( B ) U2OS cells stably expressing FYVE - RFP were co ‐ transfected with pcDNA3 . 1 or with vectors encoding TAB2C or TAB3C together with a control siRNA ( siUNR ) or with siRNAs specific for VPS34 , Beclin 1 ( BCN1 ) or TAK1 for 24 h , followed by the quantification of cells exhibiting FYVE - RFP + dots ( FYVE - RFP + ) . Results are mean values ± s . d . ( n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(B) U2OS cells stably expressing FYVE-RFP were co‐transfected with pcDNA3.1 or with vectors encoding TAB2C or TAB3C together with a control siRNA (siUNR) or with siRNAs specific for VPS34, Beclin 1 (BCN1) or TAK1 for 24 h, followed by the quantification of cells exhibiting FYVE-RFP+ dots (FYVE-RFP+). Results are mean values±s.d. (n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( C ) Inhibition of the interaction between endogenous TAB2 , TAB3 and BCN1 by C ‐ terminal fragments of TAB2 and TAB3 . Forty ‐ eight hours after transfection with pcDNA3 . 1 or plasmids coding for the indicated proteins , cells were lysed , TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected . GAPDH levels were monitored to ensure equal loading . This experiment has been done three times , yielding comparable results .

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(C) Inhibition of the interaction between endogenous TAB2, TAB3 and BCN1 by C‐terminal fragments of TAB2 and TAB3. Forty‐eight hours after transfection with pcDNA3.1 or plasmids coding for the indicated proteins, cells were lysed, TAB2 or TAB3 was immunoprecipitated and BCN1 was immunodetected. GAPDH levels were monitored to ensure equal loading. This experiment has been done three times, yielding comparable results.


( D ) Mechanisms of autophagy induction by the Beclin ‐ binding domain ( BBD ) ‐ containing C ‐ terminal fragments of TAB2 and TAB3 . HeLa cells were transfected with pcDNA3 . 1 , TAB2C ‐ or TAB3C ‐ encoding plasmids in combination with the indicated siRNAs for 24 h , followed by the quantification of GFP - LC3VAC cells ( mean values ± s . d . , n = 3 ; * P0 . 01 versus siUNR ‐ , pcDNA3 . 1 ‐ transfected cells ) .

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(D) Mechanisms of autophagy induction by the Beclin‐binding domain (BBD)‐containing C‐terminal fragments of TAB2 and TAB3. HeLa cells were transfected with pcDNA3.1, TAB2C‐ or TAB3C‐encoding plasmids in combination with the indicated siRNAs for 24 h, followed by the quantification of GFP-LC3VAC cells (mean values±s.d., n=3; *P0.01 versus siUNR‐, pcDNA3.1‐transfected cells).


( A ) Determination of the TAB ‐ binding domain ( TBD ) within Beclin 1 ( BCN1 ) . Yeast two ‐ hybrid technology was used to screen for positive ( + ) or negative interactions ( − ) between BCN1 fragments and full ‐ length TAB2 .

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(A) Determination of the TAB‐binding domain (TBD) within Beclin 1 (BCN1). Yeast two‐hybrid technology was used to screen for positive (+) or negative interactions (−) between BCN1 fragments and full‐length TAB2.


( C ) Confirmation of the TBD by immunoprecipitation . The indicated constructs were transfected into HeLa cells , followed by lysis , immunoprecipitation of HA ‐ tagged full ‐ length TAB2 or TAB3 and detection of His ‐ tagged constructs .

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(C) Confirmation of the TBD by immunoprecipitation. The indicated constructs were transfected into HeLa cells, followed by lysis, immunoprecipitation of HA‐tagged full‐length TAB2 or TAB3 and detection of His‐tagged constructs.


( D , E ) Inhibition of the interaction between endogenous BCN1 , TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD . HeLa cells were transfected with pcDNA3 . 1 ( empty vector ) or with plasmids encoding the TBD or a His - tagged Beclin 1 variant lacking the TBD ( His - BCN1ΔTBD ) , followed by co ‐ immunoprecipitation of endogenous TAB2 ( D ) or TAB3 ( E ) and detection of BCN1 .

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(D, E) Inhibition of the interaction between endogenous BCN1, TAB2 and TAB3 by a BCN1 fragment corresponding to the TBD. HeLa cells were transfected with pcDNA3.1 (empty vector) or with plasmids encoding the TBD or a His-tagged Beclin 1 variant lacking the TBD (His-BCN1ΔTBD), followed by co‐immunoprecipitation of endogenous TAB2 (D) or TAB3 (E) and detection of BCN1.


( 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 ) .

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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 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 ) .

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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 . 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 ) .

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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 - 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 .

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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 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 .

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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.


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 .

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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.


Proteins observed in multiple assembled peaks ( n = 659 ) are enriched in components of the proteasome and other known large complex 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 the CORUM complex hypotheses queried , two or more distinct subunit co - elution signals were detected ( See methods and 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 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 .

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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 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 ) .

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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 ) and size exclusion chromatographic fractionation of U2OS lysates ( Kirkwood et al . 2013 , lower panel ) , in line with the 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 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 .

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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.


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 ) .

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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 and late assembly intermediate elution ranges suggests predominant components of the intermediary species accumulating in HEK293 cells .

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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 protein sets for co - elution behavior in the SEC - SWATH - MS data , viewing chromatograms for interpretation and 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 and DDB2 suggest the partial resolution of substrate - bound and free pools of CSN holo - complex .

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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 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 ) .

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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 , 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 .

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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 . 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 ) .

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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 the Cy5 - RNA and 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 . 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 .

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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 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 .

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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 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

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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 ) 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 : * *

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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 ) 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 .

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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 ) 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 .

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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 ) 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 .

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(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 ) 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 .

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(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 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 ) .

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(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 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 .

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(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 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 .

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(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 ) 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 .

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(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 ) 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 .

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(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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 .

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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 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 .

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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 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 .

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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 Intensity profiles ( right ) of TDP - 43 Wt , 12D and 12A variants ( green ) along white 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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 , and 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 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 .

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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 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 .

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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 analysis of TRA ‐ 1 ‐ 81 and 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 analysis 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 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 .

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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 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 ) .

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

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


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 ) .

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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 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 .

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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 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 ) .

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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 ratio , , in the indicated VSMC types .

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


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

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


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

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


( 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 .

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(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 the 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 concentration in isolated mitochondria .

(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 images ( 10x ; scale bar : 100 µm ) showing calcification of treated and 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 ) , and 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 ) The calcification inhibitory capacity was calculated as the difference in calcium deposition between living and fixed cells ( 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 and 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 concentration in 34 - week - old untreated and 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 and untreated and 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 ) Body masses of 34 - week - old untreated and treated LmnaG609G / + mice .

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


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

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


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

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


( A ) ATP concentration 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 the indicated experimental mouse groups .

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


( C ) Magnesium concentration in isolated mitochondria .

(C) Magnesium concentration in isolated mitochondria.


( 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 ) .

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(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 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 ) .

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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 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 .

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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 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 .

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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 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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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.


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

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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 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 )

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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.