Representative ICa ( upper panel ) and Cm ( lower panel ) in response to a 50 ms depolarizing step to the potential eliciting Imax . ( C ' ) Exocytic ∆ Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2 - 100 ms for all respective genotypes and at [ Ca2 + ] e = 6 mM for a second set of recordings from wild - type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants . ( C " ) Magnification of the initial , short depolarizing steps ( 2 - 20 ms ) for clarity . Exocytic ∆ Cm of cultured endophilin - deficient IHCs appears strongly reduced . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis ) . The reduced Ca2 + - efficiency of exocytosis ( ∆ Cm / QCa ) in endophilin - deficient IHCs indicates that diminished Ca2 + - influx cannot fully account for the reduction of exocytosis . ( * p < 0 . 05 ; * * p < 0 . 01 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ; please also refer to Appendix Table S4 for detailed statistical analysis ) .

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Representative ICa (upper panel) and Cm (lower panel) in response to a 50 ms depolarizing step to the potential eliciting Imax. (C') Exocytic ∆Cm and corresponding QCa elicited by depolarizations of stimulus durations from 2-100 ms for all respective genotypes and at [Ca2+]e = 6 mM for a second set of recordings from wild-type IHCs to experimentally approximate the decreased ICa observed in the endophilin mutants. (C") Magnification of the initial, short depolarizing steps (2-20 ms) for clarity. Exocytic ∆Cm of cultured endophilin-deficient IHCs appears strongly reduced. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Tables S2 and S3 for detailed statistical analysis). The reduced Ca2+-efficiency of exocytosis (∆Cm/QCa) in endophilin-deficient IHCs indicates that diminished Ca2+-influx cannot fully account for the reduction of exocytosis. (*p < 0.05; **p < 0.01; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's; please also refer to Appendix Table S4 for detailed statistical analysis).


Ca2 + - current - voltage relationships in response to 10 ms step - depolarizations . ( A ' ) The peak of Ca2 + - influx was significantly reduced in endophilin 1 - SKO ( * * p = 0 . 0024 ) and 1 / 3 - DKO ( * * * p < 0 . 0001 ) when compared to Wt . ( Wt n = 30 / N = 20 ; 1 - SKO n = 15 / N = 9 ; 1 / 3 - DKO n = 39 / N = 20 ; Non - parametric K - W with post - hoc Dunn ' s correction ) .

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Ca2+-current-voltage relationships in response to 10 ms step-depolarizations. (A') The peak of Ca2+-influx was significantly reduced in endophilin 1-SKO (**p = 0.0024) and 1/3-DKO (***p < 0.0001) when compared to Wt. (Wt n = 30/ N = 20; 1-SKO n = 15/ N = 9; 1/3-DKO n = 39/ N = 20; Non-parametric K-W with post-hoc Dunn's correction).


Quantification of CtBP2 - labeled ribbons revealed a number of approximately 14 - 15 ribbon synapses per IHC in all groups , that remained comparable across all genotypes ( Wt n = 12 / N = 9 ; 1 - SKO n = 6 / N = 4 ; 1 / 3 - DKO n = 8 / N = 6 ; one - way ANOVA , p = 0 . 717 ) .

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Quantification of CtBP2-labeled ribbons revealed a number of approximately 14-15 ribbon synapses per IHC in all groups, that remained comparable across all genotypes (Wt n = 12/ N = 9; 1-SKO n = 6/ N = 4; 1/3-DKO n = 8/ N = 6; one-way ANOVA, p = 0.717).


Quantification of the cross - sectional area of CaV1 . 3 immunofluorescence revealed approx . 34 % smaller clusters in 1 - SKOs and 24 % smaller clusters in 1 / 3 - DKOs . ( Wt n = 153 / N = 2 ; 1 - SKO n = 102 / N = 2 ; 1 / 3 - DKO n = 441 / N = 4 ; * * * p < 0 . 0001 ; K - W and Dunn ' s post - hoc test ) .

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Quantification of the cross-sectional area of CaV1.3 immunofluorescence revealed approx. 34 % smaller clusters in 1-SKOs and 24 % smaller clusters in 1/3-DKOs. (Wt n = 153/ N = 2; 1-SKO n = 102/ N = 2; 1/3-DKO n = 441/ N = 4; ***p < 0.0001; K-W and Dunn's post-hoc test).


Representative maximum projections of confocal sections from organs of Corti of p15 Wt , 1 - SKO , and 1 / 3 - DKO mice stained for CtBP2 to label synaptic ribbons . Please note that CtBP2 expression is also found in the nucleus ( nuclei are highlighted by dashed circles in the individual panels ) . Scale bars : 10 µm .

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Representative maximum projections of confocal sections from organs of Corti of p15 Wt, 1-SKO, and 1/3-DKO mice stained for CtBP2 to label synaptic ribbons. Please note that CtBP2 expression is also found in the nucleus (nuclei are highlighted by dashed circles in the individual panels). Scale bars: 10 µm.


2D STED images of IHC ribbon synapses stained for CtBP2 ( magenta ) and CaV1 . 3 ( green ) . CaV1 . 3 Ca2 + - channels remain clustered at AZs of 1 - SKO and 1 / 3 - DKO IHCs . Scale bars : 1 µm .

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2D STED images of IHC ribbon synapses stained for CtBP2 (magenta) and CaV1.3 (green). CaV1.3 Ca2+-channels remain clustered at AZs of 1-SKO and 1/3-DKO IHCs. Scale bars: 1 µm.


Representative Ca2 + - currents ( upper panel ) and exocytic membrane capacitance increments ( ∆ Cm ; lower panel ) in response to a 200 ms depolarizing step to - 14 mV .

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Representative Ca2+-currents (upper panel) and exocytic membrane capacitance increments (∆Cm; lower panel) in response to a 200 ms depolarizing step to -14 mV.


Exocytic ∆ Cm ( top ) and corresponding Ca2 + - current integrals QCa ( bottom ) of Wt and endophilin - deficient IHCs in response to voltage steps from - 84 to - 14 mV of variable stimulus duration ( 5 to 200 ms ) . In 1 / 3 - DKO IHCs , our data indicate reduced sustained exocytosis to a strong depolarization ( 200 ms ; * p = 0 . 0470 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis ) . Inset : Initial ∆ Cm showed comparable RRP exocytosis in endophilin - deficient IHCs .

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Exocytic ∆Cm (top) and corresponding Ca2+-current integrals QCa (bottom) of Wt and endophilin-deficient IHCs in response to voltage steps from -84 to -14 mV of variable stimulus duration (5 to 200 ms). In 1/3-DKO IHCs, our data indicate reduced sustained exocytosis to a strong depolarization (200 ms; *p = 0.0470, K-W with Dunn's post-hoc test; please also refer to Appendix Tables S5 and S6 for detailed statistical analysis). Inset: Initial ∆Cm showed comparable RRP exocytosis in endophilin-deficient IHCs.


Ca2 + - efficiency to drive exocytosis was reduced for strong depolarizations ( 200 ms ) in 1 / 3 - DKO IHCs ; * p = 0 . 0323 , K - W with Dunn ' s post - hoc test ; please also refer to Appendix Table S7 for detailed statistical analysis .

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Ca2+-efficiency to drive exocytosis was reduced for strong depolarizations (200 ms) in 1/3-DKO IHCs; *p = 0.0323, K-W with Dunn's post-hoc test; please also refer to Appendix Table S7 for detailed statistical analysis.


Confocal maximum projections of otoferlin - immunolabeled organs of Corti from p15 Wt and 1 / 3 - DKO mice illustrated with an intensity - coded look - up table where brighter colors indicate higher intensity . Scale bars : 5 µm .

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Confocal maximum projections of otoferlin-immunolabeled organs of Corti from p15 Wt and 1/3-DKO mice illustrated with an intensity-coded look-up table where brighter colors indicate higher intensity. Scale bars: 5 µm.


Otoferlin levels were reduced by approx . 28 . 4 ± 3 . 5 % in 1 / 3 - DKO IHCs compared to Wt IHCs ( relative intensities normalized to Wt ; Wt n = 31 images ( 229 IHCs ) / N = 5 organs of Corti ( 3 animals ) ; 1 / 3 - DKO n = 20 images ( 196 IHCs ) / N = 5 organs of Corti ( 4 animals ) ; unpaired student ' s t - test ; * * * p < 0 . 001 ; box plots illustrate the median with the interquartile range , whiskers indicate 10 - 90 % of data points , the squares present the respective mean value ) .

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Otoferlin levels were reduced by approx. 28.4 ± 3.5 % in 1/3-DKO IHCs compared to Wt IHCs (relative intensities normalized to Wt; Wt n = 31 images (229 IHCs)/ N = 5 organs of Corti (3 animals); 1/3-DKO n = 20 images (196 IHCs)/ N = 5 organs of Corti (4 animals); unpaired student's t-test; ***p < 0.001; box plots illustrate the median with the interquartile range, whiskers indicate 10-90 % of data points, the squares present the respective mean value).


Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical ( 0 . 0 ) to basal ( 1 . 0 ) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1 / 3 - DKO and an overall reduction of otoferlin levels throughout the entire cell in 1 / 3 - DKOs . Traces indicate the mean pixel intensity ± SEM ; Wt n = 123 / N = 3 ; 1 / 3 - DKO n = 124 / N = 3 .

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Normalized otoferlin intensity line profiles through single IHCs of the respective genotypes at a longitudinal central plane through the nucleus from apical (0.0) to basal (1.0) revealed similar distribution patterns of otoferlin in IHCs of Wt and 1/3-DKO and an overall reduction of otoferlin levels throughout the entire cell in 1/3-DKOs. Traces indicate the mean pixel intensity ± SEM; Wt n = 123/ N = 3; 1/3-DKO n = 124/ N = 3.


Interaction of otoferlin and endophilin - A1 detected by co - IP in HeLa cells co - expressing GFP - otoferlin and RFP - endophilin - A1 . Otoferlin - GFP was immunoprecipitated ( IP ) by GFP - Trap beads , and blots were probed with a KO - validated anti - endophilin - A1 antibody .

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Interaction of otoferlin and endophilin-A1 detected by co-IP in HeLa cells co-expressing GFP-otoferlin and RFP-endophilin-A1. Otoferlin-GFP was immunoprecipitated (IP) by GFP-Trap beads, and blots were probed with a KO-validated anti-endophilin-A1 antibody.


Exogenously overexpressed GFP - otoferlin was immunoprecipitated using GFP - Trap beads and incubated with purified endophilin - A1 ( pA1 ) . IP was then followed by immunoblotting with an anti - endophilin - A1 antibody .

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Exogenously overexpressed GFP-otoferlin was immunoprecipitated using GFP-Trap beads and incubated with purified endophilin-A1 (pA1). IP was then followed by immunoblotting with an anti-endophilin-A1 antibody.


Endocytosis was assessed by determining the decrease in Cm during 20 s post - depolarization in perforated patch recordings from both Wt , 1 / 2 - DKO and 1 / 3 - DKO IHCs . The two kinetic components are presented as average ∆ Cm recordings after ( A ) 20 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 7 ; 1 / 3 - DKO n = 13 / N = 10 ) and ( B ) 200 ms ( Wt n = 12 / N = 8 ; 1 / 2 - DKO n = 13 / N = 6 ; 1 / 3 - DKO n = 14 / N = 10 ) depolarization stimuli .

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Endocytosis was assessed by determining the decrease in Cm during 20 s post-depolarization in perforated patch recordings from both Wt, 1/2-DKO and 1/3-DKO IHCs. The two kinetic components are presented as average ∆Cm recordings after (A) 20 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 7; 1/3-DKO n = 13/ N = 10) and (B) 200 ms (Wt n = 12/ N = 8; 1/2-DKO n = 13/ N = 6; 1/3-DKO n = 14/ N = 10) depolarization stimuli.


Average time of return to baseline obtained by fitting a linear function to the Cm - data following a 20 ms depolarization .

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Average time of return to baseline obtained by fitting a linear function to the Cm-data following a 20 ms depolarization.


Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses , obtained from fitting Cm - data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm - recording for 200 ms depolarizations . ( * p < 0 . 05 ; one - way ANOVA with post - hoc Tukey ' s or non - parametric K - W with post - hoc Dunn ' s ) .

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Average slope of the linear component of endocytosis for 20 ms and 200 ms depolarizing pulses, obtained from fitting Cm-data for 20 s after cessation of the 20 ms depolarizations or for the last 10 s of the Cm- recording for 200 ms depolarizations. (*p < 0.05; one-way ANOVA with post-hoc Tukey's or non-parametric K-W with post-hoc Dunn's).


Average amplitude of the exponential component obtained from fits to Cm - data following 200 ms depolarization ( nexp fit = 11 for Wt , 10 for 1 / 2 - DKO and 11 for 1 / 3 - DKO ; x data points in ( E ) correspond to IHCs with no exponential component in Cm ) . correspond to statistically identified outliers .

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Average amplitude of the exponential component obtained from fits to Cm-data following 200 ms depolarization (nexp fit = 11 for Wt, 10 for 1/2-DKO and 11 for 1/3-DKO; x data points in (E) correspond to IHCs with no exponential component in Cm). correspond to statistically identified outliers.


time constant of the exponential component obtained from fits to Cm - data following 200 ms

time constant of the exponential component obtained from fits to Cm-data following 200 ms depolarization


Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center ( exemplary for a 1 / 3 - DKO specimen ) . Graphical aids indicate small vesicles , ELVs and different subpopulations of coated structures quantified in ( B - D ) . Scale bar : 500 nm .

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Representative electron micrograph illustrating the region of interest for analysis within a radius of r = 1 µm from the ribbon center (exemplary for a 1/3-DKO specimen). Graphical aids indicate small vesicles, ELVs and different subpopulations of coated structures quantified in (B-D). Scale bar: 500 nm.


While an increased number of cytosolic vesicles can be observed at 1 - SKO AZs , reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs . RA - SVs were excluded from this quantification .

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While an increased number of cytosolic vesicles can be observed at 1-SKO AZs, reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs. RA-SVs were excluded from this quantification.


Accumulations of ELVs occur in 1 / 2 - DKO and 1 / 3 - DKO IHCs , as measured by the relative cumulative ELV area per section .

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Accumulations of ELVs occur in 1/2-DKO and 1/3-DKO IHCs, as measured by the relative cumulative ELV area per section.


Increased overall number of coated structures in endophilin mutants . ( D ' ) Prominent accumulation of coated pits can be observed in 1 / 2 - DKO and 1 / 3 - DKO IHCs alongside a shift towards more coated vacuoles ( d > 70 nm ) in 1 / 2 - DKO IHCs and 1 / 3 - DKO IHCs .

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Increased overall number of coated structures in endophilin mutants. (D') Prominent accumulation of coated pits can be observed in 1/2-DKO and 1/3-DKO IHCs alongside a shift towards more coated vacuoles (d > 70 nm) in 1/2-DKO IHCs and 1/3-DKO IHCs.


Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin - A alleles . Scale bars : 200 nm .

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Representative electron micrographs of random ultrathin sections from IHC ribbon synapses of p15 mutants lacking different endophilin-A alleles. Scale bars: 200 nm.


Reduced numbers of RA - SVs in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 . Reduced ratio of RA - SVs in the distal half of the ribbon over RA - SVs in its proximal half in 1 / 2 - DKO and 1 / 3 - DKO IHCs . K - W followed by Dunn ' s test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

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Reduced numbers of RA-SVs in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001. Reduced ratio of RA-SVs in the distal half of the ribbon over RA-SVs in its proximal half in 1/2-DKO and 1/3-DKO IHCs. K-W followed by Dunn's test; **p < 0.01; ***p < 0.001.


Reduced numbers of MP - SVs ( D ) in IHCs of endophilin mutants with the strongest reduction observed in 1 / 2 - DKO mice . One - way ANOVA followed by Tukey ' s post - hoc test ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 .

Reduced numbers of MP-SVs (D) in IHCs of endophilin mutants with the strongest reduction observed in 1/2-DKO mice. One-way ANOVA followed by Tukey's post-hoc test; *p < 0.05; **p < 0.01; ***p < 0.001.


Significantly reduced numbers of RA - SVs in 1 / 2 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0229 ) .

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Significantly reduced numbers of RA-SVs in 1/2-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0229).


Significantly reduced numbers of MP - SVs in 1 / 2 - DKO and a strong trend ( p = 0 . 0511 ) towards less MP - SVs in 1 / 3 - DKO IHCs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0138 ) .

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Significantly reduced numbers of MP-SVs in 1/2-DKO and a strong trend (p = 0.0511) towards less MP-SVs in 1/3-DKO IHCs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0138).


Increased SV diameter in 1 / 2 - DKO , but unchanged SV diameter in 1 / 3 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * p = 0 . 0154 ) .

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Increased SV diameter in 1/2-DKO, but unchanged SV diameter in 1/3-DKOs (one-way ANOVA followed by Tukey's post-hoc test; *p = 0.0154).


Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1 / 2 - DKOs ( one - way ANOVA followed by Tukey ' s post - hoc test ; * * * p = 0 . 0002 ) .

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Accumulation of ELVs with a minimal distance to the ribbon of less than 200 nm in 1/2-DKOs (one-way ANOVA followed by Tukey's post-hoc test; ***p = 0.0002).


Representative electron micrographs of Wt and 1 / 2 - DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity ( radius r = 1 µm from the center of the ribbon ) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K + . Scale bars : 200 nm .

Representative electron micrographs of Wt and 1/2-DKO specimen indicate accumulations of ELVs and high numbers of coated pits forming from the ELVs in ribbon proximity (radius r = 1 µm from the center of the ribbon) in the DKO after 15 min stimulation with extracellular solution supplemented with 50 mM K+. Scale bars: 200 nm.


Significantly reduced numbers of RA - SVs in IHCs of 1 / 2 - DKO compared to Wt after stimulation , as quantified using random ultrathin sections of ribbon - occupied AZs from several IHCs per genotype . Mann - Whitney test ; * * * p < 0 . 001 .

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Significantly reduced numbers of RA-SVs in IHCs of 1/2-DKO compared to Wt after stimulation, as quantified using random ultrathin sections of ribbon-occupied AZs from several IHCs per genotype. Mann-Whitney test; ***p < 0.001.


Numbers of MP - SVs were slightly reduced in stimulated Wt compared to stimulated 1 / 2 - DKOs , where SV numbers closely matched the ones of unstimulated 1 / 2 - DKO . Mann - Whitney test ; * p < 0 . 05 .

Numbers of MP-SVs were slightly reduced in stimulated Wt compared to stimulated 1/2-DKOs, where SV numbers closely matched the ones of unstimulated 1/2-DKO. Mann-Whitney test; *p < 0.05.


Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1 / 2 - DKOs compared to Wt after stimulation . RA - SVs were excluded from this quantification . Student ' s t - test ; * * * p < 0 . 001 .

Reduced numbers of small vesicles are present in a 1 µm radius around the ribbon of 1/2-DKOs compared to Wt after stimulation. RA-SVs were excluded from this quantification. Student's t-test; ***p < 0.001.


Stimulated 1 / 2 - DKOs exhibited accumulations of ELVs in comparison to stimulated Wt . Mann - Whitney test ; * * * p < 0 . 001 .

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Stimulated 1/2-DKOs exhibited accumulations of ELVs in comparison to stimulated Wt. Mann-Whitney test; ***p < 0.001.


Increased overall number of coated structures in stimulated endophilin 1 / 2 - DKOs . ( F ' ) Both , coated vesicles and coated pits forming at ELVs were significantly increased in 1 / 2 - DKO after stimulation . Mann - Whitney test ; * * p < 0 . 01 ; * * * p < 0 . 001 .

Increased overall number of coated structures in stimulated endophilin 1/2-DKOs. (F') Both, coated vesicles and coated pits forming at ELVs were significantly increased in 1/2-DKO after stimulation. Mann-Whitney test; **p < 0.01; ***p < 0.001.


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 ) .

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.


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 .

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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 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 .

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.


A Flow cytometric quantification of lymphocytes , monocytes , B - cells , natural killer ( NK ) cells , dendritic cells ( DCs ) and granulocytes in peripheral venous blood samples from non - diabetic ( ND ) and type - 2 diabetic ( T2D ) patients with COVID - 19 .

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A Flow cytometric quantification of lymphocytes, monocytes, B-cells, natural killer (NK) cells, dendritic cells (DCs) and granulocytes in peripheral venous blood samples from non-diabetic (ND) and type-2 diabetic (T2D) patients with COVID-19.


B Quantification of monocyte subpopulation phenotypes as classical ( CD14Hi CD16 - ) , intermediate ( CD14Hi CD16 + ) or non - classical ( CD14Lo CD16 + ) .

B Quantification of monocyte subpopulation phenotypes as classical (CD14Hi CD16-), intermediate (CD14Hi CD16+) or non-classical (CD14Lo CD16+).


C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID - 19 patients and in T2D patients without COVID - 19 .

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C Principle component analysis of lymphocyte and monocyte population frequencies in ND and T2D COVID-19 patients and in T2D patients without COVID-19.


A Quantification of CD14 expression and size ( FSC ) in monocyte populations from non diabetic ( ND ) and type - 2 diabetic ( T2D ) COVID - 19 patients .

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A Quantification of CD14 expression and size (FSC) in monocyte populations from non diabetic (ND) and type-2 diabetic (T2D) COVID-19 patients.


B Frequency of conventional ( FSCLo ) and large ( FSCHi ) monocytes in ND and T2D COVID - 19 patients .

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B Frequency of conventional (FSCLo) and large (FSCHi) monocytes in ND and T2D COVID-19 patients.


C Expression of CD16 , CD14 and HLA - DR in FSCLo and FSCHi monocytes from COVID - 19 patients .

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C Expression of CD16, CD14 and HLA-DR in FSCLo and FSCHi monocytes from COVID-19 patients.


D Proportions of classical , intermediate and non - classical monocytes within FSCLo and FSCHi monocytes from COVID - 19 patients .

D Proportions of classical, intermediate and non-classical monocytes within FSCLo and FSCHi monocytes from COVID-19 patients.


E Correlative analyses of IL8 expression in peripheral blood mononuclear cells ( PBMCs ) to monocyte FSC .

E Correlative analyses of IL8 expression in peripheral blood mononuclear cells (PBMCs) to monocyte FSC.


F IL8 , IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID - 19 patients .

F IL8, IL6 and CCL2 mRNA expression in PBMCs from ND and T2D COVID-19 patients.


G Monocyte size quantified in ND and T2D COVID - 19 patients treated in general wards ( Gen ) or in intensive care unit ( ICU ) .

G Monocyte size quantified in ND and T2D COVID-19 patients treated in general wards (Gen) or in intensive care unit (ICU).


A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non - diabetic ( ND ) and type 2 diabetic ( T2D ) COVID - 19 patients ( A ) .

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A Quantification of IRF5 and IFNB1 mRNA expression in peripheral blood mononuclear cells of non-diabetic (ND) and type 2 diabetic (T2D) COVID-19 patients (A).


B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples .

B Histograms of IRF5 expression on different populations analysed by flow cytometry on venous blood samples.


C IRF5 median fluorescence intensity ( MFI ) in monocytes of ND and T2D patients with COVID - 19 .

C IRF5 median fluorescence intensity (MFI) in monocytes of ND and T2D patients with COVID-19.


D IRF5 expression overlaid onto monocyte phenotypic gating .

D IRF5 expression overlaid onto monocyte phenotypic gating.


E IRF5 expression ( MFI ) in monocyte subtypes from ND and T2D COVID - 19 patients .

E IRF5 expression (MFI) in monocyte subtypes from ND and T2D COVID-19 patients.


F HLA - DR and IRF5 expression in monocytes from COVID - 19 patients .

F HLA-DR and IRF5 expression in monocytes from COVID-19 patients.


G IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted to intensive care unit ( ICU ) or treated exclusively in general wards ( Gen ) .

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G IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted to intensive care unit (ICU) or treated exclusively in general wards (Gen) .


H IRF5 expression in monocytes of ND or T2D COVID - 19 patients admitted ICU or treated exclusively in Gen .

H IRF5 expression in monocytes of ND or T2D COVID-19 patients admitted ICU or treated exclusively in Gen.


B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID - 19 either treated in Gen or requiring ICU admission .

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B Proportions of lymphocytes and monocytes in peripheral venous blood from T2D patients with COVID-19 either treated in Gen or requiring ICU admission.


C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID - 19 patients treated in Gen or requiring ICU treatment .

C Phenotypic analysis of lymphocyte and monocyte subpopulations in peripheral venous blood from T2D COVID-19 patients treated in Gen or requiring ICU treatment.


( B ) Quantification of SARS - CoV - 2 infection . After the siRNA knockdown of each target , viral infection was quantified as mean log2 fold - change ( log2FC ) of the percentage of SARS - CoV - 2 + cells relative to the mean of scrambled non - targeting siRNAs ( " SCRAMBLED " , green dots ; accordingly the mean log2FC value of scrambled non - targeting siRNAs was normalized to zero ) . Predicted positive targets ( " TARGETING " , grey dots ) , predicted negative targets ( " NEG " , blue dots ) and positive controls ( " POS " , red dots , including ACE2 and TMPRSS2 ) are all shown . Wilcoxon rank - sum test P value comparing the predicted positive targets to scrambled non - targeting siRNAs is given .

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(B) Quantification of SARS-CoV-2 infection. After the siRNA knockdown of each target, viral infection was quantified as mean log2 fold-change (log2FC) of the percentage of SARS-CoV-2+ cells relative to the mean of scrambled non-targeting siRNAs ("SCRAMBLED", green dots; accordingly the mean log2FC value of scrambled non-targeting siRNAs was normalized to zero). Predicted positive targets ("TARGETING", grey dots), predicted negative targets ("NEG", blue dots) and positive controls ("POS", red dots, including ACE2 and TMPRSS2) are all shown. Wilcoxon rank-sum test P value comparing the predicted positive targets to scrambled non-targeting siRNAs is given.


( C ) Quantification of cell number after each target knockdown , calculated as the mean fraction of DAPI + cells relative to the scrambled non - targeting siRNAs ( i . e . the latter was normalized to 1 ) . Colors of dots and P value are interpreted in the same way as in ( B ) .

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(C) Quantification of cell number after each target knockdown, calculated as the mean fraction of DAPI+ cells relative to the scrambled non-targeting siRNAs (i.e. the latter was normalized to 1). Colors of dots and P value are interpreted in the same way as in (B).


( D ) Representative fluorescence images from the siRNA assay showing SARS - CoV - 2 infection ( green channel , top row ) , cell number ( blue channel , middle row ) and merged ( bottom row ) . Results for scrambled non - targeting siRNA as negative control ( left column ) , knockdown of three predicted top metabolic targets ( PIKFYVE , SLC16A10 , and PIP5K1C , middle columns ) , and knockdown of positive control ( ACE2 , right column ) are shown . Scale bar = 10 μm .

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(D) Representative fluorescence images from the siRNA assay showing SARS-CoV-2 infection (green channel, top row), cell number (blue channel, middle row) and merged (bottom row). Results for scrambled non-targeting siRNA as negative control (left column), knockdown of three predicted top metabolic targets (PIKFYVE, SLC16A10, and PIP5K1C, middle columns), and knockdown of positive control (ACE2, right column) are shown. Scale bar=10 μm.


( A ) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups : control ( no virus or remdesivir treatment ) , virus ( SARS - CoV - 2 - infected ) , virus + remdesivir ( SARS - CoV - 2 - infected treated by remdesivir ) , remdesivir ( remdesivir treatment alone without virus ) . There are 3 replicates in each group .

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(A) PCA plot of the gene expression profiles for the Vero E6 samples from all experimental groups: control (no virus or remdesivir treatment), virus (SARS-CoV-2-infected), virus+remdesivir (SARS-CoV-2-infected treated by remdesivir), remdesivir (remdesivir treatment alone without virus). There are 3 replicates in each group.


( C ) PCA plot of the average metabolic flux profile computed using the iMAT algorithm ( Shlomi et al . 2008 ; Methods ) representative of each of the experimental groups , the labels are the same as ( A ) .

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(C) PCA plot of the average metabolic flux profile computed using the iMAT algorithm (Shlomi et al. 2008; Methods) representative of each of the experimental groups, the labels are the same as (A).


( a ) Survival rate of wild - type mice ( WT ) and indicated knockout mice after intranasal infection with 750 plaque forming units ( PFU ) of the PR8 virus .

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(a) Survival rate of wild-type mice (WT) and indicated knockout mice after intranasal infection with 750 plaque forming units (PFU) of the PR8 virus.


( b ) Hematoxylin and eosin ( H & amp ; amp ; E ) - stained lung sections from mice infected with PR8 virus on day 2 . Arrows indicate cellular infiltrates and necrotic debris occluding the airways . Original magnification , × 40 . ( c ) Disease severity ( ' lung score ' ) and extent of cell death ( necrosis score ) on day 2 after infection based on analysis of H & amp ; amp ; E - stained lung sections . ( d ) Necrosis score on day 7 , estimated visually from H & amp ; amp ; E - stained lung sections , and flow cytometry analysis for annexin V - stained lung cell populations .

(b) Hematoxylin and eosin (H&amp;E)-stained lung sections from mice infected with PR8 virus on day 2. Arrows indicate cellular infiltrates and necrotic debris occluding the airways. Original magnification, ×40. (c) Disease severity ('lung score') and extent of cell death (necrosis score) on day 2 after infection based on analysis of H&amp;E-stained lung sections. (d) Necrosis score on day 7, estimated visually from H&amp;E-stained lung sections, and flow cytometry analysis for annexin V-stained lung cell populations.


( e ) Immunohistochemistry staining of neutrophils occluding the airways of PR8 - infected mice . Original magnification , × 20 .

(e) Immunohistochemistry staining of neutrophils occluding the airways of PR8-infected mice. Original magnification, ×20.


( f , g ) Flow cytometry analysis of neutrophil , dendritic cell ( DC ) and macrophage cell numbers on days 2 and 7 .

(f,g) Flow cytometry analysis of neutrophil, dendritic cell (DC) and macrophage cell numbers on days 2 and 7.


( h ) Pulmonary viral titers on days 2 and 7 after infection in Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( log - rank test ( a ) and unpaired two - sided Student ' s t - test ( c , d , f , g ) . Data are pooled from three experiments ( a , f - h ; mean ± s . e . m . in f - h ; n = 10 - 12 mice total ) or are representative of three experiments ( b - e ; mean ± s . e . m . ; n = 3 - 5 mice per experiment ) .

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(h) Pulmonary viral titers on days 2 and 7 after infection in Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (log-rank test (a) and unpaired two-sided Student's t-test (c,d,f,g). Data are pooled from three experiments (a,f-h; mean ± s.e.m. in f-h; n = 10-12 mice total) or are representative of three experiments (b-e; mean ± s.e.m.; n = 3-5 mice per experiment).


( a - c ) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild - type mice ( WT ) and Ripk2 − / − mice with the PR8 virus were analyzed for IL - 6 and TNF ( day 2 ; a ) , IL - 18 and IFN - γ ( b ) , and indicated chemokines ( c ) . * P 0 . 05 , * * P 0 . 01 ( unpaired two - sided Student ' s t - test ) . Data were normalized for total protein ( t . p . ) from lung homogenates . Data are pooled from three experiments ( mean ± s . e . m . ; n = 10 - 12 mice total ) .

(a-c) Supernatants of whole lung homogenates taken on days 2 and 7 after infection of wild-type mice (WT) and Ripk2 −/− mice with the PR8 virus were analyzed for IL-6 and TNF (day 2; a), IL-18 and IFN-γ (b), and indicated chemokines (c). *P 0.05, **P 0.01 (unpaired two-sided Student's t-test). Data were normalized for total protein (t.p.) from lung homogenates. Data are pooled from three experiments (mean ± s.e.m.; n = 10-12 mice total).


( a - c ) Analysis on day 2 after infection with PR8 virus of wild - type mice ( WT ) or Ripk2 − / − mice injected intraperitoneally with control rabbit serum and Ripk2 − / − mice injected intraperitoneally with anti - IL - 18 neutralizing antiserum ( αIL - 18Ab ) 3 h before infection , for IL - 18 , IFN - γ and CCL2 amounts in whole lung homogenates ( a ; t . p . , total protein ) , and for neutrophil numbers by immunohistochemistry ( b ) and by flow cytometry ( c ) . Original magnification , × 20 .

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(a-c) Analysis on day 2 after infection with PR8 virus of wild-type mice (WT) or Ripk2−/− mice injected intraperitoneally with control rabbit serum and Ripk2−/− mice injected intraperitoneally with anti-IL-18 neutralizing antiserum (αIL-18Ab) 3 h before infection, for IL-18, IFN-γ and CCL2 amounts in whole lung homogenates (a; t.p., total protein), and for neutrophil numbers by immunohistochemistry (b) and by flow cytometry (c). Original magnification, ×20.


( d ) Analysis of survival of indicated mice .

(d) Analysis of survival of indicated mice.


( e ) Neutrophil numbers in WT , Ripk2 − / − and Ripk2 − / − Il18 − / − mice on day 2 after PR8 infection .

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(e) Neutrophil numbers in WT, Ripk2−/− and Ripk2−/−Il18−/− mice on day 2 after PR8 infection.


( f ) Survival of Ripk2 − / − Il18 − / − , Ripk2 − / − and WT mice . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ( a , c , e ) and log - rank test ( d , f ) ) . Data are representative of two independent experiments ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Survival of Ripk2−/−Il18−/−, Ripk2−/− and WT mice. *P 0.05, **P 0.01, ***P 0.001 (one-way ANOVA with Tukey's post-hoc test (a,c,e) and log-rank test (d,f)). Data are representative of two independent experiments (mean ± s.e.m.; n = 6-8 mice per experiment).


( a , b ) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL - 18 production by using IL - 18 immunohistochemistry ( a ) and IL - 18 ELISA ( b ) of whole lung homogenates . WT , wild type ; t . p . , total protein . In a , blue arrows denote IL - 18 + macrophages , and red arrows denote IL - 18 + bronchial epithelium . Original magnification , × 200 .

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(a,b) RIPK2 bone marrow chimeras were analyzed at day 2 after PR8 infection for contribution to elevated IL-18 production by using IL-18 immunohistochemistry (a) and IL-18 ELISA (b) of whole lung homogenates. WT, wild type; t.p., total protein. In a, blue arrows denote IL-18+ macrophages, and red arrows denote IL-18+ bronchial epithelium. Original magnification, ×200.


( c ) Flow cytometry analysis of NK cell counts in Ripk2 − / − and wild - type mice .

(c) Flow cytometry analysis of NK cell counts in Ripk2−/− and wild-type mice.


( d ) Analysis of wild - type and Ripk2 − / − NK cells for IFN - γ production on a per cell basis .

(d) Analysis of wild-type and Ripk2−/− NK cells for IFN-γ production on a per cell basis.


( e ) Accumulation of T and B cells in lungs .

(e) Accumulation of T and B cells in lungs.


( f ) Analysis of CD8 + T cells for IFN - γ and CD44 expression in wild - type and Ripk2 − / − mice . * P 0 . 05 , * * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( b ) and unpaired two - sided Student ' s t - test ( d , f ) ) . Data are representative of two experiments ( a , b , f ) or three experiments ( c - e ) ( mean ± s . e . m . ; n = 6 - 8 mice per experiment ) .

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(f) Analysis of CD8+ T cells for IFN-γ and CD44 expression in wild-type and Ripk2−/− mice. *P 0.05, **P 0.01 (one-way ANOVA with Tukey's post-hoc test (b) and unpaired two-sided Student's t-test (d,f)). Data are representative of two experiments (a,b,f) or three experiments (c-e) (mean ± s.e.m.; n = 6-8 mice per experiment).


( a ) Immunoblots showing active caspase - 1 ( casp - 1 ) p20 compared to GAPDH loading controls in wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs after PR8 infection .

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(a) Immunoblots showing active caspase-1 (casp-1) p20 compared to GAPDH loading controls in wild-type (WT), Ripk2−/− and Nod2−/− BMDCs after PR8 infection.


( b ) IL - 18 release in PR8 - infected WT , Ripk2 − / − and Nod2 − / − BMDCs .

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(b) IL-18 release in PR8-infected WT, Ripk2−/− and Nod2−/− BMDCs.


( c , d ) Immunoblot analysis showing active casp - 1 p20 ( c ) and IL - 18 production ( d ) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10 .

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(c,d) Immunoblot analysis showing active casp-1 p20 (c) and IL-18 production (d) in BMDCs infected for 4 h with Listeria monocytogenes at multiplicity of infection of 10.


( e , f ) IL - 18 and casp - 1 activation in Ripk2 − / − BMDCs treated with the NLRP3 inflammasome - specific inhibitor glyburide ( glyb . ) during PR8 infection .

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(e,f) IL-18 and casp-1 activation in Ripk2−/− BMDCs treated with the NLRP3 inflammasome-specific inhibitor glyburide (glyb.) during PR8 infection.


( g ) Immunoblot analysis of casp - 1 p20 during Listeria infection of WT and Aim2 − / − BMDCs . * P 0 . 01 , * * P 0 . 001 ( one - way ANOVA with Tukey ' s post - hoc test ) . Data are representative of six experiments ( a , b ) or three experiments ( c - g ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Immunoblot analysis of casp-1 p20 during Listeria infection of WT and Aim2−/− BMDCs. *P 0.01, **P 0.001 (one-way ANOVA with Tukey's post-hoc test). Data are representative of six experiments (a,b) or three experiments (c-g) (mean ± s.d.; n = 2-3 replicates per experiment).


( a ) Immunoblot analysis of wild - type ( WT ) , Ripk2 − / − and Nod2 − / − BMDCs for LC3 - II ( marker for autophagy ) after 18 h of PR8 infection .

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(a) Immunoblot analysis of wild-type (WT), Ripk2−/− and Nod2−/− BMDCs for LC3-II (marker for autophagy) after 18 h of PR8 infection.


( b , c ) Numbers of LC3 - II + puncta ( b ) , counted in at least 100 cells from five random fields from confocal images ( c ) of BMDCs after PR8 infection and / or chloroquine treatment . Original magnification , × 400 .

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(b,c) Numbers of LC3-II+ puncta (b), counted in at least 100 cells from five random fields from confocal images (c) of BMDCs after PR8 infection and/or chloroquine treatment. Original magnification, ×400.


( d , e ) Immunoblots of LC3 - II and caspase - 1 ( casp - 1 ; e ) and IL - 18 production ( d ) in wild - type and Ripk2 − / − BMDCs after treatment with rapamycin ( Rap ) .

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(d,e) Immunoblots of LC3-II and caspase-1 (casp-1; e) and IL-18 production (d) in wild-type and Ripk2−/− BMDCs after treatment with rapamycin (Rap).


( f ) Immunoblot analysis for LC3 - II in lung lysates of PR8 - infected wild - type and Ripk2 − / − mice ( 1 - 5 indicate individual mice ) .

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(f) Immunoblot analysis for LC3-II in lung lysates of PR8-infected wild-type and Ripk2−/− mice (1-5 indicate individual mice).


( g , h ) Lungneutrophil infiltration ( g ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) Lungneutrophil infiltration (g) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( g , h ) IL - 18 and IFN - γ amounts ( h ) in IAV - infected Ripk2 − / − mice treated with rapamycin , Ripk2 − / − mice and WT mice ( t . p . , total protein ) . * P 0 . 05 , * * P 0 . 01 , * * * P 0 . 001 ( Mann - Whitney test ( b ) ; one - way ANOVA with Tukey ' s post - hoc test ( d , g , h ) ) .

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(g,h) IL-18 and IFN-γ amounts (h) in IAV-infected Ripk2−/− mice treated with rapamycin, Ripk2−/− mice and WT mice (t.p., total protein). *P 0.05, **P 0.01, ***P 0.001 (Mann-Whitney test (b); one-way ANOVA with Tukey's post-hoc test (d,g,h)).


( a - c ) Flow cytometry analysis of wild - type ( WT ) and Ripk2 − / − BMDCs stained with the mitochondrial superoxide - specific stain MitoSOX during IAV or Listeria infection ( a , b ) and with the general mitochondrial stain MitoTracker Green during IAV infection ( c ) .

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(a-c) Flow cytometry analysis of wild-type (WT) and Ripk2−/− BMDCs stained with the mitochondrial superoxide-specific stain MitoSOX during IAV or Listeria infection (a,b) and with the general mitochondrial stain MitoTracker Green during IAV infection (c).


( d , e ) Analysis of IL - 18 production and immunoblotting to analyze caspase - 1 ( casp - 1 ) p20 activation in IAV - infected BMDCs that had been pretreated with the ROS inhibitor NAC .

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(d,e) Analysis of IL-18 production and immunoblotting to analyze caspase-1 (casp-1) p20 activation in IAV-infected BMDCs that had been pretreated with the ROS inhibitor NAC.


( f ) Immunoblot of casp - 1 p20 in cell lysates of IAV - infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO .

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(f) Immunoblot of casp-1 p20 in cell lysates of IAV-infected BMDCs pretreated with the mitochondrial superoxide inhibitor mitoTEMPO.


( g ) Confocal microscopy analysis of BMDCs stained for mitochondria ( red ) and LC3 ( green ) for colocalization as an indicator of mitophagy . Original magnification , × 400 ; inset ( red boxed region ) was digitally enlarged × 10 . * P 0 . 01 ( one - way ANOVA with Tukey ' s post - hoc test ( d ) and unpaired two - sided Student ' s t - test ( g ) ) . Data are representative of nine experiments ( a ) , three experiments ( b , d - g ) or six experiments ( c ) ( mean ± s . d . ; n = 2 - 3 replicates per experiment ) .

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(g) Confocal microscopy analysis of BMDCs stained for mitochondria (red) and LC3 (green) for colocalization as an indicator of mitophagy. Original magnification, ×400; inset (red boxed region) was digitally enlarged ×10. *P 0.01 (one-way ANOVA with Tukey's post-hoc test (d) and unpaired two-sided Student's t-test (g)). Data are representative of nine experiments (a), three experiments (b,d-g) or six experiments (c) (mean ± s.d.; n = 2-3 replicates per experiment).


( a , b ) IL - 18 amounts ( a ) , and caspase - 1 ( casp - 1 ) p20 and LC3 - II immunoblots ( b ) in PR8 - infected wild - type ( WT ) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580 .

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(a,b) IL-18 amounts (a), and caspase-1 (casp-1) p20 and LC3-II immunoblots (b) in PR8-infected wild-type (WT) BMDCs treated with the p38 and RIPK2 kinase inhibitor SB203580.


( c ) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2 − / − BMDCs after PR8 infection .

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(c) Immunoblots for phosphorylation of ULK1 at Ser555 in WT and Ripk2−/− BMDCs after PR8 infection.


( d ) Immunoblots for casp - 1 p20 in untreated or NAC - treated WT and Ulk1 − / − BMDCs after PR8 infection .

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(d) Immunoblots for casp-1 p20 in untreated or NAC-treated WT and Ulk1−/− BMDCs after PR8 infection.


( e ) Flow cytometry analysis of MitoSOX staining in PR8 - infected WT and Ulk1 − / − BMDCs .

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(e) Flow cytometry analysis of MitoSOX staining in PR8-infected WT and Ulk1−/− BMDCs.


( f ) MitoTracker Green staining of PR8 - infected WT and Ulk1 − / − BMDCs .

(f) MitoTracker Green staining of PR8-infected WT and Ulk1−/− BMDCs.


( G ) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration . The NDST3 KO cells with stable expression of Flag - NDST3 or Flag - EGFP ( control ) were treated with 25 nM Baf A1 for 72 h , and cell survival was measured using calcein AM staining . The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells ( n = 4 independent cultures , * * * * P < 0 . 0001 ) . Data information : Error bars represent ± standard deviation . Scale bar , 200 μm .

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(G) Baf A1 sensitivity assay for NDST3 KO cells after the NDST3 restoration. The NDST3 KO cells with stable expression of Flag-NDST3 or Flag-EGFP (control) were treated with 25 nM Baf A1 for 72 h, and cell survival was measured using calcein AM staining. The cell survival was calculated as the fluorescence intensity of the treated cells relative to the untreated cells (n = 4 independent cultures, ****P < 0.0001). Data information: Error bars represent ± standard deviation. Scale bar, 200 μm.


( C ) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow / Blue dextran staining against the pH calibration curve in ( B ) ( n = 3 independent cultures , * P = 0 . 0242 ) . ( D ) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 ( 100 nM , 1h ) ( n = 3 independent cultures , * P = 0 . 0305 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

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(C) Lysosomal pH values in WT and NDST3 KO RPE1 cells calculated from the fluorescence ratio of LysoSensor Yellow/Blue dextran staining against the pH calibration curve in (B) (n = 3 independent cultures, *P = 0.0242). (D) Lysosomal pH values measured for WT and NDST3 KO RPE1 cells after the treatment with Baf A1 (100 nM, 1h) (n = 3 independent cultures, *P = 0.0305). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( F ) Magic Red assay for Cat B proteolytic activity . WT and NDST3 KO RPE1 cells were treated with vehicle ( VEH ) or Baf A1 ( 100 nM , 1 h ) and then stained with MR - ( RR ) 2 to measure the Cathepsin B activity ( n = 48 cells in the VEH - treated WT group , n = 37 cells in the VEH - treated NDST3 KO group , n = 64 cells in the Baf A1 - treated WT group , n = 42 cells in the Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

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

(F) Magic Red assay for Cat B proteolytic activity. WT and NDST3 KO RPE1 cells were treated with vehicle (VEH) or Baf A1 (100 nM, 1 h) and then stained with MR-(RR)2 to measure the Cathepsin B activity (n = 48 cells in the VEH-treated WT group, n = 37 cells in the VEH-treated NDST3 KO group, n = 64 cells in the Baf A1-treated WT group, n = 42 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( G ) Alexa Fluor ( AF ) 488 - dextran degradation assay . WT and NDST3 KO RPE1 cells were loaded with AF488 - dextran in culture medium and observed for fluorescence . The AF488 fluorescence signals without a chase period , with a 4 - h chase period , and with the 4 - h chase period in the presence of Baf A1 ( 100 nM , 1h ) were recorded as fluorescence of uptake , chase , and chase with Baf A1 , respectively . The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods ( n = 62 cells in the VEH - treated WT group , n = 36 cells in the VEH - treated NDST3 KO group , n = 65 cells in the Baf A1 - treated WT group , n = 78 cells in the Baf A1 - treated NDST3 KO group , * * * * P < 0 . 0001 ) .

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

(G) Alexa Fluor (AF) 488-dextran degradation assay. WT and NDST3 KO RPE1 cells were loaded with AF488-dextran in culture medium and observed for fluorescence. The AF488 fluorescence signals without a chase period, with a 4-h chase period, and with the 4-h chase period in the presence of Baf A1 (100 nM, 1h) were recorded as fluorescence of uptake, chase, and chase with Baf A1, respectively. The AF488 intensity per cell was quantified and calculated into degradation percentage according to the equation in Materials and Methods (n = 62 cells in the VEH-treated WT group, n = 36 cells in the VEH-treated NDST3 KO group, n = 65 cells in the Baf A1-treated WT group, n = 78 cells in the Baf A1-treated NDST3 KO group, ****P < 0.0001).


( J ) The LC3 turnover assay after a 48 - h Baf A1 treatment . WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B - II fold change as in ( I ) ( n = 6 independent experiments , * * P = 0 . 0014 ) . Data information : Error bars represent ± standard deviation . Scale bar , 10 μm .

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

(J) The LC3 turnover assay after a 48-h Baf A1 treatment. WT and NDST3 KO RPE1 cells were cultured with 25 nM Baf A1 for 48 h and analyzed for the LC3B-II fold change as in (I) (n = 6 independent experiments, **P = 0.0014). Data information: Error bars represent ± standard deviation. Scale bar, 10 μm.


( A ) To examine the levels of the V - ATPase V1 - V0 holoenzyme in WT and NDST3 KO RPE1 cells , cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V - ATPase subunits V1A ( ATP6V1A ) , V1C1 ( ATP6V1C1 ) , and V0D ( ATP6V0D ) . LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins , respectively . ( B ) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from ( A ) ( n = 3 independent experiments , P = 0 . 7786 ) . Data information : Error bars represent ± standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) To examine the levels of the V-ATPase V1-V0 holoenzyme in WT and NDST3 KO RPE1 cells, cytosolic and membrane fractions were analyzed by immunoblotting using antibodies against the V-ATPase subunits V1A (ATP6V1A), V1C1 (ATP6V1C1), and V0D (ATP6V0D). LAMP1 and vinculin were used as loading controls for membrane proteins and cytosolic proteins, respectively. (B) Quantification of the levels of ATP6V0D relative to LAMP1 in the membrane fraction from (A) (n = 3 independent experiments, P = 0.7786). Data information: Error bars represent ± standard deviation.


( A , B ) Perinuclear enrichment and colocalization of acetylated microtubules , lysosomes , and V - ATPase V1 subunits . RPE1 cells stably expressing LAMP1 - GFP were treated with vehicle ( VEH ) or 10 μg / mL nocodazole ( Noco ) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated - α - tubulin ( Ac - α - tubulin ) . The nucleus was stained with DAPI . Representative images of LAMP1 - GFP , ATP6V1C1 , and Ac - α - tubulin immunostaining are shown in ( A ) . Representative distribution profiles of Ac - α - tubulin , LAMP1 - GFP and ATP6V1C1 were plotted by Fiji software in ( B ) .

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

(A, B) Perinuclear enrichment and colocalization of acetylated microtubules, lysosomes, and V-ATPase V1 subunits. RPE1 cells stably expressing LAMP1-GFP were treated with vehicle (VEH) or 10 μg/mL nocodazole (Noco) for 6 h before being fixed and subjected to immunofluorescence staining with antibodies against ATP6V1C1 and acetylated-α-tubulin (Ac-α-tubulin). The nucleus was stained with DAPI. Representative images of LAMP1-GFP, ATP6V1C1, and Ac-α-tubulin immunostaining are shown in (A). Representative distribution profiles of Ac-α-tubulin, LAMP1-GFP and ATP6V1C1 were plotted by Fiji software in (B).


The assembly of V - ATPase V1 - V0 holoenzymes in WT RPE1 cells over - expressing the α - tubulin deacetylase HDAC6 or EGFP as a control . Representative immunoblots for ATP6V0D , ATP6V1A , ATP6V1C1 , LAMP1 and GAPDH are shown in ( H ) .

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

The assembly of V-ATPase V1-V0 holoenzymes in WT RPE1 cells over-expressing the α-tubulin deacetylase HDAC6 or EGFP as a control. Representative immunoblots for ATP6V0D, ATP6V1A, ATP6V1C1, LAMP1 and GAPDH are shown in (H).


( M - O ) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V - ATPase V1 subunits . WT and NDST3 KO PRE1 cells stably expressing LAMP1 - GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac - α - tubulin . The relative levels of Ac - α - tubulin ( N ) ( n = 75 cells in the WT group , n = 79 cells in the NDST3 KO group , * * * * P < 0 . 0001 ) and the colocalization of LAMP1 - GFP with ATP6V1C1 ( O ) ( n = 75 cells in the WT group , n = 79 cells in the NDST3 KO group , * * * * P < 0 . 0001 ) are quantified .

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

(M-O) Loss of NDST3 enhances microtubule acetylation and the colocalization of the lysosomes with V-ATPase V1 subunits. WT and NDST3 KO PRE1 cells stably expressing LAMP1-GFP were fixed and analyzed by immunofluorescence staining with antibodies against ATP6V1C1 and Ac-α-tubulin. The relative levels of Ac-α-tubulin (N) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) and the colocalization of LAMP1-GFP with ATP6V1C1 (O) (n = 75 cells in the WT group, n = 79 cells in the NDST3 KO group, ****P < 0.0001) are quantified.


( C ) In vitro enzymatic assay quantitating the effect of NDST3 on α - tubulin acetylation . Flag - NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti - Flag beads . Flag - EGFP was used as a control . The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro , with or without 1 mM NAD + , 400 nM TSA , or 5 mM nicotinamide ( NAM ) for 2 h at room temperature . The reaction products were examined by immunoblotting against Ac - α - tubulin or total α - tubulin ( n = 3 independent experiments , * P = 0 . 0185 , * * P < 0 . 01 , * * * * P < 0 . 0001 ) . ( D ) The same in vitro enzymatic assay as in ( C ) , except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP ( n = 3 independent experiments , * * P < 0 . 01 , * * * P = 0 . 0001 ) . Data information : Error bars represent ± standard deviation . ns means non - significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) In vitro enzymatic assay quantitating the effect of NDST3 on α-tubulin acetylation. Flag-NDST3 was expressed in HEK 293 cells and immunoprecipitated by anti-Flag beads. Flag-EGFP was used as a control. The immunoprecipitated protein was incubated with purified tubulin heterodimers in vitro, with or without 1 mM NAD+, 400 nM TSA, or 5 mM nicotinamide (NAM) for 2 h at room temperature. The reaction products were examined by immunoblotting against Ac-α-tubulin or total α-tubulin (n = 3 independent experiments, *P = 0.0185, **P < 0.01, ****P < 0.0001). (D) The same in vitro enzymatic assay as in (C), except that the purified tubulin was polymerized in the presence of 20 μM Taxol and 1 mM GTP (n = 3 independent experiments, **P < 0.01, ***P = 0.0001). Data information: Error bars represent ± standard deviation. ns means non-significant.


( A , B ) NDST3 protein levels relative to β - tubulin in B lymphocytes derived from healthy controls and C9orf72 - linked ALS patients , as examined by immunoblotting ( n = 3 biologically independent samples , * * P = 0 . 0026 ) . Data information : Error bars represent ± standard deviation .

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

(A, B) NDST3 protein levels relative to β-tubulin in B lymphocytes derived from healthy controls and C9orf72-linked ALS patients, as examined by immunoblotting (n = 3 biologically independent samples, **P = 0.0026). Data information: Error bars represent ± standard deviation.


( C - F ) The protein levels of NDST3 relative to GAPDH ( D ) ( * * P = 0 . 0013 ) , Ac - α - tubulin relative to total α - tubulin ( E ) ( * P = 0 . 0379 ) , and total α - tubulin relative to GAPDH ( F ) ( P = 0 . 5700 ) , as examined by immunoblotting , in spinal cord tissues from healthy controls and C9orf72 - linked ALS patients ( n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group ) . Data information : Error bars represent ± standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F) The protein levels of NDST3 relative to GAPDH (D) (**P = 0.0013), Ac-α-tubulin relative to total α-tubulin (E) (*P = 0.0379), and total α-tubulin relative to GAPDH (F) (P = 0.5700), as examined by immunoblotting, in spinal cord tissues from healthy controls and C9orf72-linked ALS patients (n = 4 biologically independent samples in the control group and n = 5 biologically independent samples in the ALS patient group). Data information: Error bars represent ± standard deviation.


( G - I ) Immunoblotting assays showing down - regulation of NDST3 in C9orf72 shRNA ( shC9orf72 ) - treated RPE1 cells , as compared to control shRNA ( shCTRL ) - treated cells . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( H ) ( n = 4 independent experiments , * * * P = 0 . 0004 ) and ( I ) ( n = 4 independent experiments , * * * P = 0 . 0001 ) , respectively . ( J - L ) Immunoblotting assays showing up - regulation of NDST3 in RPE1 cells over - expressing Flag - C9orf72 compared to those expressing Flag - EGFP as control . Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in ( K ) ( n = 3 independent experiments , * * P = 0 . 0011 ) and ( L ) ( n = 3 independent experiments , * * P = 0 . 0033 ) , respectively . Data information : Error bars represent ± standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Immunoblotting assays showing down-regulation of NDST3 in C9orf72 shRNA (shC9orf72)-treated RPE1 cells, as compared to control shRNA (shCTRL)-treated cells. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (H) (n = 4 independent experiments, ***P = 0.0004) and (I) (n = 4 independent experiments, ***P = 0.0001), respectively. (J-L) Immunoblotting assays showing up-regulation of NDST3 in RPE1 cells over-expressing Flag-C9orf72 compared to those expressing Flag-EGFP as control. Quantification of C9orf72 and NDST3 expression relative to GAPDH is shown in (K) (n = 3 independent experiments, **P = 0.0011) and (L) (n = 3 independent experiments, **P = 0.0033), respectively. Data information: Error bars represent ± standard deviation.


( M , N ) Baf A1 sensitivity assay for WT , NDST3 KO , and Noco - treated NDST3 KO RPE1 cells expressing proline - arginine poly - dipeptide ( PR82 ) . 24 h after the transfection of PR82 , WT and NDST3 KO cells were treated with vehicle or 10 μg / mL Noco overnight . The cell survival measured using calcein AM staining at 48 h post - transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells ( n = 3 independent cultures , * P = 0 . 0369 , * * * P = 0 . 0007 ) . Scale bar , 200 μm . Data information : Error bars represent ± standard deviation .

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

(M, N) Baf A1 sensitivity assay for WT, NDST3 KO, and Noco-treated NDST3 KO RPE1 cells expressing proline-arginine poly-dipeptide (PR82). 24 h after the transfection of PR82, WT and NDST3 KO cells were treated with vehicle or 10 μg/mL Noco overnight. The cell survival measured using calcein AM staining at 48 h post-transfection was quantified as the percentage of the fluorescence intensity for treated cells relative to that of untreated cells (n = 3 independent cultures, *P = 0.0369, ***P = 0.0007). Scale bar, 200 μm. Data information: Error bars represent ± standard deviation.


Tap - 1 is required for the secretion of TseL . Pellet ( P ) and supernatant ( S ) of bacterial cultures were analyzed by SDS - PAGE . The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown . The asterisk marks an unspecific band , detected by the antiserum against TseL , present in the pre - immunized serum . One representative experiment of three independent experiments is shown .

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

Tap-1 is required for the secretion of TseL. Pellet (P) and supernatant (S) of bacterial cultures were analyzed by SDS-PAGE. The result of immunoblotting with antisera against TseL and Hcp and purified antibodies against DnaK is shown. The asterisk marks an unspecific band, detected by the antiserum against TseL, present in the pre-immunized serum. One representative experiment of three independent experiments is shown.


Tap - 1 is required for TseL - mediated killing . Wild - type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay . The y - axis indicates the number of surviving C6706 .

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

Tap-1 is required for TseL-mediated killing. Wild-type C6706 or C6706 lacking tsiV1 were exposed to the indicated V52 mutants in a killing assay. The y-axis indicates the number of surviving C6706.


Tap - 1 is required for TseL function . Killing assays in which V52Δtap - 1 was mixed with wild - type C6706 or mutants lacking tsiV1 , tsiV2 , or tsiV3 . The y - axis indicates surviving C6706 .

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

Tap-1 is required for TseL function. Killing assays in which V52Δtap-1 was mixed with wild-type C6706 or mutants lacking tsiV1, tsiV2, or tsiV3. The y-axis indicates surviving C6706.


TseL secretion depends on VgrG - 1 . Western blot analysis of pellet ( P ) and supernatant ( S ) samples of the indicated strains . Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK . The unspecific band detected by rabbit serum is marked with an asterisk . One representative of three experiments is shown .

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

TseL secretion depends on VgrG-1. Western blot analysis of pellet (P) and supernatant (S) samples of the indicated strains. Samples were immunoblotted with antisera against TseL and Hcp and purified antibodies against DnaK. The unspecific band detected by rabbit serum is marked with an asterisk. One representative of three experiments is shown.


VgrG - 1 is required for TseL - mediated killing . Killing assay with wild - type V52 or indicated mutants and wild - type C6706 or a mutant lacking the immunity gene tsiV1 . The y - axis indicates surviving C6706 .

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

VgrG-1 is required for TseL-mediated killing. Killing assay with wild-type V52 or indicated mutants and wild-type C6706 or a mutant lacking the immunity gene tsiV1. The y-axis indicates surviving C6706.


VasX - and VgrG - 3 - mediated killing but not TseL - mediated killing occurs in the absence of VgrG - 1 . V52ΔvgrG - 1 was mixed in a killing assay with wild - type C6706 or mutants lacking immunity genes tsiV1 , tsiV2 , or tsiV3 ( specific for TseL , VasX , and VgrG - 3 , respectively ) . The y - axis indicates surviving C6706 .

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

VasX- and VgrG-3-mediated killing but not TseL-mediated killing occurs in the absence of VgrG-1. V52ΔvgrG-1 was mixed in a killing assay with wild-type C6706 or mutants lacking immunity genes tsiV1, tsiV2, or tsiV3 (specific for TseL, VasX, and VgrG-3, respectively). The y-axis indicates surviving C6706.


A , B Interactions between Tap - 1 and TseL ( A ) or VgrG - 1 ( B ) . Shown are immunoblots of lysates ( total ) and immunoprecipitates with anti - FLAG affinity beads ( IP : FLAG ) of V52Δtap - 1 ( A ) or V52 vgrG - 1 : : myc ( B ) transformed with empty vector or a plasmid encoding His - tagged or FLAG - tagged Tap - 1 .

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

A, B Interactions between Tap-1 and TseL (A) or VgrG-1 (B). Shown are immunoblots of lysates (total) and immunoprecipitates with anti-FLAG affinity beads (IP:FLAG) of V52Δtap-1 (A) or V52 vgrG-1::myc (B) transformed with empty vector or a plasmid encoding His-tagged or FLAG-tagged Tap-1.


C Interaction between TseL and VgrG - 1 . Shown are immunoblots of lysates ( total ) and immunoprecipitates with an anti - FLAG affinity beads ( IP : FLAG ) of V52 or V52Δtap - 1 transformed with empty vector or a plasmid encoding HIS - tagged or FLAG - tagged VgrG - 1 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Interaction between TseL and VgrG-1. Shown are immunoblots of lysates (total) and immunoprecipitates with an anti-FLAG affinity beads (IP:FLAG) of V52 or V52Δtap-1 transformed with empty vector or a plasmid encoding HIS-tagged or FLAG-tagged VgrG-1.