-level SEC chromatograms are grouped by UniprotKB identifier to detect co-elution signals indicative of protein elution ranges/peaks. Based on external size calibration of the apparent analyte molecular weight per SEC fraction, signals can be attributed to likely assembled or monomeric state (also see Appendix Figure S4). For TCPE, three distinct elution signals, numbered 1-3, are detected, two in the assembled and one in the monomer elution range.


are observed eluting in 1 - 6 distinct peaks with a wide range of apparent vs . monomeric molecular ratios ( distributions , left panels ) . molecular ratios of three peaks detected for TCPE ( displayed in A ) are indicated . Many of eluting in a single peak ( top panel bar ) appear assembled . eluting multiple times ( 27 % of ) do so preferentially in assembled range , suggesting frequent participation in multiple differently sized macromolecular assemblies ( lower panels pie chart ) . For a list of detected peaks , see Dataset EV3 .

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Proteins are observed eluting in 1-6 distinct peaks and with a wide range of apparent vs. monomeric molecular weight ratios (distributions, left panels). The molecular weight ratios of the three peaks detected for TCPE (displayed in A) are indicated. Many of the proteins eluting in a single peak (top panel and bar) appear assembled. Proteins eluting multiple times (27 % of the proteins) do so preferentially in the assembled range, suggesting frequent participation in multiple differently sized macromolecular assemblies (lower panels and pie chart). For a list of all detected protein peaks, see Dataset EV3.


observed in multiple assembled peaks ( n = 659 ) are enriched in components of proteasome other known large assemblies .

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Proteins observed in multiple assembled peaks (n = 659) are enriched in components of the proteasome and other known large complex assemblies.


For nearly half CORUM hypotheses queried , two or more distinct subunit co - elution signals were detected ( See methods Appendix information on CCprofiler ) .

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For nearly half the CORUM complex hypotheses queried, two or more distinct subunit co-elution signals were detected (See methods and Appendix information on CCprofiler).


Among four distinct co - elution signals detected from eight canonical CSN subunits ' chromatograms ( here with CSN7A , not CSN7B ) are two distinct signals indicating distinct co - elution of two different variants .

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Among the four distinct co-elution signals detected from the eight canonical CSN subunits' chromatograms (here with CSN7A, not CSN7B) are two distinct signals indicating distinct co-elution of two different complex variants.


Distinct co - elution of holo - CSN ( observed at expected fraction 35 ) Mini - CSN CSN1 / 3 / 8 ( observed eluting offset only one fraction late , F45 , of expected fraction , F44 ) . Expected fractions are estimated from cumulative sum of one copy per component external calibration . Coloring adapted to highlight subversion components their partitioning across holo - sub - . CSN1 / 3 / 8 interact form a submodule within CSN holo - structure ( PDB accession 4D10 ) . observations are consistent between two whole workflow replicates ( see Appendix Figure S6A ) . Lower right panel , validation of distinct elution behavior of holo - CSN exclusive ( CSN7A ) shared subunit ( CSN8 ) by immunoblotting . For full immunoblotting data ( CSN1 , CSN3 , CSN4 , CSN5 , CSN7A CSN8 ) see Appendix Figure S7C ) .

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Distinct co-elution of holo-CSN (observed at the expected fraction 35) and Mini-CSN CSN1/3/8 (observed eluting offset only one fraction late, F45, of the expected fraction, F44). Expected fractions are estimated from the cumulative sum of one copy per component and external size calibration. Coloring adapted to highlight subversion components and their partitioning across holo- and sub-complex. CSN1/3/8 interact and form a submodule within the CSN holo-complex structure (PDB accession 4D10). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6A). Lower right panel, validation of distinct elution behavior of holo-CSN exclusive (CSN7A) and shared subunit (CSN8) by immunoblotting. For full immunoblotting data (CSN1, CSN3, CSN4, CSN5, CSN7A and CSN8) see Appendix Figure S7C).


CSN1 / 3 / 8 display distinct fractionation patterns in co - fractionation experiments performed in other laboratories , specifically in orthogonal ion exchange fractionation of HEK293 lysates ( Wan et al . 2015 , upper panels ) exclusion chromatographic fractionation of U2OS lysates ( Kirkwood et al . 2013 , lower panel ) , in with CSN1 / 3 / 8 as distinct entity .

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CSN1/3/8 display distinct fractionation patterns in co-fractionation experiments performed in other laboratories, specifically in orthogonal ion exchange fractionation of HEK293 lysates (Wan et al. 2015, upper panels) and size exclusion chromatographic fractionation of U2OS lysates (Kirkwood et al. 2013, lower panel), in line with the CSN1/3/8 as distinct entity.


Baculoviral co - expression of human CSN1 , CSN3 CSN8 in Sf21VM insect , with CSN8 N - terminally Strep ( II ) - CSN1 & CSN3 N - terminally His6 - tagged , followed by affinity purification SDS - PAGE displays banding pattern in with formation of a stable trimer CSN1 / 3 / 8 .

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Baculoviral co-expression of human CSN1, CSN3 and CSN8 in Sf21VM insect cells, with CSN8 N-terminally Strep(II)- and CSN1 & CSN3 N-terminally His6-tagged, followed by affinity purification and SDS-PAGE displays banding pattern in line with the formation of a stable trimer CSN1/3/8.


- level SEC chromatograms of 22 canonical 26S proteasome subunits . Vertical black lines indicate apexes of six distinct co - elution signals detected in - centric scoring ; two of which represent well - known co - occurring variants , full 26S ( i ) 20S ( ii ) particle devoid 19S lid ATPase ( Indicated by structural models , PDB accession 5GJR ) four of which , composed of predominantly 20S α β subunits , appear at reduced ( 222 - 107 kDa , fractions 39 , 40 , 42 46 ) . observations are consistent between two whole workflow replicates ( see Appendix Figure S6B ) .

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Protein-level SEC chromatograms of the 22 canonical 26S proteasome subunits. Vertical black lines indicate the apexes of six distinct co-elution signals detected in complex-centric scoring; two of which represent well-known co-occurring variants, the full 26S (i) and the 20S (ii) particle devoid the 19S lid and ATPase (Indicated by structural models, PDB accession 5GJR) and four of which, composed of predominantly 20S α and β subunits, appear at reduced size (222 - 107 kDa, fractions 39, 40, 42 and 46). The observations are consistent between the two whole workflow replicates (see Appendix Figure S6B).


Subunit mass distribution across early late assembly intermediate elution ranges suggests predominant components of intermediary species accumulating in HEK293 .

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Subunit mass distribution across early and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells.


SECexplorer web - interface for querying custom sets for co - elution behavior in SEC - SWATH - MS data , viewing chromatograms for interpretation with algorithmic assistance .

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SECexplorer web-interface for querying custom protein sets for co-elution behavior in the SEC-SWATH-MS data, viewing chromatograms for interpretation and with algorithmic assistance.


Zoom in to high MW peak shoulder of holo - COP9 Signalosome ( compare Figure 5 ) , where defined co - elution signals of CSN substrate components CUL4A DDB2 suggest partial resolution of substrate - bound free pools of CSN holo - .

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Zoom in to high MW peak shoulder of holo-COP9 Signalosome (compare Figure 5), where defined co-elution signals of CSN substrate components CUL4A and DDB2 suggest the partial resolution of substrate-bound and free pools of CSN holo-complex.


Estimation of fraction of holo - CSN in likely substrate - bound pool versus free pool , with eight measurements along eight subunits on Gaussian deconvolution of two signals underlying observed peak shoulder ( also see Appendix Figure S7 ) .

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Estimation of the fraction of holo-CSN in the likely substrate-bound pool versus the free pool, with eight measurements along the eight subunits and based on Gaussian deconvolution of two signals underlying the observed peak and shoulder (also see Appendix Figure S7).


B - G . ( B , D , F ) E histograms of RDH substrate in absence of EXD2 ( gray ) after 0 - ( black ) , 100 - ( purple ) , 200 - s ( blue ) incubation with 200 ( B ) , 100 ( D ) , 5 ( F ) nM EXD2 . ( C , E , G ) Representative smFRET trajectories showing EXD2 at 200 ( C ) , 100 ( E ) , 5 ( G ) nM cleaving RDH in real time . intensities of donor acceptor are colored green red , respectively . corresponding FRET efficiencies are shown below in black . duration between two Cambridge blue lines represents time for FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 .

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B-G. (B, D, and F) E histograms of the RDH substrate in the absence of EXD2 (gray) and after 0- (black), 100- (purple), and 200-s (blue) incubation with 200 (B), 100 (D), and 5 (F) nM EXD2. (C, E, and G) Representative smFRET trajectories showing EXD2 at 200 (C), 100 (E), and 5 (G) nM cleaving RDH in real time. The fluorescence intensities of the donor and acceptor are colored green and red, respectively. The corresponding FRET efficiencies are shown below in black. The duration between two Cambridge blue lines represents the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9.


H . FRET trajectories ' percentages show FRET values ending above 0 . 9 between 0 . 2 0 . 9 . word " Surface " represents experiment with EXD2 anchored on glass surface . I . Statistics of time for FRET efficiency to increase from ~ 0 . 2 to ~ 0 . 9 under indicated experimental conditions . In violin plots , - blue dots represent mean , black bars show interquartile range ( IQR ) ( thick bars ) 1 . 5 times IQR ( thin bars ) ( n = 122 , 91 , 78 , 101 from left to right ) .

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H. The FRET trajectories' percentages show the FRET values ending above 0.9 and between 0.2 and 0.9. The word "Surface" represents the experiment with EXD2 anchored on the glass surface. I. Statistics of the time for the FRET efficiency to increase from ~ 0.2 to ~ 0.9 under indicated experimental conditions. In violin plots, light-blue dots represent mean, and black bars show the interquartile range (IQR) (thick bars) and 1.5 times IQR (thin bars) (n = 122, 91, 78, and 101 from left to right).


D . A representative gel showing that EXD2 ( 500 nM ) cleaves Cy5 - Cy5 - RDH substrates .

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D. A representative gel showing that EXD2 (500 nM) cleaves the Cy5-RNA and Cy5-RDH substrates.


F . kinetic rates k1 k2 obtained from double exponential fitting to dwell - time histograms of Pauses 1 - 3 their combination ( Total ) . Fit parameters 95 % confidence intervals are shown .

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F. The kinetic rates k1 and k2 obtained from the double exponential fitting to the dwell-time histograms of Pauses 1-3 and their combination (Total). Fit parameters and 95% confidence intervals are shown.


A - F . ( A D ) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of GC - RDH substrate under 1 mM MnCl2 ( A ) or RDH substrate under 0 . 2 mM MnCl2 ( D ) . solid red lines are step - fitting results from an automated step - finding algorithm . ( B E ) corresponding TDP of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( B ) or RDH under 0 . 2 mM MnCl2 ( E ) . ( C F ) dwell - time histograms of combined pauses ( Total ) in process of EXD2 cleavage of GC - RDH under 1 mM MnCl2 ( C ) or RDH under 0 . 2 mM MnCl2 ( F ) . histogram fittings to a double exponential function are shown in red . R2 of double exponential function are 0 . 94 0 . 95 , respectively .

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A-F. (A and D) Representative smFRET trajectories showing a stepwise FRET increase during EXD2 cleavage of the GC-RDH substrate under 1 mM MnCl2 (A) or the RDH substrate under 0.2 mM MnCl2 (D). The solid red lines are the step-fitting results from an automated step-finding algorithm. (B and E) The corresponding TDP of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (B) or RDH under 0.2 mM MnCl2 (E). (C and F) The dwell-time histograms of combined pauses (Total) in the process of EXD2 cleavage of GC-RDH under 1 mM MnCl2 (C) or RDH under 0.2 mM MnCl2 (F). The histogram fittings to a double exponential function are shown in red. The R2 of the double exponential function are 0.94 and 0.95, respectively.


( B ) ARSA activity ( nmol / h / mg ) after treatment of MSDi with a selection of four positive hit drugs at a final of 10 µM on MSDi in 25cm2 culture flasks for three days . Data represent mean ± SD of 7 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . Difference against negative

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(B) ARSA activity quantification (nmol/h/mg) after treatment of MSDi cells with a selection of four positive hit drugs at a final concentration of 10 µM on MSDi cells in 25cm2 cell culture flasks for three days. Data represent mean ±SD of 7 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against negative control


( E ) of different retinoids ' potential to restore ARSA activity in MSDi in comparison to tazarotene after treatment for 3 days at a final 10 µM of each drug ( Adapalene 1 µM ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . Difference against DMSO control : * *

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(E) Analysis of different retinoids' potential to restore ARSA activity in MSDi cells in comparison to tazarotene after treatment for 3 days at a final concentration 10 µM of each drug (Adapalene 1 µM). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Difference against DMSO control: **


( D ) of a time dependent increase of ARSA activity in MSD fibroblasts ( variant FGE Gly247Arg homozygous ) simultaneously treated with 10 20 µM tazarotene bexarotene , respectively , up to 21 days . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . Displayed are significance for next significant difference between adjacent treatment times . # # # # p < 0 . 0001 . Difference against 0 days control : * p < 0 . 05 , * * * * p < 0 . 0001 .

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(D) Analysis and quantification of a time dependent increase of ARSA activity in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) simultaneously treated with 10 and 20 µM tazarotene and bexarotene, respectively, up to 21 days. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent treatment times. #### p<0.0001. Difference against 0 days control: * p<0.05, **** p<0.0001.


( E ) of increased sulfatase activities different to ARSA , namely ARSB , GALNS , STS in MSD fibroblasts ( variant FGE Gly247Arg homozygous ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 - 6 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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(E) Analysis and quantification of increased sulfatase activities different to ARSA, namely ARSB, GALNS, and STS in MSD primary fibroblasts (variant FGE Gly247Arg homozygous) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3-6 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001, **** p<0.0001.


( F ) of ARSA activities in MSD fibroblasts with different homozygous SUMF1 mutations ( FGE Gly247Arg , FGE Gly263Val , FGE Ala279Val , FGE Arg349Trp ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . * * * * p < 0 . 0001 .

O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE B-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Quantification of ARSA activities in MSD primary fibroblasts with different homozygous SUMF1 mutations (FGE Gly247Arg, FGE Gly263Val, FGE Ala279Val, FGE Arg349Trp) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ****p<0.0001.


( G ) of ARSA activity in MSD - derived iPSCs differentiated into NPCs unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene 5 µM bexarotene for 4 days . Data represent mean ± SD of 6 independent experiments ( biological replicates ) . Unpaired t - . * * * * p < 0 . 0001 . ( H ) of SGSH activity in MSD - derived iPSCs differentiated into NPCs unaffected control NPCs controls . Simultaneous treatment with 5 µM tazarotene 5 µM bexarotene for 4 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . Unpaired t - . * p < 0 . 05 , * * * p < 0 . 001 .

O O O O B-GENEPROD B-EXP_ASSAY O B-DISEASE O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O 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-GENEPROD B-EXP_ASSAY O B-DISEASE O O B-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Quantification of ARSA activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 6 independent experiments (biological replicates). Unpaired t-test. **** p<0.0001. (H) Quantification of SGSH activity in MSD patient-derived iPSCs differentiated into NPCs and unaffected control NPCs controls. Simultaneous treatment with 5 µM tazarotene and 5 µM bexarotene for 4 days. Data represent mean ±SD of 3 independent experiments (biological replicates). Unpaired t-test. * p<0.05, *** p<0.001.


( A ) Representative confocal of control MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green ) DAPI ( nuclei , blue ) . ( B ) Representative confocal of control MSD fibroblasts with either tazarotene / bexarotene ( 10 / 20 µM , 6 days ) . Labelling with anti - LAMP1 antibody ( green ) DAPI ( nuclei , blue ) . ( C ) of total of LAMP1 - green . N = 20 13 z - series optical sections per condition with a step - of 0 . 26 microns , displayed at maximum extension automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD compared to DMSO treated control ) , # p < 0 . 05 ( MSD DMSO vs MSD treated ) . RFU : relative units . ( D ) of LAMP1 - green vesicle ( µm ) . N = 20 13 z - series optical sections per condition with a step - of 0 . 26 microns , displayed at maximum extension automated equalization of brightness . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . * * * p < 0 . 001 ( DMSO treated MSD compared to DMSO treated control ) , # # p < 0 . 01 ( MSD DMSO vs MSD treated ) .

O O O O B-EXP_ASSAY O O B-DISEASE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O B-EXP_ASSAY O O B-DISEASE B-CELL_TYPE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O O O O O O B-DISEASE B-SMALL_MOLECULE O B-DISEASE O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-DISEASE O O B-SMALL_MOLECULE O O O O O O O O O O O O B-DISEASE B-SMALL_MOLECULE O B-DISEASE O O O

(A) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (B) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). (C) Quantification of total intensity of LAMP1- green fluorescence. N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), # p<0.05 (MSD DMSO vs MSD treated). RFU: relative fluorescence units. (D) Quantification of LAMP1- green fluorescence vesicle size (µm). N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), ## p<0.01 (MSD DMSO vs MSD treated).


( A ) ARSA activity upon treatment of MSDi with 10 µM tazarotene , 20 µM bexarotene , 10 µM tazarotene 20 µM bexarotene in combination with increasing concentrations of pan - RAR receptor antagonist AGN 193109 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 3 - 11 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s for multiple comparisons . Displayed are significance for next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 , # # # p < 0 . 001 . Difference against 0 µM DMSO control ( B ) ARSA activity upon treatment of MSDi with 10 µM tazarotene , 20 µM bexarotene , 10 µM tazarotene 20 µM bexarotene in combination with increasing concentrations of pan - RXR receptor antagonist HX 531 ( 24 h pretreatment ) for 72 hours . Data represent mean ± SD of 4 - 14 independent experiments ( biological replicates ) . Two - way ANOVA followed by Tukey ' s for multiple comparisons . Displayed are significance for next significant difference between adjacent concentrations / conditions . # p < 0 . 05 , # # p < 0 . 01 . Difference against 0 µM DMSO control : * * * * p < 0 . 0001 .

O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O

(A) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RAR receptor antagonist AGN 193109 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 3-11 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01, ### p<0.001. Difference against 0 µM DMSO control (B) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RXR receptor antagonist HX 531 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 4-14 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01. Difference against 0 µM DMSO control: **** p<0.0001.


( A ) ARSA amount after treatment of MSD fibroblasts ( variant FGE Gly247Arg homozygous ) with tazarotene , bexarotene , tazarotene / bexarotene in combination for 6 days referred to β - actin amounts normalization of ARSA activity on ARSA amount ( specific ARSA activity ) . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . Displayed are significance for next significant difference between adjacent concentrations . # p < 0 . 05 . Difference against 0 / 0 µM control : * p < 0 . 05 , * * * p < 0 . 001 .

O O O B-GENEPROD O O O O B-DISEASE B-CELL_TYPE O O B-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) ARSA protein amount quantification after treatment of MSD primary fibroblasts (variant FGE Gly247Arg homozygous) with tazarotene, bexarotene, and tazarotene/bexarotene in combination for 6 days referred to β- actin amounts and normalization of ARSA activity based on ARSA protein amount (specific ARSA activity). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0/0 µM control: * p<0.05, *** p<0.001.


( C ) of ARSA activities in CRISPR / Cas9 generated ARPE19 SUMF1 - / - appropriate controls ( ARPE19 wild type , MSD fibroblasts ( variant FGE Gly247Arg homozygous ) ) after 6 days of simultaneous treatment with tazarotene / bexarotene 10 / 20 µM for up to 21 days . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . Displayed are significance for next significant difference between adjacent concentrations . # # p < 0 . 01 . Difference against 0 days control : * * * * p < 0 . 0001 .

O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE B-GENEPROD O O O O O O B-CELL_LINE O O O B-DISEASE B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSD primary fibroblasts (variant FGE Gly247Arg homozygous)) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM for up to 21 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 days control: **** p<0.0001.


( A ) - chase - experiment in HT1080 FGE Ser155Pro after pre - treatment with tazarotene / bexarotene DMSO ( control ) for 3 days . Upper panel : representative autoradiogram of intracellular ( C ) 35S isotope labelled FGE in either condition with a chase time of 4 hours . Lower panel : of 35S isotope labelled intracellular FGE amounts . Data represent mean ± SD of 3 independent experiments ( biological replicates ) . One - way ANOVA followed by Tukey ' s for multiple comparisons . * * p < 0 . 01 , * * * p < 0 . 001 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O 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) Pulse-chase-experiment in HT1080 FGE Ser155Pro cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (C) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ** p<0.01, *** p<0.001.


B Sedimentation to quantify condensation of unmodified TDP - 43 versus in vitro phosphorylated TDP - 43 ( + CK1δ , + ATP ) controls ( CK1δ or ATP only ) ; TDP - 43 detected by Western blot ( rabbit anti - TDP - 43 N - term ) . Due to incomplete TEV cleavage , some TDP - 43 - MBP - His6 remains present co - fractionates with TDP - 43 , due to TDP - 43 self - self interaction . C of band intensities of TDP - 43 shown as means of Supernatant / ( Supernatant + Condensate ) [ S / ( S + C ) ] of three independent experimental replicates ( n = 3 ) ± SD . * * * p < 0 . 0002 by one - way ANOVA with Dunnett ´ s multiple comparison to Wt .

O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Sedimentation assay to quantify condensation of unmodified TDP-43 versus in vitro phosphorylated TDP-43 (+CK1δ, +ATP) and controls (CK1δ or ATP only); TDP-43 detected by Western blot (rabbit anti-TDP-43 N-term). Due to incomplete TEV cleavage, some TDP-43-MBP-His6 remains present and co-fractionates with cleaved TDP-43, due to TDP-43 self-self interaction. C Quantification of band intensities of cleaved TDP-43 shown as means of Supernatant/(Supernatant+Condensate) [S/(S+C)] ratio of three independent experimental replicates (n=3) ± SD. ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt.


Representative bright field microscopic of TDP - 43 condensates ( in Hepes buffer ) , Bar , 25 µm ( F ) of condensate number ( G ) Box plots show comparison of median inter - quartile range ( upper lower quartiles ) of fields of view ( FOV ) from Min to Max ( whiskers ) of two replicates ( ≥ 22 FOV per condition ) . * p < 0 . 0332 , * * p < 0 . 0021 * * * p < 0 . 0002 by one - way ANOVA with Dunnett ´ s multiple comparison to Wt , comparing respective condition ( 5 , 10 , 20 µM ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 bright field microscopic images of TDP-43 condensates (in Hepes buffer), Bar, 25 µm (F) and quantification of condensate number (G) Box plots show the comparison of median and inter-quartile range (upper and lower quartiles) of all fields of view (FOV) from Min to Max (whiskers) of two replicates (≥ 22 FOV per condition). *p < 0.0332, **p < 0.0021 and ***p < 0.0002 by one-way ANOVA with Dunnett´s multiple comparison test to Wt, comparing the respective concentration condition (5, 10, 20 µM).


B Representative of FRAP experiments at indicated time - points . Boxes indicate bleached ( half - bleach of condensate ) . Bar , 5 µm . C FRAP curves after half - bleach of freshly formed Alexa488 - labeled TDP - 43 condensates . Values represent means ± SD of three independent experimental replicates ( n = 3 ) of ≥ 9 droplets analyzed per condition . * * * p < 0 . 0002 by one - way ANOVA with Tukey ´ s multiple comparison for under curve ( AUC ) of droplets .

O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative images of FRAP experiments at indicated time-points. Boxes indicate bleached area (half-bleach of condensate). Bar, 5 µm. C FRAP curves after half-bleach of freshly formed Alexa488-labeled TDP-43 condensates. Values represent means ± SD of three independent experimental replicates (n=3) of ≥ 9 droplets analyzed per condition. ***p < 0.0002 by one-way ANOVA with Tukey´s multiple comparison test for area under the curve (AUC) of individual droplets.


D Confocal of Alexa488 - labeled TDP - 43 formed in an in vitro aggregation ( with TEV protease cleavage ) . Bar , 100 µm . Zoom shows magnified view of at 24h time point . Bar , 20 µm .

O B-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

D Confocal images of Alexa488-labeled TDP-43 aggregates formed in an in vitro aggregation assay (with TEV protease cleavage). Bar, 100 µm. Zoom shows magnified view of aggregates at the 24h time point. Bar, 20 µm.


A Hormone - inducible import , representative still of GCR2 - EGFP2 - TDP - 43 Wt , 12D 12A before during import triggered by addition of dexamethasone . were live recorded by spinning disc confocal microscopy . Bar , 20 µm . B of hormone - inducible import measured during a total time course of 50 min . Values represent mean of GCR2 - EGFP2 - TDP - 43 in cytoplasm for three independent replicates ± ( ≥ 42 per condition ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O

A Hormone-inducible nuclear import assay, representative still images of GCR2-EGFP2-TDP-43 Wt, 12D and 12A before and during import triggered by addition of dexamethasone. Images were live recorded by spinning disc confocal microscopy. Bar, 20 µm. B Quantification of the hormone-inducible nuclear import measured during a total time course of 50 min. Values represent the mean fluorescence intensity of GCR2-EGFP2-TDP-43 in the cytoplasm for three independent replicates ± SEM (≥ 42 cells per condition).


C Phosphomimetic 12D TDP - 43 is competent in autoregulating TDP - 43 expression . SDS - PAGE followed by TDP - 43 Western blot showing downregulation of endogenous TDP - 43 through autoregulation ( 60 ) after 48 h expression of Wt , 12D 12A variants in HeLa . TDP - 43 was detected using rabbit anti - TDP - 43 C - term antibody ( Proteintech ) , Histone H3 ( rabbit anti - Histone H3 antibody , Abcam ) was visualized as a loading control . * denotes an unspecific band .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-CELL_LINE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O

C Phosphomimetic 12D TDP-43 is competent in autoregulating TDP-43 expression. SDS-PAGE followed by TDP-43 Western blot showing downregulation of endogenous TDP-43 through autoregulation (60) after 48 h expression of Wt, 12D and 12A variants in HeLa cells. TDP-43 was detected using rabbit anti-TDP-43 C-term antibody (Proteintech), Histone H3 (rabbit anti-Histone H3 antibody, Abcam) was visualized as a loading control. * denotes an unspecific band.


E Splicing by RT - PCR of known TDP - 43 splice targets ( SKAR exon 3 Bim exon 3 ) in HeLa . Silencing of endogenous TDP - 43 by siRNA leads to altered splice isoforms of SKAR Bim ( second vs . first lane ) . These splicing alterations can be rescued by re - expression of TDP - 43 Wt , but also 12D or 12A variants , demonstrating that phosphomimetic TDP - 43 is fully competent in regulation splicing of these TDP - 43 splice targets .

O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

E Splicing analysis by RT-PCR of known TDP-43 splice targets (SKAR exon 3 and Bim exon 3) in HeLa cells. Silencing of endogenous TDP-43 by siRNA leads to altered splice isoforms of SKAR and Bim (second vs. first lane). These splicing alterations can be rescued by re-expression of TDP-43 Wt, but also 12D or 12A variants, demonstrating that phosphomimetic TDP-43 is fully competent in regulation splicing of these TDP-43 splice targets.


B Reduced SG association of TDP - 43 by 12D mutations or in vitro phosphorylation . Bar , 20 µm .

O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O O O O O

B Reduced SG association of TDP-43 by 12D mutations or in vitro phosphorylation. Bar, 20 µm.


F SG recruitment of different TDP - 43 - NLSmut variants in intact HeLa in absence of endogenous TDP - 43 . After TDP - 43 silencing expression of NLSmut Wt , 12D 12A variants , SGs were induced by H2O2 treatment SG recruitment of TDP - 43 was monitored by TDP - 43 G3BP1 immunostaining . For clarity , signals were converted to grey values in channels ( upper two rows ) . In merge ( lower row ) , nuclei were stained in DAPI ( turquoise ) , TDP - 43 ( green ) G3BP1 ( red ) . Bar , 40 µm .

O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O

F SG recruitment of different TDP-43-NLSmut variants in intact HeLa cells in the absence of endogenous TDP-43. After TDP-43 silencing and expression of NLSmut Wt, 12D and 12A variants, SGs were induced by H2O2 treatment and SG recruitment of TDP-43 was monitored by TDP-43 and G3BP1 immunostaining. For clarity, signals were converted to grey values in the individual channels (upper two rows). In the merge (lower row), nuclei were stained in DAPI (turquoise), TDP-43 (green) and G3BP1 (red). Bar, 40 µm.


H Recruitment of TDP - 43 into arsenite - induced bodies ( NBs ) in HeLa . After TDP - 43 silencing expression myc - TDP - 43 Wt , 12D 12A , NBs were induced by sodium arsenite treatment NB recruitment of TDP - 43 was monitored by TDP - 43 immunostaining . Bar , 20 µm .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O

H Recruitment of TDP-43 into arsenite-induced nuclear bodies (NBs) in HeLa cells. After TDP-43 silencing and expression myc-TDP-43 Wt, 12D and 12A, NBs were induced by sodium arsenite treatment and NB recruitment of TDP-43 was monitored by TDP-43 immunostaining. Bar, 20 µm.


J profiles ( right ) of TDP - 43 Wt , 12D 12A variants ( green ) along lines ( left ) . Bar , 10 µm .

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J Intensity profiles (right) of TDP-43 Wt, 12D and 12A variants (green) along white lines (left). Bar, 10 µm.


A Biochemical fractionation into RIPA - soluble ( S ) RIPA - insoluble ( I ) fractions to analyze solubility of different myc - TDP - 43 variants ( Wt , 12D 12A ) expressed in HeLa for 48 h . TDP - 43 was detected by TDP - 43 Western blot ( upper blot , rabbit anti - TDP - 43 C - term , Proteintech ) Myc Western blot ( lower blot , mouse anti - Myc 9E10 ) . B of myc - TDP - 43 variants ( Wt , 12D 12A ) in ( S ) versus ( I ) fractions extracted from TDP - 43 Western blots of 4 independent replicates ± SD , plotted as S / ( S + I ) . * p < 0 . 0332 by one way ANOVA with Dunnett ´ s multiple comparison to Wt .

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A Biochemical fractionation into RIPA-soluble (S) and RIPA-insoluble (I) fractions to analyze solubility of the different myc-TDP-43 variants (Wt, 12D and 12A) expressed in HeLa cells for 48 h. TDP-43 was detected by TDP-43 Western blot (upper blot, rabbit anti-TDP-43 C-term, Proteintech) and Myc Western blot (lower blot, mouse anti-Myc 9E10). B Quantification of myc-TDP-43 variants (Wt, 12D and 12A) in (S) versus (I) fractions extracted from TDP-43 Western blots of 4 independent replicates ± SD, plotted as S/(S+I). *p < 0.0332 by one way ANOVA with Dunnett´s multiple comparison test to Wt.


D hippocampal neurons expressing EGFP - TDP - 43 Wt , 12D or 12A with additional NLS mutation . Bar , 80 µm . Right : Zoomed of squares ( TDP - 43 signal ) . Bar , 10 µm .

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D Primary hippocampal neurons expressing EGFP-TDP-43 Wt, 12D or 12A with additional NLS mutation. Bar, 80 µm. Right: Zoomed images of white squares (TDP-43 signal). Bar, 10 µm.


E SG recruitment of EGFP - TDP - 43 NLS mutant variants ( Wt , 12D , 12A ) in hippocampal neurons . SG formation was induced by 1h heat shock at 42 ° C . SGs TDP - 43 were monitored by G3BP1 antibody EGFP , respectively . For clarity , signals were converted to grey values in channels ( first two columns ) . In merge ( third column ) , EGFP - TDP - 43 shown in green , G3BP1 in red nuclei ( DAPI ) in turquoise . Bar , 20 µm .

O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O 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-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O

E SG recruitment of EGFP-TDP-43 NLS mutant variants (Wt, 12D, 12A) in primary hippocampal neurons. SG formation was induced by 1h heat shock at 42°C. SGs and TDP-43 were monitored by G3BP1 antibody staining and EGFP fluorescence, respectively. For clarity, signals were converted to grey values in the individual channels (first two columns). In the merge (third column), EGFP-TDP-43 shown in green, G3BP1 in red and nuclei (DAPI staining) in turquoise. Bar, 20 µm.


Effects of ActA BMP4 on expression of selected pluripotency , PGC , mesodermal genes during PGC ‐ precursor induction . DM : dorsomorphin , SB : SB431542 . Samples were calibrated with iPSC values , iPSC values depict 1 . Y ‐ axes are in log2 scale .

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Effects of ActA and BMP4 on the expression of selected pluripotency, PGC, and mesodermal genes during PGC‐precursor induction. DM: dorsomorphin, SB: SB431542. Samples were calibrated with iPSC values, and iPSC values depict 1. Y‐axes are in log2 scale.


Gene expression dynamics of selected pluripotency , PGC , mesodermal genes during prolonged PGC ‐ precursor induction of up to day 7 . Y ‐ axes are in log2 scale .

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Gene expression dynamics of selected pluripotency, PGC, and mesodermal genes during prolonged PGC‐precursor induction of up to day 7. Y‐axes are in log2 scale.


Immunofluorescence for OCT4 ( red ) , BLIMP1 ( green ) , T ( ) on days 0 , 1 , 2 . Nuclei were stained with Hoechst ( blue ) . culture contained BMP4 ( 5 ng / ml ) , Activin A ( 50 ng / ml ) , bFGF ( 20 ng / ml ) . Scale bar : 100 μm .

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Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (white) on days 0, 1, and 2. Nuclei were stained with Hoechst (blue). The culture contained BMP4 (5 ng/ml), Activin A (50 ng/ml), and bFGF (20 ng/ml). Scale bar: 100 μm.


FACS of ‐ dependent effect of BMP4 on TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 4 . B25 : 25 ng / ml BMP4 ; B50 : 50 ng / ml BMP4 ; B100 : 100 ng / ml BMP4 .

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FACS analysis of the concentration‐dependent effect of BMP4 on TRA‐1‐81+/cKIT+ PGCLCs on day 4. B25: 25 ng/ml BMP4; B50: 50 ng/ml BMP4; B100: 100 ng/ml BMP4.


FACS of TRA ‐ 1 ‐ 81 cKIT during PGC induction of up to day 8 .

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FACS analysis of TRA‐1‐81 and cKIT during PGC induction of up to day 8.


Gene expression of selected PGC markers in TRA ‐ 1 ‐ 81 + / cKIT + SSEA1 + / cKIT + FACS fractions of d4 PGCLCs . Samples were calibrated with iPSC values , iPSC values depict 1 .

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Gene expression analysis of selected PGC markers in TRA‐1‐81+/cKIT+ and SSEA1+/cKIT+ FACS fractions of d4 PGCLCs. Samples were calibrated with iPSC values, and iPSC values depict 1.


Gene expression dynamics of selected pluripotency PGC genes in FACS ‐ sorted , specified during PGC induction . value for iPSCs is set as 1 , values are on log2 scale .

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Gene expression dynamics of selected pluripotency and PGC genes in FACS‐sorted, specified cells during PGC induction. The value for iPSCs is set as 1, and values are on log2 scale.


Immunofluorescence for OCT4 ( green ) , BLIMP1 ( red ) , STELLA ( yellow ) in TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs on day 6 . Nuclei were stained with Hoechst ( blue ) . Scale bar : 15 μm .

B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_TYPE O O O O B-SUBCELLULAR O O O B-EXP_ASSAY O O O O O O O O O O

Immunofluorescence analysis for OCT4 (green), BLIMP1 (red), and STELLA (yellow) in TRA‐1‐81+/cKIT+ PGCLCs on day 6. Nuclei were stained with Hoechst (blue). Scale bar: 15 μm.


Bisulfite of DMRs of imprinted genes ( PEG1 , KvDMR1 , PEG10 , NESP55 ) in iPSCs ( top ) d6 TRA ‐ 1 ‐ 81 + / cKIT + PGCLCs ( bottom ) . black circles represent unmethylated methylated CpG sequences , respectively .

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Bisulfite sequence analysis of DMRs of the imprinted genes (PEG1, KvDMR1, PEG10, and NESP55) in iPSCs (top) and d6 TRA‐1‐81+/cKIT+ PGCLCs (bottom). White and black circles represent unmethylated and methylated CpG sequences, respectively.


Immunofluorescence for 5mC in d6 PGCLCs . Nuclei were stained with Hoechst ( blue ) . Scale bar : 50 μm .

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Immunofluorescence analysis for 5mC in d6 PGCLCs.Nuclei were stained with Hoechst (blue). Scale bar: 50 μm.


Transplantation of human iPSCs PGCLCs into ovaries of recipient mice . ( i ) left ovary : control ovary without transplantation ; right ovary : teratoma from iPSC ‐ reconstituted ovary ; ( ii ) PGCLC ‐ reconstituted ovaries . Note that no teratomas had formed . Bottom panels : Immunohistochemical for NUMA in sections of iPSC ‐ PGCLC ‐ reconstituted ovaries . ( iii ) iPSC ‐ induced teratoma shown in ( i ) ; ( iv ) ovary containing NUMA + shown in ( ii ) . Insert : higher magnification of selected . Scale bars : ( i ) ( ii ) , 0 . 5 cm ; ( iii ) ( iv ) , 50 μm .

O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O B-ORGANISM O O O O O B-TISSUE O O B-TISSUE O O O O B-TISSUE O O O B-CELL_TYPE O O B-TISSUE O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-CELL_TYPE O B-CELL_TYPE O O B-TISSUE O O O O B-CELL_TYPE O O O O O O O O O O O O B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Transplantation of human iPSCs and PGCLCs into ovaries of recipient mice. (i) left ovary: control ovary without transplantation; right ovary: teratoma from iPSC‐reconstituted ovary; (ii) PGCLC‐reconstituted ovaries. Note that no teratomas had formed. Bottom panels: Immunohistochemical analysis for NUMA in sections of iPSC‐ and PGCLC‐reconstituted ovaries. (iii) iPSC‐induced teratoma shown in (i); (iv) ovary containing NUMA+ cells shown in (ii). Insert: higher magnification of selected area. Scale bars: (i) and (ii), 0.5 cm; (iii) and (iv), 50 μm.


Effects of KSR WNT3A during PGC ‐ precursor induction on expression of selected pluripotency , PGC , a mesodermal gene of d2 cultures . value for iPSCs is set as 1 , values are on log2 scale . Data are presented as means ± SD ( n = 3 ) .

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Effects of KSR and WNT3A during PGC‐precursor induction on the expression of selected pluripotency, PGC, and a mesodermal gene of d2 cultures. The value for iPSCs is set as 1, and values are on log2 scale. Data are presented as means ± SD (n = 3).


Effects of KSR WNT3A on induction of d6 PGCLCs as analyzed by FACS gated for TRA ‐ 1 ‐ 81 cKIT .

O O B-SMALL_MOLECULE B-GENEPROD O O O O B-CELL_TYPE O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O

Effects of KSR and WNT3A on the induction of d6 PGCLCs as analyzed by FACS gated for TRA‐1‐81 and cKIT.


Gene expression of selected PGC hematopoietic markers in d6 PGCLCs that were cultured in 0 % ( control ) or 20 % KSR ( KSR ) condition during PGC ‐ precursor induction . Samples were calibrated with iPSC values , iPSC values depict 1 . Data are presented as means ± SD ( n = 3 ) .

B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE O O O O B-CELL_TYPE O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O B-CELL_TYPE O O O O O O O O O O O O O O O O O

Gene expression analysis of selected PGC and hematopoietic markers in d6 PGCLCs that were cultured in 0% (control) or 20% KSR (KSR) condition during PGC‐precursor induction. Samples were calibrated with iPSC values, and iPSC values depict 1. Data are presented as means ± SD (n = 3).


Immunofluorescence for OCT4 ( red ) , BLIMP1 ( green ) , T ( yellow ) in d2 precursors cultured in increasing KSR concentrations . Nuclei were stained with Hoechst ( blue ) . Scale bar : 100 μm .

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Immunofluorescence analysis for OCT4 (red), BLIMP1 (green), and T (yellow) in d2 precursors cultured in increasing KSR concentrations. Nuclei were stained with Hoechst (blue). Scale bar: 100 μm.


Effect of knockdown of BLIMP1 PRDM14 on PGCLC induction . Knockdown of BLIMP1 ( F ) PRDM14 ( G ) was done using lentiviral system with two shRNA vectors for each gene . knockdown efficiencies were assessed by qPCR ( left panel , each ) . induction of d4 PGCLCs analyzed by FACS , gated for TRA ‐ 1 ‐ 81 cKIT ( right panel , each ) . Data are presented as means ± SD ( n = 3 ) .

O O O O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-CELL_TYPE O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

Effect of knockdown of BLIMP1 and PRDM14 on PGCLC induction. Knockdown of BLIMP1 (F) and PRDM14 (G) was done using the lentiviral system with two individual shRNA vectors for each gene. The knockdown efficiencies were assessed by qPCR (left panel, each). The induction of d4 PGCLCs analyzed by FACS, gated for TRA‐1‐81 and cKIT (right panel, each). Data are presented as means ± SD (n = 3).


( A ) Oxygen consumption , , in indicated VSMC types .

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(A) Oxygen consumption ratio, , in the indicated VSMC types.


( B ) mitochondrial ATP synthesis in indicated VSMC types .

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(B) mitochondrial ATP synthesis in the indicated VSMC types.


( C ) mitochondrial potential ( MP ) , in indicated VSMC types .

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(C) mitochondrial membrane potential (MP), in the indicated VSMC types.


( A ) ATP synthesis in indicated VSMC types , measured by incorporation of phosphate - 32 ( 32Pi ) into ADP . [ 32Pi ] - ATP was separated from 32Pi using molybdate method , as explained in Methods section .

O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O

(A) Cytosolic ATP synthesis in the indicated VSMC types, measured by incorporation of phosphate-32 (32Pi) into ADP. [32Pi]-ATP was separated from 32Pi using the molybdate method, as explained in the Methods section.


(B) Intracellular lactate concentration


( C ) external acidification in indicated VSMC types .

(C) external acidification in the indicated VSMC types.


( D ) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi / mL calcium - 45 ( 45Ca2 + ) as a radiotracer .

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(D) Calcium accumulation in mitochondria after 24 h of incubation in MEM media containing 10 µCi/mL calcium-45 (45Ca2+) as a radiotracer.


( E ) Magnesium in isolated mitochondria .

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(E) Magnesium concentration in isolated mitochondria.


( B ) Representative time - course of 2 mmol / L phosphate on calcification of LmnaG609G / + VSMCs ( up ) . Calcification was visualized with Alizarin red . Representative microscopic ( 10x ; scale bar : 100 µm ) showing calcification of treated untreated LmnaG609G / + VSMCs ( down ) .

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(B) Representative time-course of 2 mmol/L phosphate on calcification of LmnaG609G/+ VSMCs (up). Calcification was visualized with Alizarin red. Representative microscopic images (10x; scale bar: 100 µm) showing calcification of treated and untreated LmnaG609G/+ VSMCs (down).


( C - F ) Measures of calcium in treated living LmnaG609G / + VSMCs ( C ) , treated fixed LmnaG609G / + VSMCs ( D ) , untreated living LmnaG609G / + VSMCs ( E ) , untreated fixed LmnaG609G / + VSMCs ( F ) .

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(C-F) Measures of calcium in treated living LmnaG609G/+ VSMCs (C), treated fixed LmnaG609G/+ VSMCs (D), untreated living LmnaG609G/+ VSMCs (E), and untreated fixed LmnaG609G/+ VSMCs (F).


( G ) calcification inhibitory capacity was calculated as difference in calcium deposition between living fixed ( ΔCa2 + ) .

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(G) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells (ΔCa2+).


( A ) Magnesium intake in 34 - week - old untreated treated LmnaG609G / + mice ( n = 16 ) .

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(A) Magnesium intake in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( B ) Plasma magnesium in 34 - week - old untreated treated LmnaG609G / + mice ( n = 16 ) .

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(B) Plasma magnesium concentration in 34-week-old untreated and treated LmnaG609G/+ mice (n=16).


( C ) Calcium content of aortas obtained from 34 - week - old wild - type mice untreated treated LmnaG609G / + mice ( n = 20 ) .

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(C) Calcium content of aortas obtained from 34-week-old wild-type mice and untreated and treated LmnaG609G/+ mice (n=20).


( D ) masses of 34 - week - old untreated treated LmnaG609G / + mice .

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(D) Body masses of 34-week-old untreated and treated LmnaG609G/+ mice.


( E ) Kaplan - Meier graph for untreated treated LmnaG609G / + mice ( n = 16 ) .

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(E) Kaplan-Meier graph for untreated and treated LmnaG609G/+ mice (n=16).


( F ) Representative of 40 - wk - old wild - type , untreated treated LmnaG609G / + mice .

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(F) Representative photographs of 40-wk-old wild-type, untreated and treated LmnaG609G/+ mice.


( A ) ATP in liver homogenates obtained from 34 - week - old wild - type , untreated or treated LmnaG609G / + mice .

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(A) ATP concentration in liver homogenates obtained from 34-week-old wild-type, untreated or treated LmnaG609G/+ mice.


( B ) Mitochondrial calcium measured in liver mitochondria isolated from indicated experimental mouse groups .

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(B) Mitochondrial calcium measured in liver mitochondria isolated from the indicated experimental mouse groups.


( C ) Magnesium in isolated mitochondria .

(C) Magnesium concentration in isolated mitochondria.


( D ) Activities of indicated mitochondrial complexes ( I , II , III , IV , V ) in absence or presence of rotenone ( 2 µmol / L ) , 2 - thenoyltrifluoroacetone ( TTFA ; 1 mmol / L ) , antimycin A ( 10 µmol / L ) , potassium cyanide ( KCN ; 1 mmol / L ) , or oligomycin ( 10 µmol / L ) .

O O O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O

(D) Activities of the indicated mitochondrial complexes (I, II, III, IV, and V) in the absence or presence of rotenone (2 µmol/L), 2-thenoyltrifluoroacetone (TTFA; 1 mmol/L), antimycin A (10 µmol/L), potassium cyanide (KCN; 1 mmol/L), or oligomycin (10 µmol/L).


A ) Infection susceptibility ( top ) to VSVG Spike - typed lentiviruses of a panel of lung cancer lines with or without ( Empty vector ) overexpression of ACE2 . ACE2 were determined by Western - blot ( bottom ) .

O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD O O B-ORGANISM O O O O B-DISEASE I-DISEASE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O

A) Infection susceptibility (top) to VSVG and Spike-typed lentiviruses of a panel of lung cancer cell lines with or without (Empty vector) overexpression of ACE2. ACE2 protein levels were determined by Western-blot (bottom).


C ) Gene - level scores obtained by robust ranking algorithm of sgRNA relative abundances between infected with Spike - typed lentiviruses uninfected . Genes were ordered alphabetically in x - axis top hits ( log10 ( enrichment - score ) < - 5 ) were colored labelled . Note that CTSL CTSL1 are different gene isoforms .

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C) Gene-level scores obtained by robust ranking algorithm analysis of the sgRNA relative abundances between cells infected with Spike-typed lentiviruses and uninfected cells. Genes were ordered alphabetically in the x-axis and the top hits (log10(enrichment-score) < -5) were colored and labelled. Note that CTSL and CTSL1 are different gene isoforms.


A ) Scheme depicting screen validation strategy ( top ) representative density plots ( bottom ) of flow cytometry data from infected with VSVG - or Spike - typed lentivirus . infected population is labeled with a darker color .

O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O

A) Scheme depicting the screen validation strategy (top) and representative density plots (bottom) of flow cytometry data from cells infected with VSVG- or Spike-typed lentivirus. The infected population is labeled with a darker color.


B ) Susceptibility to infection with Spike - typed VSVG - typed lentiviruses in Calu1ACE2 CRISPR KO lines with loss - of - function of selected screen candidates . NT596 NT764 are two non - targeting CRISPR controls . ACE2 are Calu1ACE2 transduced with a CRISPR vector against ACE2 . Bars represent average standard error of mean ( ) of of infected in each condition ( three biological replicates ) , normalized to non - targeting control NT596 . t - p - value between relative of infection with Spike - typed VSVG - typed for each is indicated .

O O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O B-CELL_LINE B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O 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-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O O O O O O O

B) Susceptibility to infection with Spike-typed and VSVG-typed lentiviruses in Calu1ACE2 CRISPR KO cell lines with loss-of-function of selected screen candidates. NT596 and NT764 are two non-targeting CRISPR controls. ACE2 are Calu1ACE2 cells transduced with a CRISPR vector against ACE2. Bars represent the average and standard error of the mean (SEM) of the percentage of infected cells in each condition (three biological replicates), normalized to the non-targeting control cell line NT596. The t-test p-value between the relative percentage of infection with Spike-typed and VSVG-typed for each cell line is indicated.


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C) Confocal microscopy images of SPNS1-KO1 and PLAC8-KO1 Calu1ACE2 cells overexpressing CRISPR-resistant SPNS1- or GFP-PLAC8, respectively.


D ) Rescue experiments in PLAC8 SPNS1 Calu1ACE2 KO lines : bar plot showing average ( three biological replicates ) of infection ( normalized to NT596 with GFP overexpression ) of Spike - typed VSVG - typed lentiviruses in PLAC8 - KO1 SPNS1 - KO1 lines that overexpress CRISPR - resistant PLAC8 - or GFP - SPNS1 constructs . significance above each bar represents t - p - value between each condition control ( NT596 with GFP overexpression )

O O O O O B-GENEPROD I-GENEPROD B-CELL_LINE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O B-ORGANISM O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O

D) Rescue experiments in PLAC8 and SPNS1 Calu1ACE2 KO cell lines: bar plot showing the average (three biological replicates) and SEM percentage of infection (normalized to NT596 with GFP overexpression) of Spike-typed and VSVG-typed lentiviruses in PLAC8-KO1 and SPNS1-KO1 cell lines that overexpress CRISPR-resistant PLAC8- or GFP-SPNS1 constructs. The significance above each bar represents the t-test p-value between each condition and the control cell line (NT596 with GFP overexpression)


A , B ) Susceptibility to infection with SARS - CoV - 2 in Calu1ACE2 ( A ) H1299ACE2 ( B ) lines with loss - of - function of indicated genes . Left : representative immunofluorescence of SARS - CoV - 2 nucleocapsid ( green ) nuclei ( red ) in indicated lines . Scale bar : 20 µm . Right : immunofluorescence of number of infected at 24 h post - infection in different loss - of - function lines infected with SARS - CoV - 2 . Bars represent average of of infected relative to CRISPR non - targeting transduced ( NT596 ) in three biological replicates ( three different CRISPR KO lines infected independently ) . significance above each bar represents one - tail t - p - value between each condition CRISPR non - targeting transduced ( NT596 ) .

O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O B-CELL_LINE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

A, B) Susceptibility to infection with SARS-CoV-2 viruses in Calu1ACE2 (A) and H1299ACE2 (B) cell lines with loss-of-function of the indicated genes. Left: representative immunofluorescence images of SARS-CoV-2 nucleocapsid protein (green) and cell nuclei (red) in the indicated cell lines. Scale bar: 20 µm. Right: immunofluorescence quantification of the number of infected cells at 24 h post-infection in the different loss-of-function cell lines infected with SARS-CoV-2. Bars represent the average and SEM of the percentage of infected cells relative to CRISPR non-targeting transduced (NT596) cells in three biological replicates (three different CRISPR KO cell lines infected independently). The significance above each bar represents the one-tail t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.


C ) Western - blot analyses of SPNS1 PLAC8 in different lung cancer lines tested in Fig 1 with or without ACE2 overexpression . Note that PLAC8 SPNS1 immunodetection was performed on same blots than in Figure 1A , that ACE2 Actin panels from Figure 1A were included here to allow comparison .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O B-DISEASE I-DISEASE O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O

C) Western-blot analyses of SPNS1 and PLAC8 protein levels in the different lung cancer cell lines tested in Fig 1 with or without ACE2 overexpression. Note that PLAC8 and SPNS1 immunodetection was performed on the same blots than in Figure 1A, and that ACE2 and Actin panels from Figure 1A were included here to allow comparison.


D ) Susceptibility to Spike - typed lentiviruses in indicated ACE2 overexpressing cancer lines that were transduced with GFP , GFP - PLAC8 or GFP - SPNS1 . Bars represent average of of infected in three technical replicates ( independent infections of same ) relative to Calu1ACE2 - GFP . significance above each bar represents t - between GFP - PLAC8 or GFP - SPNS1 GFP for each .

O O O O B-GENEPROD O O B-ORGANISM O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O O B-CELL_LINE O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O O

D) Susceptibility to Spike-typed lentiviruses in the indicated ACE2 overexpressing cancer cell lines that were transduced with GFP, GFP-PLAC8 or GFP-SPNS1. Bars represent the average and SEM of the percentage of infected cells in three technical replicates (independent infections of the same cell line) relative to Calu1ACE2-GFP cells. The significance above each bar represents the t-test between GFP-PLAC8 or GFP-SPNS1 and GFP cells for each cell line.


E ) Susceptibility to infection with SARS - CoV - 2 in lines that overexpress ACE2 GFP - SPNS1 or GFP - PLAC8 . Bars represent immunofluorescence of number of SARS - CoV - 2 infected at 24 h post - infection . Representative of SARS - CoV - 2 nucleocapsid immunofluorescence ( green ) nuclei ( red ) in GFP GFP - PLAC8 are shown right to each plot .

O O O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD B-EXP_ASSAY O O O B-SUBCELLULAR O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O

E) Susceptibility to infection with SARS-CoV-2 viruses in cell lines that overexpress ACE2 and GFP-SPNS1 or GFP-PLAC8. Bars represent the immunofluorescence quantification of the number of SARS-CoV-2 infected cells at 24 h post-infection. Representative images of SARS-CoV-2 nucleocapsid immunofluorescence (green) and cell nuclei (red) in GFP and GFP-PLAC8 cells are shown right to each plot.


A ) Binding endocytosis studies with Alexa Fluor 647 - conjugated S - RBD or transferrin in Calu1 - ACE2 - RFP deficient in PLAC8 SPNS1 . Left : scatterplots of flow cytometry data showing cutoffs used to define ACE2 - RFP Alexa Fluor 647 positive . Right : bar plots showing of positive for Alexa Fluor 647 in either ACE2 - RFP positive ( red ) or negative ( blue ) in binding endocytosis experiments with S - RBD or transferrin . Bars represent average of three biological replicates ( three different CRISPR KO lines ) . significance above each bar represents t - p - value between each condition CRISPR non - targeting transduced ( NT596 ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O

A) Binding and endocytosis studies with Alexa Fluor 647-conjugated S-RBD or transferrin in Calu1-ACE2-RFP cells deficient in PLAC8 and SPNS1. Left: scatterplots of flow cytometry data showing the cutoffs used to define ACE2-RFP and Alexa Fluor 647 positive cells. Right: bar plots showing the ratio of cells positive for Alexa Fluor 647 in either ACE2-RFP positive (red) or negative (blue) cells in binding and endocytosis experiments with S-RBD or transferrin. Bars represent the average and SEM of three biological replicates (three different CRISPR KO cell lines). The significance above each bar represents the t-test p-value between each condition and CRISPR non-targeting transduced (NT596) cells.


B - E ) Endocytosis experiments with S - RBD in PLAC8 - or SPNS1 - KO Calu1 - ACE2 - RFP with overexpression of GFP - PLAC8 or GFP - SPNS1 , respectively . were incubated with S - RBD for 1 h at 37 ºC fixed with 4 % p - formaldehyde after an acid wash to remove non - internalized S - RBD . Representative wide - field ( B ) of mean intensities of each fluorophore ( C ) in each show that S - RBD signal correlates with ACE2 - RFP expression but not with GFP - PLAC8 or GFP - SPNS1 . Pearson correlation coefficient ( r ) correlation p - value ( p ) is indicated within each scatterplot . Each dot represents a measurement from different microphotographs of a single replicate . Scale bar : 20 µm . Representative confocal microphotographs ( D ) spot colocalization analyses using ComDet plugin ( E ) demonstrate colocalization of S - RBD endosomes with ACE2 - RFP GFP - PLAC8 or GFP - SPNS1 . ( D ) Red ovals defining detected S - RBD dots are overlaid in each channel to assess colocalization with ACE2 - RFP , GFP - PLAC8 or GFP - SPNS1 . Inlets show a magnification of region defined by squares . Scale bar : 20 µm . ( E ) Boxplots representing of internalized S - RBD that colocalize with ACE2 - RFP , GFP - PLAC8 , GFP - SPNS1 or their combination ( ACE2 - RFP GFP - PLAC8 or ACE2 - RFP GFP - SPNS1 ) . central band represents median , box represents interquartile range ( IQR ) between 25th 75th percentile , whiskers extend from box to 1 . 5 * IQR . Each dot represents a measurement from different microphotographs of a single replicate .

O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD O O B-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O 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-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O 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-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O

B-E) Endocytosis experiments with S-RBD in PLAC8- or SPNS1-KO Calu1-ACE2-RFP cells with overexpression of GFP-PLAC8 or GFP-SPNS1, respectively. Cells were incubated with S-RBD for 1 h at 37 ºC and fixed with 4% p-formaldehyde after an acid wash to remove non-internalized S-RBD. Representative wide-field fluorescence images (B) and quantification of mean fluorescence intensities of each fluorophore (C) in each cell show that S-RBD signal correlates with ACE2-RFP expression but not with GFP-PLAC8 or GFP-SPNS1. The Pearson correlation coefficient (r) and correlation test p-value (p) is indicated within each scatterplot. Each dot represents a cell measurement from different microphotographs of a single replicate. Scale bar: 20 µm. Representative fluorescence confocal microphotographs (D) and spot colocalization analyses using ComDet plugin (E) demonstrate colocalization of S-RBD endosomes with ACE2-RFP and GFP-PLAC8 or GFP-SPNS1. (D) Red ovals defining the detected S-RBD dots are overlaid in each channel to assess the colocalization with ACE2-RFP, GFP-PLAC8 or GFP-SPNS1. Inlets show a magnification of the region defined by the white squares. Scale bar: 20 µm. (E) Boxplots representing the percentage of internalized S-RBD that colocalize with ACE2-RFP, GFP-PLAC8, GFP-SPNS1 or their combination (ACE2-RFP and GFP-PLAC8 or ACE2-RFP and GFP-SPNS1). The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate.


C ) Representative ( left ) ( right ) of number of acidic particles in PLAC8 - SPNS1 - KO Calu1ACE2 compared to CRISPR non - targeting control ( NT596 ) using live - lysotracker . two samples Wilcoxon p - value of pooled observations compared to NT596 is indicated above each condition . central band represents median , box represents interquartile range ( IQR ) between 25th 75th percentile , whiskers extend from box to 1 . 5 * IQR . Each dot represents a measurement from different microphotographs of a single replicate . Three biological replicates per condition were used .

O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O

C) Representative images (left) and quantification (right) of the number of acidic particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using live-cell lysotracker staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.


D ) Lysosomal degradation studies in Calu1ACE2 using fluorogenic substrate DQ - Red - BSA . Left : density plots representing shift in in NT596 untreated or treated with DQ - Red - BSA under growth or serum starvation conditions . Right : bar plot representing mean of median values ( MFI ) of three biological replicates of PLAC8 - SPNS1 - KO CRISPR non - targeting in growth serum starvation conditions .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-EXP_ASSAY B-TISSUE O O O

D) Lysosomal degradation studies in Calu1ACE2 cells using the fluorogenic substrate DQ-Red-BSA. Left: density plots representing the shift in fluorescence intensity in NT596 cells untreated or treated with DQ-Red-BSA under growth or serum starvation conditions. Right: bar plot representing the mean and SEM of the median fluorescence intensity values (MFI) of three biological replicates of PLAC8- and SPNS1-KO and CRISPR non-targeting cells in growth and serum starvation conditions.


E ) Representative ( left ) ( right ) of number of LC3B particles in PLAC8 - SPNS1 - KO Calu1ACE2 compared to CRISPR non - targeting control ( NT596 ) using LC3B immunofluorescence . two samples Wilcoxon p - value of pooled observations compared to NT596 is indicated above each condition . central band represents median , box represents interquartile range ( IQR ) between 25th 75th percentile , whiskers extend from box to 1 . 5 * IQR . Each dot represents a measurement from different microphotographs of a single replicate . Three biological replicates per condition were used .

O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O

E) Representative images (left) and quantification (right) of the number of LC3B particles in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596) using LC3B immunofluorescence staining. The two samples Wilcoxon Test p-value of the pooled observations compared to NT596 cells is indicated above each condition. The central band represents the median, the box represents the interquartile range (IQR) between the 25th and 75th percentile, and the whiskers extend from the box to 1.5*IQR. Each dot represents a cell measurement from different microphotographs of a single replicate. Three biological replicates per condition were used.


F ) Autophagic flux analyses using reporter GFP - LC3 - RFP - LC3ΔG in PLAC8 - SPNS1 - KO Calu1ACE2 compared to CRISPR non - targeting control ( NT596 ) . Left : density plots showing shift in GFP under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 . Right : bar plots showing mean of median GFP / RFP of three biological replicates in different lines conditions .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O

F) Autophagic flux analyses using the fluorescence reporter GFP-LC3-RFP-LC3ΔG in PLAC8- and SPNS1-KO Calu1ACE2 cells compared to CRISPR non-targeting control cells (NT596). Left: density plots showing the shift in GFP intensity under starvation conditions compared to control growth conditions in NT596 Calu1ACE2 cells. Right: bar plots showing the mean and SEM of the median GFP/RFP intensity ratio of three biological replicates in the different cell lines and conditions.


Induction of UAS constructs EPs by ap promoter directed Gal4 expression within dorsal compartment of wing imaginal disc : ( A ) ap - Gal4 control ; ( B , E , H , J , K ) UAS - dS6K ; ( C , F , I ) UAS - S6K1dE / D3E ; ( D , E , F ) DHR3 - EP ; ( G , H , I , K ) UAS - DHR3 - RNAi ( J ) unidirectional DHR3 - EP . bending down of wing indicates a slight overgrowth of dorsal compartment , whereas a bending up reveals a slight growth deficit of this compartment .

O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-TISSUE I-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O

Induction of UAS constructs and EPs by the ap promoter directed Gal4 expression within the dorsal compartment of the wing imaginal disc: (A) ap-Gal4 control; (B, E, H, J, K) UAS-dS6K; (C, F, I) UAS-S6K1dE/D3E; (D, E, F) DHR3-EP; (G, H, I, K) UAS-DHR3-RNAi and (J) unidirectional DHR3-EP. The bending down of the wing indicates a slight overgrowth of the dorsal compartment, whereas a bending up reveals a slight growth deficit of this compartment.


Induction of UAS constructs DHR3 - EP by eyeless promoter - directed Gal4 expression in developing eye : ( A ) eye - Gal4 control ; ( B , D , E ) DHR3 - EP ; ( C , D ) UAS - dTsc1 UAS - dTsc2 ; ( E , F ) UAS - dPTEN .

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Induction of UAS constructs and DHR3-EP by the eyeless promoter-directed Gal4 expression in the developing eye: (A) eye-Gal4 control; (B, D, E) DHR3-EP; (C, D) UAS-dTsc1 and UAS-dTsc2; (E, F) UAS-dPTEN.


( B - G ) Adult wings in which ap - Gal4 induces expression of following UAS constructs : ( B ) ap - Gal4 control ; ( C ) UAS - dS6K ; ( D ) UAS - DHR3 - RS ; ( E ) both UAS - dS6K UAS - DHR3 - RS ; ( F ) UAS - DHR3 - RS in a dS6Kl - 1 mutant escaper ; ( G ) UAS - dS6K , UAS - DHR3 - RS , UAS - DHR3 - RNAi together .

O O O O O O B-TISSUE O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O O O O

(B-G) Adult wings in which the ap-Gal4 induces expression of the following UAS constructs: (B) ap-Gal4 control; (C) UAS-dS6K; (D) UAS-DHR3-RS; (E) both UAS-dS6K and UAS-DHR3-RS; (F) UAS-DHR3-RS in a dS6Kl-1 mutant escaper; (G) UAS-dS6K, UAS-DHR3-RS, and UAS-DHR3-RNAi together.


( H ) Upper panel , H2B substrate phosphorylation by dS6K from larval extracts . Ubiquitous induction of UAS - DHR3 RNAi , using either a daughterless - Gal4 ( da > Ri ) or an actin - Gal4 ( ac > Ri ) driver , provoked a drop in dS6K activity , as compared with control drivers alone ( da , ac ) . Middle lower panels are western blots detecting dS6K α - tubulin , respectively .

O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O

(H) Upper panel, H2B substrate phosphorylation by dS6K from larval protein extracts. Ubiquitous induction of UAS-DHR3 RNAi, using either a daughterless-Gal4 (da>Ri) or an actin-Gal4 (ac>Ri) driver, provoked a drop in dS6K activity, as compared with control drivers alone (da, ac). Middle and lower panels are western blots detecting dS6K and α-tubulin, respectively.