Figure 6 ( A ) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B ( CT - B ) conjugated to Alexa Fluor 488 ( green ) . Islets are co - stained with DAPI ( blue ) ( scale bar = 50μm . ) . ( B ) FACS profiling of dispersed islets from 12 week old mice stained with insulin and CT - B . ( C ) Confocal images of rafts ( using CTB - Alexa Fluor 647 ) in MIN6 cells transfected with Fluorescein - 488 - labelled siRNAScr or Dy547 - labelled siRNAABCA12 ( scale bar = 10μm ) . ( D ) Quantitation by confocal microscopy of the effect of ABCA12 - deficincy on CTB binding to MIN6 cells ( mean ± SEM , * p = < 0 . 05 , Students t - test , n = 4 biological replicates , 50 - 100 cells for each ) . ( E ) Quantitation by flow cytometry of the effect of ABCA12 - deficiency on CTB binding to MIN6 cells ( mean ± SEM , n = 3 biological replicates , * p = < 0 . 05 , Students t - test ) . ( F ) The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 ( incubation for 5 or 10 minutes as indicated , mean ± SEM , n = 4 ( biological replicates ) , * p = < 0 . 05 , Students t - test ) . ( G ) Western blot of the abundance of CDC42 in ABCA12 deficient MIN6 cells . ( H ) The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells ( BDK , 100 ng / ml , 4 min ; * p = < 0 . 05 , Students t - test , significance versus ABCA12 - deficient BDK activated cells is indicated , n = 4 biological replicates , mean ± SEM ) . ( I , J ) ( I ) Confocal images of F - actin in ABCA12 - deficient MIN6 cells ( scale bar = 20μm ) and ( J ) quantification of abundance showing the effect of ABCA12 - deficiency ( mean ± SEM , * p = < 0 . 05 , Students t - test , n = 4 biological replicates , 50 - 100 cells for each ) . ( K ) F - actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 ( mean ± SEM , n = 3 biological replicates , * p = < 0 . 05 , Students t - test ) . ( L , M ) ( L ) Representative image of F - actin using LifeAct staining of purified islets isolated from Abca12tm1d versus control mice ( scale bar = 10μm ) and ( M ) quantitation of fluorescent signals from these samples ( n = 3 biological replicates , mean ± SEM , * * * p = < 0 . 001 , * * * * p = < 0 . 0001 Students t - test ) . ( N ) Colocalization between ABCA12 ( red ) and CDC42 ( green ) in MIN6 cells ( scale bar = 10μm ) . O ) Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 cells with ABCA12 - deficiency ( mean ± SEM , n = 4 biological replicates , * p = < 0 . 05 , Students t - test ) . ( P ) The effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12 - deficiency . Treatments are defined in the table below . ( Q ) Effects of Jasplakinolide on F - actin levels ( green ) in siRNA treated cells ( scale bar = 10μm ) . ( R ) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose ( mean ± SEM , n = 4 biological replicates , * p = < 0 . 05 , * * p = < 0 . 01 , significance relative to untreated cells , Students t - test )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Detection of lipid rafts in 8 week old islets with recombinant cholera toxin subunit B (CT-B) conjugated to Alexa Fluor 488 (green). Islets are co-stained with DAPI (blue) (scale bar = 50μm.).(B) FACS profiling of dispersed islets from 12 week old mice stained with insulin and CT-B.(C) Confocal images of rafts (using CTB-Alexa Fluor 647) in MIN6 cells transfected with Fluorescein-488-labelled siRNAScr or Dy547-labelled siRNAABCA12 (scale bar=10μm). (D) Quantitation by confocal microscopy of the effect of ABCA12-deficincy on CTB binding to MIN6 cells (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each). (E) Quantitation by flow cytometry of the effect of ABCA12-deficiency on CTB binding to MIN6 cells (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test). (F) The effect 10 mM MβCD on GSIS in MIN6 cells depleted or not for ABCA12 (incubation for 5 or 10 minutes as indicated, mean±SEM, n=4 (biological replicates), *p=<0.05, Students t-test).(G) Western blot of the abundance of CDC42 in ABCA12 deficient MIN6 cells.(H) The effect of ABCA12 deficiency on activation of CDC42 by bradykinin in MIN6 cells (BDK, 100 ng/ml, 4 min; *p=<0.05, Students t-test, significance versus ABCA12-deficient BDK activated cells is indicated, n=4 biological replicates, mean±SEM).(I,J) (I) Confocal images of F-actin in ABCA12-deficient MIN6 cells (scale bar = 20μm) and (J) quantification of abundance showing the effect of ABCA12-deficiency (mean±SEM, *p=<0.05, Students t-test, n=4 biological replicates, 50-100 cells for each).(K) F-actin levels assessed by FACS sorting of labelled MIN6 cells transfected with siRNAABCA12 (mean±SEM, n=3 biological replicates, *p=<0.05, Students t-test).(L,M) (L) Representative image of F-actin using LifeAct staining of purified islets isolated from Abca12tm1d versus control mice (scale bar = 10μm) and (M) quantitation of fluorescent signals from these samples (n=3 biological replicates, mean±SEM, ***p=<0.001, ****p=<0.0001 Students t-test).(N) Colocalization between ABCA12 (red) and CDC42 (green) in MIN6 cells (scale bar = 10μm).O) Effects of overexpression of a constitutively active form of CDC42 on GSIS from MIN6 cells with ABCA12-deficiency (mean±SEM, n=4 biological replicates, *p=<0.05, Students t-test).(P) The effect of overexpression of consitutively active CDC42 on ABCA1 abundance in MIN6 cells with ABCA12-deficiency. Treatments are defined in the table below.(Q) Effects of Jasplakinolide on F-actin levels (green) in siRNA treated cells (scale bar=10μm).(R) Effects of Abca12 knockdown and Jasplakinolide treatment on MIN6 cell in low and high glucose (mean±SEM, n=4 biological replicates, *p=<0.05, **p=<0.01, significance relative to untreated cells, Students t-test)


Figure 7 ( A ) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively ; COH = cholesterol , CE = cholesteryl ester ) . ( B ) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively , * * p = < 0 . 01 Students t - test ) ; CE = cholesteryl ester , Cer = ceramide , DHC ( Hex2Cer ) = dihexosylceramide , MHC ( HexCer ) = monohexosylceramide , SM = sphingomyelin , GM3 = GM3 ganglioside , dhCer = dihydroceramide , DG = diacylglyerol , TG = triacylglycerol ) . ( C ) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age ( mean ± SEM , n = 10 , 4 and 5 mice per genotype respectively , * p = < 0 . 05 , * * p = < 0 . 01 , Students t - test ) ( D ) Immunohistochemical staining of levels IL - 1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age ( bar = 25μm , islets outlined ) . ( E ) Quantification of islet specific IL1β staining from sections of Abca12tm1d , Cre and wild type mice ( mean ± SEM , n = 3 , 3 and 4 mice of each genotype respectively ; * p = < 0 . 05 , Students t - test ) . Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing β - cell mass ( F ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) . Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing cleaved caspase 3 ( G ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) . Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon ( H ) ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) . Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing F480 + macrophage populations ( I ) ( mean ± SEM , n = 3 - 9 mice per genotype , * * p = < 0 . 01 , * * * p = < 0 . 001 , Students t - test ) . ( J ) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes ( mean ± SEM , n = 3 , 4 and 5 mice per genotype respectively , * p = < 0 . 05 , Students t - test ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) Mass spectroscopy analysis of levels of cholesterol and cholesteryl esters in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively; COH=cholesterol, CE=cholesteryl ester).(B) Mass spectroscopy analysis of lipids families in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, **p=<0.01 Students t-test); CE=cholesteryl ester, Cer=ceramide, DHC (Hex2Cer)=dihexosylceramide, MHC (HexCer)=monohexosylceramide, SM=sphingomyelin, GM3=GM3 ganglioside, dhCer=dihydroceramide, DG=diacylglyerol, TG=triacylglycerol).(C) Mass spectroscopy analysis of triglyceride species in purified islets from Abca12tm1d mice versus control animals at 16 weeks of age (mean±SEM, n=10, 4 and 5 mice per genotype respectively, *p=<0.05, **p=<0.01, Students t-test)(D) Immunohistochemical staining of levels IL-1β expression in sections of pancreas from Abca12tm1d mice versus control animals at 24 weeks of age (bar = 25μm, islets outlined).(E) Quantification of islet specific IL1β staining from sections of Abca12tm1d, Cre and wild type mice (mean±SEM, n=3, 3 and 4 mice of each genotype respectively; *p=<0.05, Students t-test).Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing β-cell mass (F) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing cleaved caspase 3 (G) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing glucagon (H) (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).Analysis of Abca12tm1d pancreata from mice at 24 weeks of age compared to control mice assessing F480+ macrophage populations (I)(mean±SEM, n=3-9 mice per genotype, **p=<0.01, ***p=<0.001, Students t-test).(J) Fasting blood glucose levels coincident with the emergence of inflammatory phenotypes (mean±SEM, n=3, 4 and 5 mice per genotype respectively, *p=<0.05, Students t-test).


Figure 1Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP , and FLAG - TCHP at N - and C - terminal . total lysates were immunoprecipitated with anti - FLAG antibodies , and blots were probed sequentially with anti - PCM1 , anti - FLAG , and anti - ACTIN antibodies . IgG light chains are indicated with Red Ponceau staining . The input totals were analyzed by parallel immunoblotting as a control for the level of expression . Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP - FLAG C - terminal or constructs with deletions of the coiled - coil domain 1 ( TCHP ∆ 1 ) and 2 ( TCHP ∆ 2 ) , as indicated in the scheme . total lysates were immunoprecipitated with anti - FLAG antibodies , and blots were probed sequentially with anti - PCM1 , anti - FLAG , and anti - ACTIN antibodies . IgG light chains are indicated with Red Ponceau staining . The input totals were analyzed by parallel immunoblotting as a control for the level of expression . Anti - PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot . Co - localization of TCHP - V5 and centriolar satellite proteins . HUVECs were transduced with TCHP - V5 lentivirus and stained for anti - PCM1 , anti - CEP290 , anti - CEP72 and anti - V5 antibodies . Scale bars , 25 µm and 5 µm in the inset . Right panel : the two - channel intensity correlation of pixels corresponding to regions identified with TCHP - V5 and satellite markers ( n = 90 cells ; Pearson colocalization coefficient ) . TCHP knock - down or control cells were stained for anti - PCM1 antibody . Panel below : quantification ( n = 50 cells ; unpaired t - test ; * * p = 0 . 004 vs control ) . Scale bars , 50 µm and 5 µm in the inset .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP, and FLAG-TCHP at N- and C- terminal. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.Immunoblot analysis showed HEK293 transfected for 48 hours with expression vectors for TCHP-FLAG C-terminal or constructs with deletions of the coiled-coil domain 1 (TCHP ∆1) and 2 (TCHP ∆2), as indicated in the scheme. total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-PCM1, anti-FLAG, and anti-ACTIN antibodies. IgG light chains are indicated with Red Ponceau staining. The input totals were analyzed by parallel immunoblotting as a control for the level of expression.Anti-PCM1 immunoprecipitation from HUVECs cells and TCHP and PCM1 immunoblot.Co-localization of TCHP-V5 and centriolar satellite proteins. HUVECs were transduced with TCHP-V5 lentivirus and stained for anti-PCM1, anti-CEP290, anti-CEP72 and anti-V5 antibodies. Scale bars, 25 µm and 5 µm in the inset. Right panel: the two-channel intensity correlation of pixels corresponding to regions identified with TCHP-V5 and satellite markers (n=90 cells; Pearson colocalization coefficient).TCHP knock-down or control cells were stained for anti-PCM1 antibody. Panel below: quantification (n=50 cells; unpaired t-test; **p=0.004 vs control). Scale bars, 50 µm and 5 µm in the inset.


Figure 2Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) and MG132 treatments for the indicated number of hours before immunoblotting . PCM1 levels were analysed in the insoluble fraction . Laminin has been used as a loading control . Below panel : Quantification of PCM1 degradation . ( one - way ANOVA ; * p = 0 . 0180 vs control time 0 ) . Control or TCHP knock - down HUVECs were subjected to cycloheximide ( CHX ) and MG132 treatments for the indicated number of hours before immunoblotting . Condition as in ( A ) , Western blot for anti - GABARAP and anti - ACTIN antibodies . Below panel : Quantification of GABARAP degradation . ( one - way ANOVA ; * p = 0 . 0215 vs control time 0 ) . Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting . Western blot was probed for anti - PCM1 and anti - V5 antibodies . Laminin has been used as a loading control . Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting . Western blot was probed for anti - V5 , anti - GABARAP and anti - ACTIN antibodies . Below panels : Quantification of C ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0003 vs control CHX ) and D ( one - way ANOVA ; * * p < 0 . 0001 vs control time 0 ; # # p = 0 . 0056 vs control CHX ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. PCM1 levels were analysed in the insoluble fraction. Laminin has been used as a loading control. Below panel: Quantification of PCM1 degradation. (one-way ANOVA; *p=0.0180 vs control time 0).Control or TCHP knock-down HUVECs were subjected to cycloheximide (CHX) and MG132 treatments for the indicated number of hours before immunoblotting. Condition as in (A), Western blot for anti-GABARAP and anti-ACTIN antibodies. Below panel: Quantification of GABARAP degradation. (one-way ANOVA; *p =0.0215 vs control time 0).Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-PCM1 and anti-V5 antibodies. Laminin has been used as a loading control.Control or TCHP overexpressing HUVECs were subjected to CHX treatment for the indicated number of hours prior to immunoblotting. Western blot was probed for anti-V5, anti-GABARAP and anti-ACTIN antibodies. Below panels: Quantification of C (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0003 vs control CHX) and D (one-way ANOVA; **p<0.0001 vs control time 0; ##p=0.0056 vs control CHX).


Figure 3Immunofluorescent staining for LC3 and p62 in TCHP knock - down and control cells . Scale bars , 25μm . Lower panel : quantification . ( n = 90 cells , unpaired t - test ; LC3 : * * p < 0 . 0001 vs control ; p62 : * * p < 0 . 0001 vs control ) . Western blot of p62 and LC3 during under normal culture condition or starved condition ( HBSS ) or the presence of BafA1 ( 2 hours ) in TCHP knock - down or control cells . Lower panels : p62 quantification . ( one - way ANOVA ; * * p = 0 . 0003 vs control , # # p = 0 . 0093 vs shTCHP ) ; LC3 quantification . ( one - way ANOVA ; * * p = 0 . 0008 vs control , # # p = 0 . 0011 vs shTCHP ) . HUVECs were transduced with the tandem mCherry - EGFP - LC3 and with shRNA TCHP or control vectors . Left panels : Representative picture of mCherrry - EGFP - LC3 reporters . Scale bars , 25μm . Right panel : Quantification of the number of mCherry - only ( red bars , autolysosomes ) or double - positive ( mCherry + / EGFP + ; yellow bars , autophagosomes ) . ( n = 80 cells , one - way ANOVA ; * p = 0 . 0447 vs control ; # # p = 0 . 0007 vs shTCHP ) . Ratiometric flow cytometric analysis of mCherrry - EGFP - LC3 reporters as in C . ( one - way ANOVA ; * p = 0 . 0247 vs control ; # # p = 0 . 0088 vs shTCHP ) . Quantification of long - lived protein degradation assay in TCHP knock - down and control cells by flow cytometry ( one - way ANOVA ; * * p = 0 . 0018 vs control , # # p < 0 . 0001 vs shTCHP ) . HUVECs were transduced with TCHP - V5 and control vectors . Western blot for anti - V5 and anti - p62 antibody during under normal culture condition or HBSS or BafA1 or Torin - 1 treatment . Lower panel : quantification . ( one - way ANOVA ; * * p = 0 . 0044 ; # p = 0 . 0117 vs TCHP - V5 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Immunofluorescent staining for LC3 and p62 in TCHP knock-down and control cells. Scale bars, 25μm. Lower panel: quantification. (n=90 cells, unpaired t-test; LC3: **p<0.0001 vs control; p62: **p<0.0001 vs control).Western blot of p62 and LC3 during under normal culture condition or starved condition (HBSS) or the presence of BafA1 (2 hours) in TCHP knock-down or control cells. Lower panels: p62 quantification. (one-way ANOVA; **p=0.0003 vs control, ##p=0.0093 vs shTCHP); LC3 quantification. (one-way ANOVA; **p=0.0008 vs control, ##p=0.0011 vs shTCHP).HUVECs were transduced with the tandem mCherry-EGFP-LC3 and with shRNA TCHP or control vectors. Left panels: Representative picture of mCherrry-EGFP-LC3 reporters. Scale bars, 25μm. Right panel: Quantification of the number of mCherry-only (red bars, autolysosomes) or double-positive (mCherry+/EGFP+; yellow bars, autophagosomes). (n=80 cells, one-way ANOVA; *p=0.0447 vs control; ##p=0.0007 vs shTCHP).Ratiometric flow cytometric analysis of mCherrry-EGFP-LC3 reporters as in C. (one-way ANOVA; *p=0.0247 vs control; ##p=0.0088 vs shTCHP).Quantification of long-lived protein degradation assay in TCHP knock-down and control cells by flow cytometry (one-way ANOVA; **p=0.0018 vs control, ##p<0.0001 vs shTCHP).HUVECs were transduced with TCHP-V5 and control vectors. Western blot for anti-V5 and anti-p62 antibody during under normal culture condition or HBSS or BafA1 or Torin-1 treatment. Lower panel: quantification. (one-way ANOVA; **p=0.0044; #p=0.0117 vs TCHP-V5).


Figure 4The postnuclear fraction ( PNS ) from TCHP knock - down and control HUVECs in the presence or absence of BafA1 was separated into ( low - speed pellet ) LSP and ( high - speed pellet ) HSP fractions and then analysed by immunoblots using anti p62 , LC3 and GABARAP antibodies . The sub - fractions were treated with proteinase K ( Prot . K ) with or without Triton X - 100 ( TX - 100 ) . Quantification of the Western blot : the p62 , LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non - treated samples . Representative TEM images of autophagosomes in control and TCHP knock - down HUVECs in the complete medium after 3h incubation with BafA1 ( Scale bars , 600nm ) : i , quantification of mean autophagic vesicles ( AVs ) and autophagosome ( APs ) area and ii , distribution of the cross - section areas of the analysed vesicles expressed in percentage . ( n = 7 cells ; > 100 vesicles per sample ; AVs : unpaired t - test ; * * p < 0 . 0001 vs control ; APs : unpaired t - test * * p < 0 . 0001 vs control ) . Left panels : Representative single - channel and merged images of HUVECs expressing GFP - LC3 and immunostained for STX17 . Scale bars , 25 µm and 2 µm in the inset . Right panels : Quantification of LC3 / STX17 positive cells in the presence or absence of BafA1 ( one - way ANOVA ; * * p = 0 . 0060 vs control ; # p = 0 . 0136 vs control BafA1 ) and quantification LC3 / STX17 positive puncta per cells ( n = 50 cells ; one - way ANOVA ; * * p < 0 . 0001 vs shTCHP BafA1 ) .

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Figure 4The postnuclear fraction (PNS) from TCHP knock-down and control HUVECs in the presence or absence of BafA1 was separated into (low-speed pellet) LSP and (high-speed pellet) HSP fractions and then analysed by immunoblots using anti p62, LC3 and GABARAP antibodies. The sub-fractions were treated with proteinase K (Prot. K) with or without Triton X- 100 (TX-100). Quantification of the Western blot: the p62, LC3 and GABARAP levels relative to respective GAPDH were quantified using densitometry analysis and normalized to the value of non-treated samples.Representative TEM images of autophagosomes in control and TCHP knock-down HUVECs in the complete medium after 3h incubation with BafA1 (Scale bars, 600nm): i, quantification of mean autophagic vesicles (AVs) and autophagosome (APs) area and ii, distribution of the cross-section areas of the analysed vesicles expressed in percentage. (n=7 cells; >100 vesicles per sample; AVs: unpaired t-test; **p<0.0001 vs control; APs: unpaired t-test **p<0.0001 vs control).Left panels: Representative single-channel and merged images of HUVECs expressing GFP-LC3 and immunostained for STX17. Scale bars, 25 µm and 2 µm in the inset. Right panels: Quantification of LC3/STX17 positive cells in the presence or absence of BafA1 (one-way ANOVA; **p=0.0060 vs control; #p=0.0136 vs control BafA1) and quantification LC3/STX17 positive puncta per cells (n=50 cells; one-way ANOVA; **p<0.0001 vs shTCHP BafA1).


Figure 5Expression of TCHP , p62 , IL - 1β , IL - 8 , and IL - 6 in ECs from vessels wall from patients with CAD ( n = 8 patients per group , unpaired t - test ; TCHP * * p < 0 . 0001 ; p62 * * p = 0 . 0007 ; IL - 1β * * p < 0 . 0001 ; IL - 8 * * p < 0 . 0001 ; and IL - 6 * * p < 0 . 0001 vs . Healthy subject ) . Staining , and quantification of p62 in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . Scale bars , 50 µm ; ( n = 3 patients per group ; n = 80 cells ; one - way ANOVA ; * * p < 0 . 0001 vs healthy DMSO ; # # p = 0 . 003 vs patients DMSO ) . Expression of IL - 6 , IL - 8 and IL - 1β ( n = 3 patients per group ; one - way ANOVA ; IL - 6 : * p = 0 . 0135 vs healthy DMSO ; # p = 0 . 0180 vs patients DMSO ; IL - 8 : * p = 0 . 0139 vs healthy DMSO ; # # p = 0 . 0024 vs patients DMSO ; IL - 1β : * p = 0 . 0104 vs healthy DMSO ; # # p = 0 . 0069 vs patients DMSO ) . migration speed , in ECs from healthy subject and patients either treated with vehicle ( DMSO ) or BAY . ( n = 3 patients per group ; one - way ANOVA ; * p = 0 . 0325 vs healthy DMSO ; # p = 0 . 0336 vs patients DMSO ) . Left panels : representative images of the heart of wild - type and Tchp knock - out mice stained for CD31 ( green ) and p62 ( magenta ) . Scale bars , 100µm , and 50µm for the inset . Right panels : Quantification of the percentage of cardiac area ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 083 vs wild - type ) or vessels positive for p62 ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 058 vs wild - type ) and vessel density ( n = 4 mice per group ; unpaired t - test ; * * p = 0 . 0097 vs wild - type ) .

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Figure 5Expression of TCHP, p62, IL-1β, IL-8, and IL-6 in ECs from vessels wall from patients with CAD (n=8 patients per group, unpaired t-test; TCHP **p <0.0001; p62 **p = 0.0007; IL-1β **p <0.0001; IL-8 **p <0.0001; and IL-6 **p <0.0001 vs. Healthy subject).Staining, and quantification of p62 in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. Scale bars, 50 µm; (n=3 patients per group; n=80 cells; one-way ANOVA; **p<0.0001 vs healthy DMSO; ##p=0.003 vs patients DMSO).Expression of IL-6, IL-8 and IL-1β (n=3 patients per group; one-way ANOVA; IL-6: *p = 0.0135 vs healthy DMSO; #p = 0.0180 vs patients DMSO; IL-8: *p = 0.0139 vs healthy DMSO; ##p = 0.0024 vs patients DMSO; IL-1β: *p = 0.0104 vs healthy DMSO; ##p = 0.0069 vs patients DMSO).migration speed, in ECs from healthy subject and patients either treated with vehicle (DMSO) or BAY. (n=3 patients per group; one-way ANOVA; *p=0.0325 vs healthy DMSO; #p=0.0336 vs patients DMSO).Left panels: representative images of the heart of wild-type and Tchp knock-out mice stained for CD31 (green) and p62 (magenta). Scale bars, 100µm, and 50µm for the inset. Right panels: Quantification of the percentage of cardiac area (n=4 mice per group; unpaired t-test; **p=0.083 vs wild-type) or vessels positive for p62 (n=4 mice per group; unpaired t-test; **p=0.058 vs wild-type) and vessel density (n=4 mice per group; unpaired t-test; **p=0.0097 vs wild-type).


Figure 1b . Multiplexed Error Robust Fluorescent in situ hybridization ( MERFISH ) and DAPI stained nuclei in the mouse hippocampus . Each gene is represented by a different color . For the entire hippocampus ( left ) , only the mRNA spots are shown with a scale bar of 500 microns . On the zoomed - in section ( right ) , each gene is represented by a different color dot , and the DAPI intensity is displayed in white . The scale bar is 20 microns .

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Figure 1b. Multiplexed Error Robust Fluorescent in situ hybridization (MERFISH) and DAPI stained nuclei in the mouse hippocampus. Each gene is represented by a different color. For the entire hippocampus (left), only the mRNA spots are shown with a scale bar of 500 microns. On the zoomed-in section (right), each gene is represented by a different color dot, and the DAPI intensity is displayed in white. The scale bar is 20 microns.


Figure 6a . Normalized expression of Tox in CA1sp1 , Leng8 in CA3sp3 , Hecw1 in DG3 , and Thra in Astro1 show variable expression throughout the hippocampus . Scale bar is 500 microns . spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell ( sub ) type neighborhoods .

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Figure 6a. Normalized expression of Tox in CA1sp1, Leng8 in CA3sp3, Hecw1 in DG3, and Thra in Astro1 show variable expression throughout the hippocampus. Scale bar is 500 microns. spDEGs were computed by comparing the true variance in gene expression between cell subtype neighborhoods to that of randomly permuted cell (sub)type neighborhoods.


Figure 1 ( C ) HeLa GFP - LC3 cells were transfected with indicated siRNAs and infected with DsRed - labeled Salmonella . The percentage of GFP - LC3 + Salmonella at 1 hr post - infection is shown . Data are shown as means ± SD ; n = 75 cells per condition from three biological replicates . Data are representative of three independent experiments . ( D ) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in ( C ) . The scale bars represent 10 μm . ( E ) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D / PepA . Data are representative of three independent experiments . ( G ) HeLa GFP - LC3 cells were transfected with control siRNA , CLEC12A siRNA1 , or CLEC12A siRNA2 as well as CLEC12A rescue constructs ( R1 or R2 ) and infected for 1 hr with DsRed - labeled Salmonella . Data are shown as mean ± SD ; n = 75 per condition . Data are representative of three independent experiments . ( H ) HeLa cells from ( G ) were lysed , and HA - CLEC12A expression levels were assessed by western blot . Data are representative of three independent experiments .

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Figure 1(C) HeLa GFP-LC3 cells were transfected with indicated siRNAs and infected with DsRed-labeled Salmonella. The percentage of GFP-LC3+ Salmonella at 1 hr post-infection is shown. Data are shown as means ± SD; n = 75 cells per condition from three biological replicates. Data are representative of three independent experiments.(D) Representative epifluorescent microscopy images of HeLa cells used to generate data shown in (C). The scale bars represent 10 μm.(E) Western blot of HeLa cells transfected with the indicated siRNAs and treated with Torin E64D/PepA. Data are representative of three independent experiments.(G) HeLa GFP-LC3 cells were transfected with control siRNA, CLEC12A siRNA1, or CLEC12A siRNA2 as well as CLEC12A rescue constructs (R1 or R2) and infected for 1 hr with DsRed-labeled Salmonella. Data are shown as mean ± SD; n = 75 per condition. Data are representative of three independent experiments.(H) HeLa cells from (G) were lysed, and HA-CLEC12A expression levels were assessed by western blot. Data are representative of three independent experiments.


Figure 2 ( A ) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA . The scale bars represent 10 μm . ( B ) Percentage of LC3 - Salmonella colocalization in WT HeLa cells , ATG16L1 KO HeLa cells , and ATG16L1 KO HeLa cells stably expressing ATG16L1 ∗ 300T or ∗ 300A alleles in the presence of control siRNA or CLEC12A siRNA . Data are shown as means ± SD ; n = 75 . Data shown are representative of three independent experiments .

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Figure 2(A) Representative epifluorescent images showing impaired antibacterial autophagy in ATG16L1 KO HeLa cells complemented with the indicated ATG16L1 protein in the presence of control siRNA or CLEC12A siRNA. The scale bars represent 10 μm. (B) Percentage of LC3-Salmonella colocalization in WT HeLa cells, ATG16L1 KO HeLa cells, and ATG16L1 KO HeLa cells stably expressing ATG16L1∗300T or ∗300A alleles in the presence of control siRNA or CLEC12A siRNA. Data are shown as means ± SD; n = 75. Data shown are representative of three independent experiments.


Figure 3 ( A ) Confocal micrographs of LC3 - Listeria colocalization in WT and Clec12a − / − BMDMs at 1 hr post - infection . The scale bars represent 10 μm . ( B ) Quantification of LC3 - Listeria colocalization in WT and Clec12a − / − BMDMs . Data shown represent mean ± SD of n = 3 independent experiments ; unpaired t test . ∗ ∗ p < 0 . 01 . ( C and D ) Salmonella cfus quantified per gram of stool ( C ) and per organ ( D ) at 4 days post - infection are displayed . Data are shown as mean ± SD ; ∗ ∗ p ≤ 0 . 01 . Data are representative of at least two independent experiments ( n = 9 for WT ; n = 7 or 8 for Clec12a − / − ) . ( E ) Clinical score for infected mice at 4 days post - infection . Data are shown as mean ± SD . Data are representative of at least two independent experiments . ∗ ∗ ∗ ∗ p < 0 . 0001 ; unpaired t test . ( n = 14 for WT ; n = 13 for Clec12a − / − ) . ( F ) Survival curve for infected WT and Clec12a − / − mice ( n = 9 mice per genotype ) .

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Figure 3(A) Confocal micrographs of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs at 1 hr post-infection. The scale bars represent 10 μm. (B) Quantification of LC3-Listeria colocalization in WT and Clec12a−/− BMDMs. Data shown represent mean ± SD of n = 3 independent experiments; unpaired t test. ∗∗p < 0.01.(C and D) Salmonella cfus quantified per gram of stool (C) and per organ (D) at 4 days post-infection are displayed. Data are shown as mean ± SD; ∗∗p ≤ 0.01. Data are representative of at least two independent experiments (n = 9 for WT; n = 7 or 8 for Clec12a−/−).(E) Clinical score for infected mice at 4 days post-infection. Data are shown as mean ± SD. Data are representative of at least two independent experiments. ∗∗∗∗p < 0.0001; unpaired t test. (n = 14 for WT; n = 13 for Clec12a−/−).(F) Survival curve for infected WT and Clec12a−/− mice (n = 9 mice per genotype).


Figure 4 ( A ) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs . Data are shown as mean ± SD ; n = 6 . Data are representative of four independent experiments . ( B ) Quantitation of CLEC12A - bacteria and LC3 - bacteria colocalization at indicated time points . Data are shown as mean ± SD ; n = 3 independent experiments . ( C ) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP - CLEC12A at 20 , 40 , 60 , and 80 min post - infection . The scale bars represent 10 μm .

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Figure 4(A) Gentamicin protection assay in HeLa cells showing fold replication of Salmonella in cells treated with the indicated siRNAs. Data are shown as mean ± SD; n = 6. Data are representative of four independent experiments.(B) Quantitation of CLEC12A-bacteria and LC3-bacteria colocalization at indicated time points. Data are shown as mean ± SD; n = 3 independent experiments. (C) Representative confocal micrographs of HeLa cells showing association of intracellular bacteria with GFP-CLEC12A at 20, 40, 60, and 80 min post-infection. The scale bars represent 10 μm.


Figure 5 ( A - C ) Representative epifluorescent images of HeLa cells showing colocalization of ( A ) CLEC12A ( green ) and galectin 8 or galectin 3 ( red ) around intracellular Salmonella ( blue ) , ( B ) GFP - CLEC12A ( green ) and NDP52 ( red ) around Salmonella ( blue ) , and ( C ) endogenous CLEC12A ( red ) and GFP - LC3 ( green ) around Salmonella ( blue ) . ( D ) CLEC12A or GFP - CLEC12A ( green ) and GABARAPL2 or ATG16L1 ( red ) around intracellular Salmonella ( blue ) . Images are representative of three or more independent experiments . ( E ) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed - labeled Salmonella for 1 hr . ( F ) Quantitation of ubiquitin - bacteria colocalization at 1 hr post - infection in cells shown in ( E ) . Data are shown as means ± SEM from three independent experiments . ( G ) Representative epifluorescent images of HeLa cells transfected and infected as in ( E ) . ( H ) Quantitation of NDP52 - bacteria colocalization at 1 hr post - infection in cells shown in ( G ) . Data are shown as means ± SEM from three independent experiments . ( I ) Representative confocal micrographs of HeLa cells expressing GFP - CLEC12A . Cells were untreated or exposed to hypertonic media containing PEG 1000 , followed by hypotonic shock .

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Figure 5(A-C) Representative epifluorescent images of HeLa cells showing colocalization of (A) CLEC12A (green) and galectin 8 or galectin 3 (red) around intracellular Salmonella (blue), (B) GFP-CLEC12A (green) and NDP52 (red) around Salmonella (blue), and (C) endogenous CLEC12A (red) and GFP-LC3 (green) around Salmonella (blue).(D) CLEC12A or GFP-CLEC12A (green) and GABARAPL2 or ATG16L1 (red) around intracellular Salmonella (blue). Images are representative of three or more independent experiments.(E) Representative epifluorescent images of HeLa cells transfected with a control siRNA or a siRNA against CLEC12A and infected with DsRed-labeled Salmonella for 1 hr. (F) Quantitation of ubiquitin-bacteria colocalization at 1 hr post-infection in cells shown in (E). Data are shown as means ± SEM from three independent experiments.(G) Representative epifluorescent images of HeLa cells transfected and infected as in (E). (H) Quantitation of NDP52-bacteria colocalization at 1 hr post-infection in cells shown in (G). Data are shown as means ± SEM from three independent experiments.(I) Representative confocal micrographs of HeLa cells expressing GFP-CLEC12A. Cells were untreated or exposed to hypertonic media containing PEG 1000, followed by hypotonic shock.


Figure 6 ( E ) HeLa GFP - LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed - labeled Salmonella for 1 hr . The fraction of GFP - positive intracellular Salmonella is shown . Data are mean ± SD and are representative of three independent experiments . ( F ) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1 , KLHL9 , NEDD8 , or KLHL13 . Data shown as mean ± SD ; n = 4 . Data are representative of three independent experiments .

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Figure 6(E) HeLa GFP-LC3 cells were transfected with indicated siRNAs for 48 hr and then infected with DsRed-labeled Salmonella for 1 hr. The fraction of GFP-positive intracellular Salmonella is shown. Data are mean ± SD and are representative of three independent experiments.(F) Gentamicin protection assay in HeLa cells showing increased intracellular bacterial replication after 48 hr of knockdown of ATG16L1, KLHL9, NEDD8, or KLHL13. Data shown as mean ± SD; n = 4. Data are representative of three independent experiments.


Figure 1B . Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS - CoV - 2 at a multiplicity of infection ( MOI ) of 0 . 1 after 24 h . The numbers of viral RNA copies were quantified with RT - qPCR . The negative control siRNA served as the control and is abbreviated as " Ctrl . " C1 - C11 represent the final candidate sequences after selection . The siRNAs capable of inhibiting up to 99 % of viral envelope gene expression with P values < 0 . 005 compared with Ctrl siRNA are marked with * . P value by Student t test . E . IC50 of C6 and the fully modified C6G25S . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6 or C6G25S before virus infection at an MOI of 0 . 1 . The viral RNA was quantified by RT - qPCR at 24 h after infection . F . IC50 data for viral RdRp inhibition by C6G25S . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before virus infection at an MOI of 0 . 1 . The viral RNA was quantified by RT - qPCR at 24 h after infection .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Vero E6 cells were transfected with 10 nM of siRNA before infection by SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 after 24 h. The numbers of viral RNA copies were quantified with RT-qPCR. The negative control siRNA served as the control and is abbreviated as "Ctrl." C1-C11 represent the final candidate sequences after selection. The siRNAs capable of inhibiting up to 99% of viral envelope gene expression with P values < 0.005 compared with Ctrl siRNA are marked with *. P value by Student t test.E. IC50 of C6 and the fully modified C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6 or C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. F. IC50 data for viral RdRp inhibition by C6G25S. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before virus infection at an MOI of 0.1. The viral RNA was quantified by RT-qPCR at 24 h after infection. D


Figure 2B . IC50 for C6G25S against different variants . Vero E6 cells were transfected with 10 , 2 , 0 . 4 , 0 . 08 , or 0 . 016 nM of C6G25S before infection with different strains of SARS - CoV - 2 . The viral RNA was quantified by RT - qPCR at 24 h after infection . Data are presented as mean ± SD of three biological replicates .

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Figure 2B. IC50 for C6G25S against different variants. Vero E6 cells were transfected with 10, 2, 0.4, 0.08, or 0.016 nM of C6G25S before infection with different strains of SARS-CoV-2. The viral RNA was quantified by RT-qPCR at 24 h after infection. Data are presented as mean ± SD of three biological replicates.


Figure 3A , B , C . K18 - hACE2 - transgenic mice treated with C6G25S by 1 . 48 mg / L of AI for 30 min ( A ) , 50 μL of saline containing 50 μg of C6G25S by IN ( B ) , or PBS ( C ) ( n = 5 per group ) . C6G25S distribution in lungs was visualized by in situ hybridization ( ISH ) staining with C6G25S - specific probe ( red color ) . Bronchi ( i ) and bronchioles ( ii ) marked with the boxes are enlarged on the right . E , F . C57 / B6 mice ( n = 3 per group ) were treated by AI with C6G25S 1 . 48 mg / L for 30 min ( E ) or 50 μL of saline containing 50 μg of C6G25S by IN administration ( F ) . The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem - loop RT - qPCR . Quantification data represent mean ± SD . P value by Student t test .

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Figure 3A, B, C. K18-hACE2-transgenic mice treated with C6G25S by 1.48 mg/L of AI for 30 min (A), 50 μL of saline containing 50 μg of C6G25S by IN (B), or PBS (C) (n = 5 per group). C6G25S distribution in lungs was visualized by in situ hybridization (ISH) staining with C6G25S-specific probe (red color). Bronchi (i) and bronchioles (ii) marked with the boxes are enlarged on the right.E, F. C57/B6 mice (n = 3 per group) were treated by AI with C6G25S 1.48 mg/L for 30 min (E) or 50 μL of saline containing 50 μg of C6G25S by IN administration (F). The C6G25S deposited in whole lungs and nasal cavities was quantified after whole tissue homogenization followed by stem-loop RT-qPCR. Quantification data represent mean ± SD. P value by Student t test.


Figure 4A . K18 - hACE2 - transgenic mice ( Winkler et al . , 2020 ) were treated once daily for 3 days before intranasal challenge with 104 plaque - forming units ( PFU ) of the original virus . Prophylactic treatment consists of 30 min of AI ( 1 . 48 mg / l of C6G25S ) , followed by IN of 50 μg C6G25S . Mice receiving vehicle control ( saline ) for both AI and IN are annotated as control . Viral RNA ( left ) and infectious virions ( right ) in lungs were quantified with RT - qPCR and plaque forming assay , respectively , at 2 days post - infection ( dpi ) . B . Mice were challenged intranasally with 104 PFU of virus and co - treatment with 1 . 48 mg / L of C6G25S or vehicle control ( saline ) by AI for 30 min on day 0 ( right after infection ) and day 1 . Viral RNA and infectious virions were quantified at 2 dpi .

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Figure 4A. K18-hACE2-transgenic mice (Winkler et al., 2020) were treated once daily for 3 days before intranasal challenge with 104 plaque-forming units (PFU) of the original virus. Prophylactic treatment consists of 30 min of AI (1.48 mg/l of C6G25S), followed by IN of 50 μg C6G25S. Mice receiving vehicle control (saline) for both AI and IN are annotated as control. Viral RNA (left) and infectious virions (right) in lungs were quantified with RT-qPCR and plaque forming assay, respectively, at 2 days post-infection (dpi). B. Mice were challenged intranasally with 104 PFU of virus and co-treatment with 1.48 mg/L of C6G25S or vehicle control (saline) by AI for 30 min on day 0 (right after infection) and day 1. Viral RNA and infectious virions were quantified at 2 dpi. C


Figure 5A . Immunohistochemical ( IHC ) staining of viral spike proteins in lung sections from K18 - hACE2 mice ( Winkler et al . , 2020 ) at day 5 post - infection . Spike proteins were detected with anti - spike antibody and stained brown . Images of bronchial epithelium of vehicle control treated ( i and ii ) and C6G25S treated ( iii and iv ) , alveoli of vehicle control treated ( v and vi ) and C6G25S treated ( vii and viii ) , bronchiole and blood vessel of vehicle control treated ( ix and x ) and C6G25S treated ( xi and xii ) group were shown . Syncytial cell was indicated by green arrow in ( xi ) and thrombosis in ( x ) . B . ISH staining of viral RNA in lungs of vehicle control group ( left ) and C6G25S treated group ( right ) at day 2 post - infection . Viral RNA was stained brown as green arrows indicated . Images are representative of 5 animals for each group . C . Images of IHC staining of Ly6G + Cells ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection . D . Images of IHC staining of F4 / 80 + Cells ( Brown color ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection . E . Images of IHC staining of CD3 + T Cells ( Brown color , arrows indicated ) in lungs of vehicle control ( left ) and C6G25S treated group ( right ) at day 2 post - infection .

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Figure 5A. Immunohistochemical (IHC) staining of viral spike proteins in lung sections from K18-hACE2 mice (Winkler et al., 2020) at day 5 post-infection. Spike proteins were detected with anti-spike antibody and stained brown. Images of bronchial epithelium of vehicle control treated (i and ii) and C6G25S treated (iii and iv), alveoli of vehicle control treated (v and vi) and C6G25S treated (vii and viii), bronchiole and blood vessel of vehicle control treated (ix and x) and C6G25S treated (xi and xii) group were shown. Syncytial cell was indicated by green arrow in (xi) and thrombosis in (x).B. ISH staining of viral RNA in lungs of vehicle control group (left) and C6G25S treated group (right) at day 2 post-infection. Viral RNA was stained brown as green arrows indicated. Images are representative of 5 animals for each group.C. Images of IHC staining of Ly6G+ Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection.D. Images of IHC staining of F4/80+ Cells (Brown color) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. E. Images of IHC staining of CD3 + T Cells (Brown color, arrows indicated) in lungs of vehicle control (left) and C6G25S treated group (right) at day 2 post-infection. F


Figure 1B HSATIII ASO - mediated depletion of nSBs . Thermal stress - exposed HeLa cells ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) were visualized by HSATIII - FISH and immunofluorescence using an anti - SAFB antibody or anti - HNRNPM antibody . The nuclei were stained with DAPI . Scale bar : 10 μm . C qRT - PCR - validation of HSATIII knockdown . The graph shows the qRT - PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions : 37 ° C , 42 ° C for 2 h , and thermal stress followed by recovery at 37 ° C for 1 h ( Recovery ) . Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions ( 42 ° C for 2 h ) . Data are shown as the mean ± SD ( n = 3 ) . HSATIII RNAs ratios ( % ) are indicated . D MA - plot ( log2 fold - change over the average expression level ) of all detected introns in HSATIII KD and control cells ( n = 3 ) . The introns with significant changes in their expression levels ( adjusted p - value < 0 . 01 ) are shown as green dots . Among these introns , up - regulated ( log2 fold - change > 1 ) and down - regulated ( log2 fold - change < - 1 ) introns are shown with magenta and light blue dots , respectively . Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol - i ( intron ) # . Total numbers of introns and genes utilized were 144769 and 32623 , respectively . Examples of RNA - seq read maps of HSATIII target RNAs . Affected introns and exons are indicated by blue ( down - regulated upon HSATIII knockdown ) G Examples of RNA - seq read maps of HSATIII target RNAs . Affected introns and exons are indicated by blue ( down - regulated upon HSATIII knockdown ) and magenta ( up - regulated ) boxes .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B HSATIII ASO-mediated depletion of nSBs. Thermal stress-exposed HeLa cells (42°C for 2 h and recovery for 1 h at 37°C) were visualized by HSATIII-FISH and immunofluorescence using an anti-SAFB antibody or anti-HNRNPM antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.C qRT-PCR-validation of HSATIII knockdown. The graph shows the qRT-PCR level of HSATIII RNAs in control and HSATIII knockdown cells under three conditions: 37°C, 42°C for 2 h, and thermal stress followed by recovery at 37°C for 1 h (Recovery). Expression levels were calculated as ratios to GAPDH mRNA and were normalized to the levels in control cells under thermal stress conditions (42°C for 2 h). Data are shown as the mean±SD (n=3). HSATIII RNAs ratios (%) are indicated.D MA-plot (log2 fold-change over the average expression level) of all detected introns in HSATIII KD and control cells (n=3). The introns with significant changes in their expression levels (adjusted p-value < 0.01) are shown as green dots. Among these introns, up-regulated (log2 fold-change > 1) and down-regulated (log2 fold-change < -1) introns are shown with magenta and light blue dots, respectively. Genes and introns that were experimentally validated in subsequent experiments are indicated as gene symbol-i (intron) #. Total numbers of introns and genes utilized were 144769 and 32623, respectively.Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown)G Examples of RNA-seq read maps of HSATIII target RNAs. Affected introns and exons are indicated by blue (down-regulated upon HSATIII knockdown) and magenta (up-regulated) boxes.


Figure 2Validation of HSATIII target introns by qRT - PCR . The graphs show the relative amounts of the intron - retaining ( IR ) ( upper ) and spliced ( lower ) forms in control and HSATIII knockdown cells under three conditions : 37 ° C ( normal ) , 42 ° C for 2 h ( thermal stress ) , and thermal stress followed by recovery at 37 ° C for 1 h . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions ( 37 ° C ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) . B Nuclear localization of the intron - retaining RNAs . The relative amounts of intron - retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT - PCR and are represented as the ratio ( % of the total ) . GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls , respectively . Data are shown as the mean ± SD ( n = 3 ) . D , E The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal , as determined by qRT - PCR . The graphs show the changes in the expression levels of the intron - retaining ( IR ) ( D ) and spliced ( E ) forms . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( multiple t - test modified by Holm - Sidak ' s method ) .

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Figure 2Validation of HSATIII target introns by qRT-PCR. The graphs show the relative amounts of the intron-retaining (IR) (upper) and spliced (lower) forms in control and HSATIII knockdown cells under three conditions: 37°C (normal), 42°C for 2 h (thermal stress), and thermal stress followed by recovery at 37°C for 1 h. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the levels in control cells under normal conditions (37°C). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).B Nuclear localization of the intron-retaining RNAs. The relative amounts of intron-retaining RNAs in the nuclear and cytoplasmic fractions were quantified by qRT-PCR and are represented as the ratio (% of the total). GAPDH mRNA and U1 snRNA were used as cytoplasmic and nuclear controls, respectively. Data are shown as the mean±SD (n=3).D, E The levels of HSATIII target introns in newly synthesized RNAs within 1 h after thermal stress removal, as determined by qRT-PCR. The graphs show the changes in the expression levels of the intron-retaining (IR) (D) and spliced (E) forms. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the level in the control cells. Data are shown as the mean±SD (n=3); *p<0.05 (multiple t-test modified by Holm-Sidak's method).


Figure 3A Splicing isoforms of the CLK1 pre - mRNA . The retained introns are indicated by red lines . An asterisk indicates the position of the premature termination codon in the nonsense - mediated mRNA decay - targeted isoform . B Time course analysis of the splicing pattern of CLK1 pre - mRNAs in control and HSATIII knockdown ( HSATIII KD ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control . C Quantification of the data shown in ( B ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) . D Thermal stress - induced expression of HSATIII in CHO ( His9 ) cells . The cells were visualized by HSATIII - FISH ( green ) , and the nuclei were stained with DAPI ( blue ) . Scale bar : 10 μm . E Ectopic HSATIII - induced nSB assembly in CHO ( His9 ) cells . The nSBs ( yellow arrowheads ) were visualized by HSATIII - FISH and immunofluorescence using an anti - SRSF1 antibody . The nuclei were stained with DAPI . Scale bar : 10 μm . F Time course analysis of the splicing pattern of Chinese hamster Clk1 pre - mRNAs in control ( CHO ) and CHO ( His9 ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control . Time course analysis of the splicing pattern of Chinese hamster Clk1 pre - mRNAs in control ( CHO ) and CHO ( His9 ) cells by semi - quantitative RT - PCR . Arrows indicate the positions of PCR primers . The GAPDH mRNA was used as an internal control . G Quantification of the data shown in ( F ) . Data are shown as the mean ± SD ( n = 3 ) ; * p < 0 . 05 ( Sidak ' s multiple comparison test ) . H The effect of HSATIII knockdown on pooling of intron - retaining RNAs in control ( CHO ) and CHO ( His9 ) cells ( 42 ° C for 2 h and recovery for 2 h at 37 ° C ) . Arrows indicate the positions of PCR primers .

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Figure 3A Splicing isoforms of the CLK1 pre-mRNA. The retained introns are indicated by red lines. An asterisk indicates the position of the premature termination codon in the nonsense-mediated mRNA decay-targeted isoform. B Time course analysis of the splicing pattern of CLK1 pre-mRNAs in control and HSATIII knockdown (HSATIII KD) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. C Quantification of the data shown in (B). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test). D Thermal stress-induced expression of HSATIII in CHO (His9) cells. The cells were visualized by HSATIII-FISH (green), and the nuclei were stained with DAPI (blue). Scale bar: 10 μm.E Ectopic HSATIII-induced nSB assembly in CHO (His9) cells. The nSBs (yellow arrowheads) were visualized by HSATIII-FISH and immunofluorescence using an anti-SRSF1 antibody. The nuclei were stained with DAPI. Scale bar: 10 μm.F Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control.Time course analysis of the splicing pattern of Chinese hamster Clk1 pre-mRNAs in control (CHO) and CHO (His9) cells by semi-quantitative RT-PCR. Arrows indicate the positions of PCR primers. The GAPDH mRNA was used as an internal control. G Quantification of the data shown in (F). Data are shown as the mean±SD (n=3); *p<0.05 (Sidak's multiple comparison test).H The effect of HSATIII knockdown on pooling of intron-retaining RNAs in control (CHO) and CHO (His9) cells (42°C for 2 h and recovery for 2 h at 37°C). Arrows indicate the positions of PCR primers.


Figure 4B RT - PCR validation of the specific precipitation of HSATIII by ChIRP . The NEAT1 ncRNA and GAPDH mRNA were used as negative controls . Input : 100 % . C Silver staining of the coprecipitated proteins . The predicted mobilities of five known nSB proteins are indicated . Input : 0 . 1 % . D Western blot analyses of proteins identified by the HSATIII - ChIRP experiment . Known nSB proteins ( SAFB , SRSF1 , SRSF7 , SRSF9 , and HNRNPM ) were used as positive controls , and ELAVL1 , PSPC1 and α - tubulin were used as negative controls . SRSF3 , SRSF5 , TRA2B and THRAP3 are newly identified nSB components . Colocalization of novel nSB proteins with HSATIII . Endogenous TRA2B ( E ) were stained with anti - TRA2B HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm . Colocalization of novel nSB proteins with HSATIII . THRAP3 ( F ) , were stained with anti - THRAP3 , HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm . Colocalization of novel nSB proteins with HSATIII . FLAG - tagged PPHLN1 ( G ) were stained with anti - FLAG antibodies HSATIII was visualized by RNA - FISH using a dig - labeled HSATIII ASO , and the nuclei were stained with DAPI . Scale bar : 10 μm .

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Figure 4B RT-PCR validation of the specific precipitation of HSATIII by ChIRP. The NEAT1 ncRNA and GAPDH mRNA were used as negative controls. Input: 100%.C Silver staining of the coprecipitated proteins. The predicted mobilities of five known nSB proteins are indicated. Input: 0.1%.D Western blot analyses of proteins identified by the HSATIII-ChIRP experiment. Known nSB proteins (SAFB, SRSF1, SRSF7, SRSF9, and HNRNPM) were used as positive controls, and ELAVL1, PSPC1 and α-tubulin were used as negative controls. SRSF3, SRSF5, TRA2B and THRAP3 are newly identified nSB components.Colocalization of novel nSB proteins with HSATIII. Endogenous TRA2B (E) were stained with anti-TRA2B HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.Colocalization of novel nSB proteins with HSATIII. THRAP3 (F), were stained with anti-THRAP3, HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.Colocalization of novel nSB proteins with HSATIII. FLAG-tagged PPHLN1 (G) were stained with anti-FLAG antibodies HSATIII was visualized by RNA-FISH using a dig-labeled HSATIII ASO, and the nuclei were stained with DAPI. Scale bar: 10 μm.


Figure 5Specific interaction of CLK1 with HSATIII during thermal stress recovery . HSATIII - ChIRP at various time points during and after thermal stress exposure . Endogenous CLK1 , SRSF7 , and SRSF9 were detected using anti - CLK1 , anti - SRSF7 , and anti - SRSF9 antibodies , respectively . HSATIII was detected by ChIRP - RNA purification , followed by RT - PCR . Input : 0 . 1 % for CLK1 , 1 % for SRSF7 and SRSF9 , and 100 % for HSATIII . Specific interaction of HSATIII with CLK1 proteins during stress recovery . HSATIII - ChIRP / western blotting was performed using HeLa cells expressing FLAG - CLK1 WT or the KR mutant . FLAG - tagged CLK1 and SRSF9 were detected by western blotting using an anti - FLAG and an anti - SRSF9 antibody , respectively . HSATIII was detected by ChIRP followed by RT - PCR . Input : 1 % for western blotting , 100 % for RT - PCR . Re - translocation of FLAG - CLK1 to nSBs after thermal stress removal . HeLa cells were transfected with a FLAG - CLK1 expression vector , cultured for 16 h , and then exposed to thermal stress ( 42 ° C for 2 h ) with or without recovery ( 37 ° C for 1 h ) . FLAG - CLK1 was visualized with an anti - FLAG antibody . Nuclear stress bodies and nuclei were visualized by HSATIII - FISH and DAPI staining , respectively . Scale bar : 10 μm . Box plot of colocalization correlation coefficients between HSATIII and FLAG - CLK1 WT . The first and third quartiles are the ends of the box , the median is indicated by the vertical line in the box , and the minimum and maximum are the ends of the whiskers . Recovery phase - specific interaction of HSATIII with the CLK1 N - terminal region . HSATIII - ChIRP / western blotting was performed on HeLa cells expressing FLAG - CLK1 ∆ C or FLAG - CLK1 ∆ N Asterisk : an unidentified partial fragment of FLAG - CLK1 ∆ N . Dependency of SRSF9 on the interaction of CLK1 ( G ) with HSATIII . HSATIII - ChIRP was performed using control and SRSF9 - depleted cells expressing FLAG - CLK1 proteins ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) . Input : 1 % . Graphs represent coprecipitation ratios of FLAG - CLK1 proteins to HSATIII in the control and SRSF9 - depleted cells . H Dependency of SRSF9 on the interaction of CLK1 ∆ C ( H ) with HSATIII . HSATIII - ChIRP was performed using control and SRSF9 - depleted cells expressing FLAG - CLK1 proteins ( 42 ° C for 2 h and recovery for 1 h at 37 ° C ) . Input : 1 % . Graphs represent coprecipitation ratios of FLAG - CLK1 proteins to HSATIII in the control and SRSF9 - depleted cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Specific interaction of CLK1 with HSATIII during thermal stress recovery. HSATIII-ChIRP at various time points during and after thermal stress exposure. Endogenous CLK1, SRSF7, and SRSF9 were detected using anti-CLK1, anti-SRSF7, and anti-SRSF9 antibodies, respectively. HSATIII was detected by ChIRP-RNA purification, followed by RT-PCR. Input: 0.1% for CLK1, 1% for SRSF7 and SRSF9, and 100% for HSATIII.Specific interaction of HSATIII with CLK1 proteins during stress recovery. HSATIII-ChIRP/western blotting was performed using HeLa cells expressing FLAG-CLK1 WT or the KR mutant. FLAG-tagged CLK1 and SRSF9 were detected by western blotting using an anti-FLAG and an anti-SRSF9 antibody, respectively. HSATIII was detected by ChIRP followed by RT-PCR. Input: 1% for western blotting, 100% for RT-PCR.Re-translocation of FLAG-CLK1 to nSBs after thermal stress removal. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and then exposed to thermal stress (42°C for 2 h) with or without recovery (37°C for 1 h). FLAG-CLK1 was visualized with an anti-FLAG antibody. Nuclear stress bodies and nuclei were visualized by HSATIII-FISH and DAPI staining, respectively. Scale bar: 10 μm.Box plot of colocalization correlation coefficients between HSATIII and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated by the vertical line in the box, and the minimum and maximum are the ends of the whiskers.Recovery phase-specific interaction of HSATIII with the CLK1 N-terminal region. HSATIII-ChIRP/western blotting was performed on HeLa cells expressing FLAG-CLK1∆C or FLAG-CLK1∆N Asterisk: an unidentified partial fragment of FLAG-CLK1∆N.Dependency of SRSF9 on the interaction of CLK1 (G) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells.H Dependency of SRSF9 on the interaction of CLK1∆C (H) with HSATIII. HSATIII-ChIRP was performed using control and SRSF9-depleted cells expressing FLAG-CLK1 proteins (42°C for 2 h and recovery for 1 h at 37°C). Input: 1%. Graphs represent coprecipitation ratios of FLAG-CLK1 proteins to HSATIII in the control and SRSF9-depleted cells.


Figure 6A Localization of SRSF9 with HSATIII during thermal stress and recovery . B Recovery phase - specific colocalization of CLK1 and SRSF9 . HeLa cells were transfected with a FLAG - CLK1 expression vector , cultured for 16 h , and exposed to thermal stress ( 42 ° C for 2 h ) followed by recovery ( 37 ° C for 1 h ) . FLAG - CLK1 and SRSF9 were co - stained using anti - FLAG and anti - SRSF9 antibodies , respectively , and the nuclei were stained with DAPI . C Box plot of colocalization correlation coefficients between SRSF9 and FLAG - CLK1 WT . The first and third quartiles are the ends of the box , the median is indicated with a vertical line in the box , and the minimum and maximum are the ends of the whiskers . F The effect of HSATIII knockdown on time course - dependent changes in SRSF9 phosphorylation . The phosphorylation states were estimated by Phos - tag SDS - PAGE , followed by western blotting using an anti - SRSF9 antibody . SRSF9 detected by western blotting of a normal SDS - PAGE gel is shown in the bottom panel . The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress - induced hypo - and de - phosphorylated forms , respectively . F The effect of HSATIII knockdown on time course - dependent changes in SRSF9 phosphorylation . The phosphorylation states were estimated by Phos - tag SDS - PAGE , followed by western blotting using an anti - SRSF9 antibody . SRSF9 detected by western blotting of a normal SDS - PAGE gel is shown in the bottom panel . The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress - induced hypo - and de - phosphorylated forms , respectively . G Quantification of the data shown in ( F ) . Graphs show the time - course changes in four representative SRSF9 phosphorylated forms in ( F ) . Relative levels of band intensity were calculated as the ratio of each band to α - tubulin and were normalized to those of control at 37 ° C ( bands ( a ) and ( b ) ) and 42 ° C ( bands ( c ) and ( d ) ) . Since band ( d ) partially overlapped with the other band ( asterisk in ( F ) ) at 37 ° C and recovery 4h , band ( d ) at those time points was not measured . H The re - phosphorylation status of SRSF9 within nSBs . Phosphorylated ( arrows ) and hypo - and de - phosphorylated ( arrowheads ) SRSF9 in the nSB fractions ( lanes 4 , 6 , and 8 ) were analyzed by western blotting on Phos - tag gels . KH - CB19 ( 10 μM ) or DMSO as a control was administrated during the recovery period . The lower panel shows a shorter exposure image of the phosphorylated forms . The asterisks indicate bands different from band ( a ) or ( d ) . I Quantification of the data shown in ( H ) . Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS - PAGE , and were normalized to those at 42 ° C .

O B-PANEL_START O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A Localization of SRSF9 with HSATIII during thermal stress and recovery.B Recovery phase-specific colocalization of CLK1 and SRSF9. HeLa cells were transfected with a FLAG-CLK1 expression vector, cultured for 16 h, and exposed to thermal stress (42°C for 2 h) followed by recovery (37°C for 1 h). FLAG-CLK1 and SRSF9 were co-stained using anti-FLAG and anti-SRSF9 antibodies, respectively, and the nuclei were stained with DAPI.C Box plot of colocalization correlation coefficients between SRSF9 and FLAG-CLK1 WT. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum are the ends of the whiskers.F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively.F The effect of HSATIII knockdown on time course-dependent changes in SRSF9 phosphorylation. The phosphorylation states were estimated by Phos-tag SDS-PAGE, followed by western blotting using an anti-SRSF9 antibody. SRSF9 detected by western blotting of a normal SDS-PAGE gel is shown in the bottom panel. The arrows and arrowheads indicate the normal phosphorylated forms and thermal stress-induced hypo- and de-phosphorylated forms, respectively. G Quantification of the data shown in (F). Graphs show the time-course changes in four representative SRSF9 phosphorylated forms in (F). Relative levels of band intensity were calculated as the ratio of each band to α-tubulin and were normalized to those of control at 37°C (bands (a) and (b)) and 42°C (bands (c) and (d)). Since band (d) partially overlapped with the other band (asterisk in (F)) at 37°C and recovery 4h, band (d) at those time points was not measured. H The re-phosphorylation status of SRSF9 within nSBs. Phosphorylated (arrows) and hypo- and de-phosphorylated (arrowheads) SRSF9 in the nSB fractions (lanes 4, 6, and 8) were analyzed by western blotting on Phos-tag gels. KH-CB19 (10 μM) or DMSO as a control was administrated during the recovery period. The lower panel shows a shorter exposure image of the phosphorylated forms. The asterisks indicate bands different from band (a) or (d). I Quantification of the data shown in (H). Relative band intensities were calculated as the ratios of each band to total SRSF9 detected by western blotting of a standard SDS-PAGE, and were normalized to those at 42°C.


Figure 7A , B Validation of the effect of SRSF9 on retention / splicing of HSATIII target introns by qRT - PCR . The relative expression levels of intron - retaining ( IR ) forms ( A ) and spliced forms ( B ) of newly transcribed RNAs during the recovery period are shown . Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control . C , D Time course analysis of the splicing patterns of CLK1 and TAF1D intron - retaining RNAs in control and HSATIII knockdown cells by semi - quantitative RT - PCR . Arrows indicate the positions of primers for RT - PCR . GAPDH mRNA was used as an internal control . The experiment was performed in triplicate . Quantification of the data shown in ( C ) , F Validation of the requirement of CLK1 catalytic activity for promotion of intron retention by qRT - PCR . Data are shown as the mean ± SD ( n = 3 ) . G The diagrams show the domain structures of wild - type ( WT ) and mutant ( mut ) SRSF9 , in which all SR / SP dipeptides were replaced by AR / AP . FLAG - SRSF9 - WT and FLAG - SRSF9 - mut were extracted from transfected HeLa cells under normal ( 37 ° C ) and thermal stress ( 42 ° C for 2 h ) conditions , separated in a Phos - tag gel , and detected by western blot using an anti - FLAG antibody . The arrowhead indicates the thermal stress - induced de - phosphorylated form of FLAG - SRSF9 - WT . The role of phosphorylation of SRSF9 on splicing of the TAF1D ( H ) splicing reporter plasmid was transfected into HeLa cells along with a siRNA ( control or SRSF9 - specific ) and a siRNA - resistant SRSF9 - WT or SRSF9 - mut expression plasmid ( or pEGFP - C1 vector as the empty vehicle ( - ) ) 48 h before the assay . Semi - quantitative RT - PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress - exposed cells ( 42 ° C for 2 h and 37 ° C for 1 h ) . The positions of the PCR primers are indicated by arrows . Graphs represent the relative changes in the spliced / unspliced ratios , normalized to the control sample with control siRNA and empty plasmid ( - ) . I The role of phosphorylation of SRSF9 on splicing of the DNAJB9 ( I ) reporters . Each splicing reporter plasmid was transfected into HeLa cells along with a siRNA ( control or SRSF9 - specific ) and a siRNA - resistant SRSF9 - WT or SRSF9 - mut expression plasmid ( or pEGFP - C1 vector as the empty vehicle ( - ) ) 48 h before the assay . Semi - quantitative RT - PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress - exposed cells ( 42 ° C for 2 h and 37 ° C for 1 h ) . The positions of the PCR primers are indicated by arrows . Graphs represent the relative changes in the spliced / unspliced ratios , normalized to the control sample with control siRNA and empty plasmid ( - ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B Validation of the effect of SRSF9 on retention/splicing of HSATIII target introns by qRT-PCR. The relative expression levels of intron-retaining (IR) forms (A) and spliced forms (B) of newly transcribed RNAs during the recovery period are shown. Expression levels were calculated as the ratio of each RNA to GAPDH mRNA and were normalized to the control.C, D Time course analysis of the splicing patterns of CLK1 and TAF1D intron-retaining RNAs in control and HSATIII knockdown cells by semi-quantitative RT-PCR. Arrows indicate the positions of primers for RT-PCR. GAPDH mRNA was used as an internal control. The experiment was performed in triplicate. Quantification of the data shown in (C),F Validation of the requirement of CLK1 catalytic activity for promotion of intron retention by qRT-PCR. Data are shown as the mean±SD (n=3).G The diagrams show the domain structures of wild-type (WT) and mutant (mut) SRSF9, in which all SR/SP dipeptides were replaced by AR/AP. FLAG-SRSF9-WT and FLAG-SRSF9-mut were extracted from transfected HeLa cells under normal (37°C) and thermal stress (42°C for 2 h) conditions, separated in a Phos-tag gel, and detected by western blot using an anti-FLAG antibody. The arrowhead indicates the thermal stress-induced de-phosphorylated form of FLAG-SRSF9-WT.The role of phosphorylation of SRSF9 on splicing of the TAF1D (H) splicing reporter plasmid was transfected into HeLa cells along with a siRNA (control or SRSF9-specific) and a siRNA-resistant SRSF9-WT or SRSF9-mut expression plasmid (or pEGFP-C1 vector as the empty vehicle (-)) 48 h before the assay. Semi-quantitative RT-PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress-exposed cells (42°C for 2 h and 37°C for 1 h). The positions of the PCR primers are indicated by arrows. Graphs represent the relative changes in the spliced/unspliced ratios, normalized to the control sample with control siRNA and empty plasmid (-).I The role of phosphorylation of SRSF9 on splicing of the DNAJB9 (I) reporters. Each splicing reporter plasmid was transfected into HeLa cells along with a siRNA (control or SRSF9-specific) and a siRNA-resistant SRSF9-WT or SRSF9-mut expression plasmid (or pEGFP-C1 vector as the empty vehicle (-)) 48 h before the assay. Semi-quantitative RT-PCR analyses revealed the splicing patterns of the reporter RNAs purified from the thermal stress-exposed cells (42°C for 2 h and 37°C for 1 h). The positions of the PCR primers are indicated by arrows. Graphs represent the relative changes in the spliced/unspliced ratios, normalized to the control sample with control siRNA and empty plasmid (-).


Figure 1C Quantitative analysis of lysosomal movement from kymographs . Results were expressed as percent of total lysosomes ( N = 44 , 49 , and 16 neurons for shSc , shLAMTOR1 , and shLAMTOR + rLAMTOR1 respectively from 3 - 10 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by two - way ANOVA with Tukey ' s * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 n . s . , not significant . G FRAP analysis of dendritic lysosome movement . Quantitative analysis for F ( N = 7 , 7 , and 9 neurons for shSc , shLAMTOR1 , and shLAMTOR + rLAMTOR1 , respectively , from 3 independent experiments ) . Data information : Data with error bars are represented as means ± SEM . Statistical significance was assessed by Dunnett ' s post - test * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , as compared to shSc ; # P < 0 . 05 , # # # P < 0 . 001 , as compared to shLAMTOR1 ; n . s . , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1C Quantitative analysis of lysosomal movement from kymographs. Results were expressed as percent of total lysosomes (N = 44, 49, and 16 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1 respectively from 3-10 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by two-way ANOVA with Tukey's *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 n.s., not significant.G FRAP analysis of dendritic lysosome movement. Quantitative analysis for F (N = 7, 7, and 9 neurons for shSc, shLAMTOR1, and shLAMTOR+rLAMTOR1, respectively, from 3 independent experiments). Data information: Data with error bars are represented as means ± SEM. Statistical significance was assessed by Dunnett's post-test *P < 0.05, **P < 0.01, ***P < 0.001, as compared to shSc; #P < 0.05, ###P < 0.001, as compared to shLAMTOR1 ; n.s., not significant.