C , D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9 + or CCR9 − M579 - A2tumors in TIL - treated mice ( C ) or the PBS alone group ( D ) . Statistical difference was calculated using the unpaired one - sided Mann - Whitney U - test .

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C, D Tumorgrowth curves showing mean ± SEMtumor volume of CCR9+ or CCR9−M579-A2tumors in TIL-treated mice (C) or the PBS alone group (D). Statistical difference was calculated using the unpaired one-sided Mann-Whitney U-test.


A ) SH - SY5Y neuroblastoma cells were either cultivated in RPMI + 10 % FCS or starved in HBSS with or without addition of LY294002 or rapamycin . After 16 h PINK1 mRNA expression was determined by RT - qPCR and the PINK1 mRNA content of cells kept in RPMI + 10 % FCS was set as 1 . Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression , comparable to the positive control LY294002 ; n = 4 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : LY294002 : p < 0 . 01 ; rapamycin : p < 1 × 10 − 4 ; HBSS : p < 0 . 005 ; HBSS + LY294002 : p < 5 × 10 − 6 ; HBSS + rapamycin : p < 0 . 0005 ; # expression changes compared to the PINK1 expression in RPMI + 10 % FCS + LY294002 : p < 0 . 05 ; + expression changes compared to the PINK1 expression in RPMI + 10 % FCS + rapamycin : p < 0 . 0005 .

O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A) SH-SY5Y neuroblastoma cells were either cultivated in RPMI+10% FCS or starved in HBSS with or without addition of LY294002 or rapamycin. After 16 h PINK1 mRNA expression was determined by RT-qPCR and the PINK1 mRNA content of cells kept in RPMI+10% FCS was set as 1. Induction of autophagy by starvation or rapamycin resulted in an induction of PINK1 expression, comparable to the positive control LY294002; n = 4; * expression changes compared to the PINK1 expression in RPMI+10% FCS: LY294002: p<0.01; rapamycin: p<1×10−4; HBSS: p<0.005; HBSS+LY294002: p<5×10−6; HBSS+ rapamycin: p<0.0005; # expression changes compared to the PINK1 expression in RPMI+10% FCS+LY294002: p<0.05; + expression changes compared to the PINK1 expression in RPMI+10% FCS+rapamycin: p<0.0005.


B ) SH - SY5Y cells were either stably transduced with a control ( nt ) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5 % or 10 % FCS . Their PINK1 mRNA content was determined by RT - qPCR and PINK1 mRNA content of nt cells kept in medium with 10 % FCS was set as 1 . Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig . 1A , while stable PINK1 knockdown ( kd ) reduced PINK1 content under both conditions ; n = 3 ; * expression changes compared to the PINK1 expression in RPMI + 10 % FCS : PINK1 kd 10 % FCS p < 0 . 0005 ; PINK1 induction by 5 % FCS : p < 0 . 05 ; # expression changes compared to the PINK1 expression in RPMI + 5 % FCS : PINK1 kd 5 % FCS : p < 0 . 005 .

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

B) SH-SY5Y cells were either stably transduced with a control (nt) shRNA or a shRNA directed against PINK1 and cultivated in RPMI medium containing 5% or 10% FCS. Their PINK1 mRNA content was determined by RT-qPCR and PINK1 mRNA content of nt cells kept in medium with 10% FCS was set as 1. Serum reduction increased PINK1 mRNA in nt cells in accordance with the data shown in Fig. 1A, while stable PINK1 knockdown (kd) reduced PINK1 content under both conditions; n = 3; * expression changes compared to the PINK1 expression in RPMI+10% FCS: PINK1 kd 10% FCS p<0.0005; PINK1 induction by 5% FCS: p<0.05; # expression changes compared to the PINK1 expression in RPMI+5% FCS: PINK1 kd 5% FCS: p<0.005.


C ) SH - SY5Y cells without ( nt ) or with stable PINK1 knockdown ( kd ) were kept in medium with 5 % FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1 . Afterwards the PINK1 63 kDa protein ( arrowhead ) and actin protein levels were determined by western blotting ( see representative gel on the right ) . The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells .

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 B-CONTROLLED_VAR O O O B-MEASURED_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 B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O

C) SH-SY5Y cells without (nt) or with stable PINK1 knockdown (kd) were kept in medium with 5% FCS and either untreated or treated for 2 h with CCCP to stabilize PINK1. Afterwards the PINK1 63 kDa protein (arrowhead) and actin protein levels were determined by western blotting (see representative gel on the right). The quantification revealed a reduction of PINK1 protein under both conditions in PINK1 kd cells.


2A ) SH - SY5Y cells were starved for 2 h in HBSS with Bafilomycin ( + Baf ) or left untreated in RPMI + 5 % FCS ( - Baf ) . The LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right , showing only LC3 - II bands in the Bafilomycin - treated samples . The LC3 - II bands of Bafilomycin treated samples were normalized to actin and the relative LC3 - II content of nt cells was set as 1 . Cells with stable PINK1 knockdown exhibited a reduced LC3 - II / actin ratio compared to the control ( nt ) cells ; n = 4 , p < 0 . 005 .

O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

2A) SH-SY5Y cells were starved for 2 h in HBSS with Bafilomycin (+Baf) or left untreated in RPMI+5% FCS (- Baf). The LC3-II and actin content were determined by western blotting. A representative blot is shown on the right, showing only LC3-II bands in the Bafilomycin-treated samples. The LC3-II bands of Bafilomycin treated samples were normalized to actin and the relative LC3-II content of nt cells was set as 1. Cells with stable PINK1 knockdown exhibited a reduced LC3-II/actin ratio compared to the control (nt) cells; n = 4, p<0.005.


2B ) Cortical neurons from 3 different isolations ( 10 - 20 DIV ) of WT and PINK1 KO mice were either non - starved or starved for 2 h in HBSS and the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of WT and PINK1 KO mice , respectively , was set as 1 . Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3 - II / actin ratio compared to the WT cells ; n = 5 , p < 0 . 001 .

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

2B) Cortical neurons from 3 different isolations (10-20 DIV) of WT and PINK1 KO mice were either non-starved or starved for 2 h in HBSS and the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of WT and PINK1 KO mice, respectively, was set as 1. Primary neurons showed a strong upregulation of autophagy in reaction to starvation but cells derived from PINK1 KO mice exhibited a reduced LC3-II/actin ratio compared to the WT cells; n = 5, p<0.001.


2C ) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA . After 48 h the LC3 - II and actin content was determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with scrambled siRNA was set as 1 . Transient PINK1 knockdown resulted in a reduced LC3 - II / actin ratio compared to the cells transfected with scrambled siRNA ; n = 6 ; p < 0 . 005 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

2C) HeLa cells were transiently transfected with scrambled siRNA or PINK1 siRNA. After 48 h the LC3-II and actin content was determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with scrambled siRNA was set as 1. Transient PINK1 knockdown resulted in a reduced LC3-II/actin ratio compared to the cells transfected with scrambled siRNA; n = 6; p<0.005.


2D ) HeLa cells were transiently transfected with PINK1 - GFP , PINK1G309D - GFP or GFP . After 24 h cells were starved for 2 h in HBSS . Afterwards the LC3 - II and actin content were determined by western blotting . A representative blot is shown on the right . The relative LC3 - II content of cells transfected with GFP was set as 1 . Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3 - II / actin ratio compared to cells transfected with GFP ; n = 3 , PINK1 : p < 0 . 01 ; PINK1G309D : p < 0 . 05 .

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

2D) HeLa cells were transiently transfected with PINK1-GFP, PINK1G309D-GFP or GFP. After 24 h cells were starved for 2 h in HBSS. Afterwards the LC3-II and actin content were determined by western blotting. A representative blot is shown on the right. The relative LC3-II content of cells transfected with GFP was set as 1. Transient PINK1 or PINK1G309D overexpression resulted in an increased LC3-II/actin ratio compared to cells transfected with GFP; n = 3, PINK1: p<0.01; PINK1G309D: p<0.05.


3A ) Regular , non - transduced SH - SY5Y cells were cultivated either in RPMI + 10 % FCS or starved in HBSS medium for 24 and 40 h . The LAMP - 2 and actin content were determined by western blotting and the relative LAMP - 2 content of untreated cells was set as 1 . Starvation enhanced LAMP - 2protein levels ; n = 3 , p < 0 . 05 .

O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O

3A) Regular, non-transduced SH-SY5Y cells were cultivated either in RPMI+10% FCS or starved in HBSS medium for 24 and 40 h. The LAMP-2 and actin content were determined by western blotting and the relative LAMP-2 content of untreated cells was set as 1. Starvation enhanced LAMP-2protein levels; n = 3, p<0.05.


3B ) Transduced nt and PINK1 knockdown ( kd ) SH - SY5Y cells were cultivated either in RPMI + 5 % FCS or starved for 24 h and 40 h in HBSS . The LAMP - 2 and actin content were determined by western blotting . A representative blot after 40 h starvation in HBSS is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP - 2 / actin ratio compared to the nt cells ; n = 4 , p < 0 . 05 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

3B) Transduced nt and PINK1 knockdown (kd) SH-SY5Y cells were cultivated either in RPMI+5% FCS or starved for 24 h and 40 h in HBSS. The LAMP-2 and actin content were determined by western blotting. A representative blot after 40 h starvation in HBSS is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 40 h a reduced LAMP-2/actin ratio compared to the nt cells; n = 4, p<0.05.


3C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp - 2 . Micrographs were taken with constant microscopical settings . LAMP - 2 positive signals in each cell were quantified with ImageJ as described in Material and Methods . Knockdown of PINK1 resulted in a decreased amount of LAMP - 2 positive stainings / cell after starvation ; n = 2 , at least 8 fields of view / condition , 89 - 124 cells / condition ; 24 h : p < 0 . 001 ; 40 h : p < 0 . 05 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

3C) nt and PINK1 knockdown (kd) SH-SY5Y cells were starved for the indicated times with HBSS and afterwards stained for Lamp-2. Micrographs were taken with constant microscopical settings. LAMP-2 positive signals in each cell were quantified with ImageJ as described in Material and Methods. Knockdown of PINK1 resulted in a decreased amount of LAMP-2 positive stainings/cell after starvation; n = 2, at least 8 fields of view/condition, 89-124 cells/condition; 24 h: p<0.001; 40 h: p<0.05.


3D ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were stained with the photo - reactive dye MTR and either irradiated for 45 min or left untreated . 24 h and 48 h after irradiation the LAMP - 2 and actin content were determined by western blotting . A representative blot 48 h after irradiation is shown on the right . The relative LAMP - 2 content of untreated cells was set as 1 . Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp - 2 / actin ratio compared to the control ( nt ) cells ; n = 5 , p < 0 . 001 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

3D) nt and PINK1 knockdown (kd) SH-SY5Y cells were stained with the photo-reactive dye MTR and either irradiated for 45 min or left untreated. 24 h and 48 h after irradiation the LAMP-2 and actin content were determined by western blotting. A representative blot 48 h after irradiation is shown on the right. The relative LAMP-2 content of untreated cells was set as 1. Cells with stable PINK1 knockdown exhibited after 48 h a reduced Lamp-2/actin ratio compared to the control (nt) cells; n = 5, p<0.001.


3E ) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown ( kd ) SH - SY5Y cells cultivated in RPMI medium with 5 % FCS . The phosphatase activity was measured and normalized to the protein content . The phosphatase activity of nt cells was set as 1 . PINK1 knockdown mediated a reduction of lysosomal activity ; n = 3 ; p < 0 . 05 .

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3E) Acid phosphatase activity as parameter for lysosomal activity was quantified in nt and PINK1 knockdown (kd) SH-SY5Y cells cultivated in RPMI medium with 5% FCS. The phosphatase activity was measured and normalized to the protein content. The phosphatase activity of nt cells was set as 1. PINK1 knockdown mediated a reduction of lysosomal activity; n = 3; p<0.05.


4A ) Oxygen consumption rate ( OCR ) was measured in nt and PINK1 knockdown ( kd ) SH - SY5Y cells without ( nt ) or with PINK1 knockdown ( kd ) cultivated in RPMI medium with 5 % FCS . Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd , no significant changes were observed ; n = 1 ( quadruplicates ) .

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

4A) Oxygen consumption rate (OCR) was measured in nt and PINK1 knockdown (kd) SH-SY5Y cells without (nt) or with PINK1 knockdown (kd) cultivated in RPMI medium with 5% FCS. Although oxygen consumption appeared to be slightly impaired in cells with PINK1 kd, no significant changes were observed; n = 1 (quadruplicates).


4B ) Energy charge ( EC ) of nt and PINK1 knockdown ( kd ) SH - SY5Y cells grown in RPMI + 5 % FCS was determined as detailed in Material Methods . PINK1 knockdown resulted in a lower EC compared to nt cells ; n = 5 , p0 . 05 .

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

4B) Energy charge (EC) of nt and PINK1 knockdown (kd) SH-SY5Y cells grown in RPMI+5% FCS was determined as detailed in Material Methods. PINK1 knockdown resulted in a lower EC compared to nt cells; n = 5, p0.05.


4C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were grown for 2 months in RPMI + 5 % FCS and their population doublings were determined . Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth ; n = 1 ( triplicates ) ; * p < 0 . 05 ( day 12 ) - p < 1 × 10 − 6 ( day 61 ) .

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 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

4C) nt and PINK1 knockdown (kd) SH-SY5Y cells were grown for 2 months in RPMI+5% FCS and their population doublings were determined. Starting from day 12 cells with PINK1 kd exhibited impaired cell population growth; n = 1 (triplicates); * p<0.05 (day 12) - p<1×10−6 (day 61).


4D ) SH - SY5Y cells were grown in RPMI + 5 % FCS and the amount of nt and PINK1 knockdown ( kd ) cells in S - phase was determined by BrdU incorporation . No differences in cell proliferation were visible between cells without ( nt ) and with PINK1 kd ; n = 3 .

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 B-CONTROLLED_VAR O O O O O O O O

4D) SH-SY5Y cells were grown in RPMI+5% FCS and the amount of nt and PINK1 knockdown (kd) cells in S-phase was determined by BrdU incorporation. No differences in cell proliferation were visible between cells without (nt) and with PINK1 kd; n = 3.


5A ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS or starved with HBSS for 12 h . DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to the protein content . Cells with PINK1 knockdown demonstrated a significantly increased caspase - 3 activity under both conditions ; n = 4 ; RPMI + 5 % FCS : p < 0 . 05 ; HBSS : p < 0 . 01 .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

5A) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS or starved with HBSS for 12 h. DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to the protein content. Cells with PINK1 knockdown demonstrated a significantly increased caspase-3 activity under both conditions; n = 4; RPMI+5% FCS: p<0.05; HBSS: p<0.01.


5B ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing .

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 B-MEASURED_VAR O O O O O O O O O O O O O

5B) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. In the representative western blot the appearance of the PARP 89 kDa cleaved product is depicted as well as GAPDH for normalizing.


5C ) nt and PINK1 knockdown ( kd ) SH - SY5Y cells were either kept in RPMI medium with 5 % FCS for 48 h or starved with HBSS for the indicated time points . For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH . Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation ; n = 4 ; 24 h : p < 0 . 005 .

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 B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O B-MEASURED_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

5C) nt and PINK1 knockdown (kd) SH-SY5Y cells were either kept in RPMI medium with 5% FCS for 48 h or starved with HBSS for the indicated time points. For quantification the cleaved PARP 89 kDa product was normalized to the full length 113 kDa PARP and the resulting value normalized to GAPDH. Cells with PINK1 knockdown demonstrated increased PARP cleavage that was significant after 24 h starvation; n = 4; 24 h: p<0.005.


5D ) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP - Parkin or PINK1 - GFP or PINK1G309D - GFP . After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined . The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1 . Starvation - mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D - GFP but not by GFP or Parkin ; GFP : n = 5 , p < 0 . 005 ; Parkin : n = 3 , p < 0 . 05 ; PINK1 : n = 5 , ns ; PINK1G309D - GFP : n = 3 , ns .

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5D) HeLa cells were transfected with scrambled siRNA or PINK1 siRNA and GFP or GFP-Parkin or PINK1-GFP or PINK1G309D-GFP. After transfection cells were starved for additional 24 h with HBSS and afterwards the amount of adherent cells was determined. The number of adherent cells transfected with scrambled siRNA and the indicated plasmid was set as 1. Starvation-mediated increased cell loss after PINK1 knockdown could be rescued by PINK1 or PINK1G309D-GFP but not by GFP or Parkin; GFP: n = 5, p<0.005; Parkin: n = 3, p<0.05; PINK1: n = 5, ns; PINK1G309D-GFP: n = 3, ns.


5E ) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA . After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase - 3 activity was analyzed and normalized to 1 , 000 , 000 cells . PINK1 knockdown increased caspase - 3 activity significantly , which was prevented by LC3 overexpression ; n = 3 ; p < 0 . 05 .

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5E) HeLa cells and HeLa cells stably overexpressing LC3 were transfected with scrambled siRNA or PINK1 siRNA. After 48 h cells were starved in HBSS for additional 24 h and afterwards DEVD cleavage as parameter for caspase-3 activity was analyzed and normalized to 1,000,000 cells. PINK1 knockdown increased caspase-3 activity significantly, which was prevented by LC3 overexpression; n = 3; p<0.05.


5F ) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA . 48 h post transfection cells were either left untreated ( + medium ) or starved ( + HBSS ) for additional 24 h and afterwards the amount of adherent cells was determined . The amount of non - starved cells was set as 1 . PINK1 knockdown resulted in elevated cell loss , which was prevented by LC3 overexpression ; HeLa : n = 6 , p < 0 . 05 ; HeLa LC3 : n = 5 .

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5F) HeLa cells and HeLa cells stably expressing LC3 were transfected with scrambled or PINK1 siRNA. 48 h post transfection cells were either left untreated (+ medium) or starved (+HBSS) for additional 24 h and afterwards the amount of adherent cells was determined. The amount of non-starved cells was set as 1. PINK1 knockdown resulted in elevated cell loss, which was prevented by LC3 overexpression; HeLa: n = 6, p<0.05; HeLa LC3: n = 5.


Cytoplasmic chromatin fragments in senescent cells . ( A ) Cytoplasmic chromatin fragments ( CCFs ) in senescent cells are strongly positive for histone H3 . Yellow arrow marks CCF . ( B ) Increased proportion of cells with CCFs in RS or OIS . Mean ± SEM , n = 3 ; P < 0 . 01 ( OIS ) , P < 0 . 002 ( RS ) . Assays were performed 10 d after induction with tamoxifen ( OIS ) or 4 wk after the last passage and > 90 % SA β - gal + ( RS ) .

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Cytoplasmic chromatin fragments in senescent cells. (A) Cytoplasmic chromatin fragments (CCFs) in senescent cells are strongly positive for histone H3. Yellow arrow marks CCF. (B) Increased proportion of cells with CCFs in RS or OIS. Mean ± SEM, n = 3; P < 0.01 (OIS), P < 0.002 (RS). Assays were performed 10 d after induction with tamoxifen (OIS) or 4 wk after the last passage and >90% SA β-gal+ (RS).


( C ) CCFs in OIS cells are lamin A / C negative . ( D ) Cells from C were scored for lamin A / C + and lamin A / C − CCFs . Note that the majority of micronuclei in control cells are lamin A / C positive . Mean ± SEM , n = 3 ; P < 0 . 0006 for lamin A / C negative CCF con vs . OIS ; P < 0 . 6 for lamin A / C positive CCF con vs . OIS .

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(C) CCFs in OIS cells are lamin A/C negative. (D) Cells from C were scored for lamin A/C+ and lamin A/C− CCFs. Note that the majority of micronuclei in control cells are lamin A/C positive. Mean ± SEM, n = 3; P < 0.0006 for lamin A/C negative CCF con vs. OIS; P < 0.6 for lamin A/C positive CCF con vs. OIS.


( E ) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac . ( F ) Cells from E were scored for indicated histone modifications in CCFs . A single representative experiment is shown and at least 100 cells were scored per slide . Note the majority of micronuclei in control cells are positive for both marks .

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(E) CCFs in OIS are strongly positive for heterochromatic histone mark H3K27me3 but not for euchromatic mark H3K9ac. (F) Cells from E were scored for indicated histone modifications in CCFs. A single representative experiment is shown and at least 100 cells were scored per slide. Note the majority of micronuclei in control cells are positive for both marks.


( G ) CCFs in RS contain γ - H2AX but not 53BP1 ( top ) . Intranuclear γ - H2AX foci colocalize with 53BP1 ( bottom ) . ( H ) CCF + and CCF − cells from G were scored for colocalizing intranuclear γ - H2AX and 53BP1 foci . Mean ± SEM , n = 3 ; P < 0 . 019 . Bars ( in IF panels ) , 10 µm .

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(G) CCFs in RS contain γ-H2AX but not 53BP1 (top). Intranuclear γ-H2AX foci colocalize with 53BP1 (bottom). (H) CCF+ and CCF− cells from G were scored for colocalizing intranuclear γ-H2AX and 53BP1 foci. Mean ± SEM, n = 3; P < 0.019. Bars (in IF panels), 10 µm.


Nuclear - to - cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1 . ( A ) Extrusion of GFP - H2B - positive chromatin fragment from nonmitotic RS nucleus . Confocal time - lapse microscopy of transiently transfected GFP - H2B - expressing cell , 60 × original magnification , 150 nm optical section , time in mins . Bars : 10 µm ; ( insert ) 2 µm .

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Nuclear-to-cytoplasm chromatin blebbing in senescent cells associated with depletion of lamin B1. (A) Extrusion of GFP-H2B-positive chromatin fragment from nonmitotic RS nucleus. Confocal time-lapse microscopy of transiently transfected GFP-H2B-expressing cell, 60× original magnification, 150 nm optical section, time in mins. Bars: 10 µm; (insert) 2 µm.


( B ) Chromatin fragments blebbing out of nucleus are γ - H2AX positive . Bars : ( top ) 10 µm ; ( bottom ) 5 µm .

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(B) Chromatin fragments blebbing out of nucleus are γ-H2AX positive. Bars: (top) 10 µm; (bottom) 5 µm.


( C ) Down - regulation of lamin B1 and lamin B receptor ( LBR ) in RS cells , assayed by Western blot . See Materials and methods for time - course details .

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(C) Down-regulation of lamin B1 and lamin B receptor (LBR) in RS cells, assayed by Western blot. See Materials and methods for time-course details.


( D ) Depletion of lamin B1 from OIS cells , assayed by immunofluorescence . ( E ) A line - scan of lamin B1 fluorescence intensity along the arrows in D . Data shown are from a single representative experiment out of three repeats .

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(D) Depletion of lamin B1 from OIS cells, assayed by immunofluorescence. (E) A line-scan of lamin B1 fluorescence intensity along the arrows in D. Data shown are from a single representative experiment out of three repeats.


. ( F ) Depletion of lamin B1 in BRAFV600E - induced OIS melanocytes in vitro . Bars , 10 µm . ( G ) Quantitative immunofluorescence of lamin B1 in melanocytes from F . A representative experiment out of two repeats is shown . At least 150 randomly selected cells were assessed .

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. (F) Depletion of lamin B1 in BRAFV600E-induced OIS melanocytes in vitro. Bars, 10 µm. (G) Quantitative immunofluorescence of lamin B1 in melanocytes from F. A representative experiment out of two repeats is shown. At least 150 randomly selected cells were assessed.


( H ) Senescent nevusmelanocytes ( n ) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis ( e ) . White dotted line shows the basement membrane between the dermis and epidermis . Bottom panels ( Epi and Nevus ) are expanded from white boxed areas on top . Confocal microscopy for DAPI ( blue ) , MelanA ( red ) , and lamin B1 ( green ) stained tissue . Bars , 20 µm . ( I ) Quantitation of results from H for three different nevi . Epifluorescence images of lamin B1 - and MelanA - stained nevi were obtained and epidermal or nevusMelanA - positive cells were scored for the presence ( positive ) of a characteristic lamin B1 " ring . " At least 50 MelanA - expressing epidermalmelanocytes and at least 100 nevusmelanocytes were scored . Average of three different nevi ± SEM ; P < 0 . 0005 .

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(H) Senescent nevusmelanocytes (n) stain less intensely for lamin B1 as compared with nonsenescent melanocytes in epidermis (e). White dotted line shows the basement membrane between the dermis and epidermis. Bottom panels (Epi and Nevus) are expanded from white boxed areas on top. Confocal microscopy for DAPI (blue), MelanA (red), and lamin B1 (green) stained tissue. Bars, 20 µm. (I) Quantitation of results from H for three different nevi. Epifluorescence images of lamin B1- and MelanA-stained nevi were obtained and epidermal or nevusMelanA-positive cells were scored for the presence (positive) of a characteristic lamin B1 "ring." At least 50 MelanA-expressing epidermalmelanocytes and at least 100 nevusmelanocytes were scored. Average of three different nevi ± SEM; P < 0.0005.


Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity . ( A ) Herniation of H3K27me3 - positive chromatin into the cytoplasm of OIS cells . Yellow boxed area is magnified in bottom panels . Bars : ( top panels ) 10 µm ; ( bottom panels ) 5 µm . ( B ) Quantitation of lamin B1 gaps or herniations in OIS . Mean ± SEM , n = 2 ; P < 0 . 01 .

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Blebbing of chromatin from nucleus to cytoplasm in senescent cells is associated with loss of nuclear envelope integrity. (A) Herniation of H3K27me3-positive chromatin into the cytoplasm of OIS cells. Yellow boxed area is magnified in bottom panels. Bars: (top panels) 10 µm; (bottom panels) 5 µm. (B) Quantitation of lamin B1 gaps or herniations in OIS. Mean ± SEM, n = 2; P < 0.01.


( C ) Isolated nuclei of senescent cells are permeable to both 70 - and 500 - kD dextrans , indicating impairment of the barrier function of nuclear envelope . Dark nuclei exclude fluorescent dextran . Bars , 10 µm . ( D ) Quantitation of results from C . Mean ± SD , n = 4 ; P < 0 . 000001 for both 70 - and 500 - kD dextrans .

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(C) Isolated nuclei of senescent cells are permeable to both 70- and 500-kD dextrans, indicating impairment of the barrier function of nuclear envelope. Dark nuclei exclude fluorescent dextran. Bars, 10 µm. (D) Quantitation of results from C. Mean ± SD, n = 4; P < 0.000001 for both 70- and 500-kD dextrans.


( E ) Representative OIS SAHF + nucleus showing dextran permeability . Bars , 10 µm . ( F ) Quantitation of dextran 70 uptake in SAHF + and SAHF − OIS cells . P < 1 . 9 × 10 − 5 . ( G ) In individual nuclei , chromatin extrusion and dextran intrusion occurred in close physical proximity . Right - hand panels are expanded from yellow boxed areas on the left . Bars : ( left ) 10 µm ; ( right ) 2 µm .

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(E) Representative OIS SAHF+ nucleus showing dextran permeability. Bars, 10 µm. (F) Quantitation of dextran 70 uptake in SAHF+ and SAHF− OIS cells. P < 1.9 × 10−5. (G) In individual nuclei, chromatin extrusion and dextran intrusion occurred in close physical proximity. Right-hand panels are expanded from yellow boxed areas on the left. Bars: (left) 10 µm; (right) 2 µm.


Cytoplasmic histone is processed by a lysosomal / autophagy pathway . ( A ) Close juxtaposition of senescence - associated CCF ( RS ) with p62 nuclear bodies . Bars : 10 µm ; ( inset ) 1 µm . ( B ) Quantitation of control and OIS cells with CCF overlapping p62 ( p62 + ) or not ( p62 − ) . Mean ± SEM , n = 3 .

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Cytoplasmic histone is processed by a lysosomal/autophagy pathway. (A) Close juxtaposition of senescence-associated CCF (RS) with p62 nuclear bodies. Bars: 10 µm; (inset) 1 µm. (B) Quantitation of control and OIS cells with CCF overlapping p62 (p62+) or not (p62−). Mean ± SEM, n = 3.


( C ) Quantitation of proliferating and RS cells with CCF overlapping p62 ( p62 + ) or not ( p62 − ) and protein ubiquitination ( FK2 + ) or not ( FK2 − ) . Mean ± SEM , n = 3 ; P < 0 . 0008 for FK2 + / p62 + CCF , comparing proliferating and RS cells . ( D ) Ubiquitinated proteins appear to line chromatin surface in CCF . Yellow boxed areas are magnified in right - hand panels . Bars : ( left panels ) 10 µm ; ( right panels ) 1 µm .

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(C) Quantitation of proliferating and RS cells with CCF overlapping p62 (p62+) or not (p62−) and protein ubiquitination (FK2+) or not (FK2−). Mean ± SEM, n = 3; P < 0.0008 for FK2+/p62+ CCF, comparing proliferating and RS cells. (D) Ubiquitinated proteins appear to line chromatin surface in CCF. Yellow boxed areas are magnified in right-hand panels. Bars: (left panels) 10 µm; (right panels) 1 µm.


( E ) Apparent activation of cathepsin L in RS IMR90 cells . In contrast to RS cells , the majority of cathepsin L in proliferating control cells exists in its inactive pro - cathepsin L form . Loading was normalized by cell number and lamin A / C was used as a loading control . See Materials and methods for details of time course .

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(E) Apparent activation of cathepsin L in RS IMR90 cells. In contrast to RS cells, the majority of cathepsin L in proliferating control cells exists in its inactive pro-cathepsin L form. Loading was normalized by cell number and lamin A/C was used as a loading control. See Materials and methods for details of time course.


( F ) Accumulation of H3cs . 1 , a specific N - terminal cleavage product of H3 , in OIS cells . See Materials and methods for details of time course . Western blotting was performed using the same cellular lysates as in Fig . S4 B , and the same lamin A / C panel is shown in both figures .

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(F) Accumulation of H3cs.1, a specific N-terminal cleavage product of H3, in OIS cells. See Materials and methods for details of time course. Western blotting was performed using the same cellular lysates as in Fig. S4 B, and the same lamin A/C panel is shown in both figures.


( G ) Accumulation of H3cs . 1 and corresponding high mobility H3 in OIS .

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(G) Accumulation of H3cs.1 and corresponding high mobility H3 in OIS.


( H ) Predominant cytoplasmic localization of H3cs . 1 in senescent cells . α - Tubulin and lamin A / C were used as fractionation controls for cytoplasmic and nuclear fractions , respectively .

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(H) Predominant cytoplasmic localization of H3cs.1 in senescent cells. α-Tubulin and lamin A/C were used as fractionation controls for cytoplasmic and nuclear fractions, respectively.


Senescent cells in vitro and in vivo contain reduced histone content . ( A ) Immunofluorescent confocal images of histone H3 in control or OIS cells ( 11 d after activation of ER - RASG12V ) . Yellow arrows indicate cells with most pronounced SAHF . ( B ) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells . Representative of two independent experiments .

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Senescent cells in vitro and in vivo contain reduced histone content. (A) Immunofluorescent confocal images of histone H3 in control or OIS cells (11 d after activation of ER-RASG12V). Yellow arrows indicate cells with most pronounced SAHF. (B) Quantitative immunofluorescence analysis of histone H3 in RS and OIS cells. Representative of two independent experiments.


( C ) Progressive loss of core histones in senescent IMR90 cells in replicative senescence ( RS ) . Lysates were normalized by cell number and an equal number of cells was loaded per each lane . Lamin A / C was used as a confirmatory loading control . See Materials and methods for details of time course .

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(C) Progressive loss of core histones in senescent IMR90 cells in replicative senescence (RS). Lysates were normalized by cell number and an equal number of cells was loaded per each lane. Lamin A/C was used as a confirmatory loading control. See Materials and methods for details of time course.


( D ) Immunohistochemistry for histone H3in human dermal nevus ( n ) and adjacent epidermis ( e ) . Right - hand panels are expanded from boxed areas on the left . Note decreased nevus staining for H3 ( right bottom ) as compared with epidermis ( right top ) . Bars : ( left ) 100 µm ; ( right ) 50 µm .

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(D) Immunohistochemistry for histone H3in human dermal nevus (n) and adjacent epidermis (e). Right-hand panels are expanded from boxed areas on the left. Note decreased nevus staining for H3 (right bottom) as compared with epidermis (right top). Bars: (left) 100 µm; (right) 50 µm.


( E ) Immunofluorescence of histone H3 ( red ) and S100 ( melanocytes [ green ] ) in human benign nevus and adjacent epidermis . Bars , 20 µm .

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(E) Immunofluorescence of histone H3(red) and S100 (melanocytes [green]) in human benign nevus and adjacent epidermis. Bars, 20 µm.


( F ) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus ( bottom panels ) , compared with epidermalmelanocytes ( top panels ) . Bars , 20 µm . ( G ) Quantitative immunofluorescence of H3 in epidermal and nevusmelanocytes . Images were obtained in blue ( DAPI ) , green ( S100 ) , and red ( H3 ) channels and then H3 intensity was measured in either S100 + epidermal cells , strictly adjacent to the basal membrane , or in S100 + nevusmelanocytes . H3 intensity histograms represent fluorescence intensity distribution , combined from three individual nevi . At least 150 epidermal ( 50 per nevus ) and 300 nevus ( 100 per nevus ) melanocytes were assessed .

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(F) Higher magnification of boxed regions in E shows reduced staining for H3 in nevus (bottom panels), compared with epidermalmelanocytes (top panels). Bars, 20 µm. (G) Quantitative immunofluorescence of H3 in epidermal and nevusmelanocytes. Images were obtained in blue (DAPI), green (S100), and red (H3) channels and then H3 intensity was measured in either S100+epidermal cells, strictly adjacent to the basal membrane, or in S100+nevusmelanocytes. H3 intensity histograms represent fluorescence intensity distribution, combined from three individual nevi. At least 150 epidermal (50 per nevus) and 300 nevus (100 per nevus) melanocytes were assessed.


Senescence - associated loss of histones is V - ATPase dependent . ( A ) Bafilomycin A1 ( BafA1 ) blocks the loss of nuclearhistone H3content in cells undergoing OIS . BafA1 was added to cells at 50 nM on day 5 after RASG12V induction . Cells were harvested 24 h later , and stained for histone H3 . Bars , 10 µm . ( B ) Quantitative histone H3immunofluorescence in cells from A . Single representative experiment out of three repeats .

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Senescence-associated loss of histones is V-ATPase dependent. (A) Bafilomycin A1 (BafA1) blocks the loss of nuclearhistone H3content in cells undergoing OIS. BafA1 was added to cells at 50 nM on day 5 after RASG12V induction. Cells were harvested 24 h later, and stained for histone H3. Bars, 10 µm. (B) Quantitative histone H3immunofluorescence in cells from A. Single representative experiment out of three repeats.


( C ) RASG12V - induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot . LC3 I / II has been used as a control for BafA1 activity . Lysates from 10 , 000 cells were loaded per well .

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(C) RASG12V-induced OIS cells were treated with BafA1 as described in A and then assessed for histones by Western blot. LC3 I/II has been used as a control for BafA1 activity. Lysates from 10,000 cells were loaded per well.


( D ) BafA1 blocks the accumulation of H3cs . 1 in cells undergoing OIS .

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(D) BafA1 blocks the accumulation of H3cs.1 in cells undergoing OIS.


( E ) BafA1 and concanamycin A ( Con A ) block the accumulation of H3cs . 1 in RS cells .

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(E) BafA1 and concanamycin A (Con A) block the accumulation of H3cs.1 in RS cells.


HEK293T cells were transfected with 200 nM siRNAs per well . After 24 h , the cells were infected with HIV - luc / 2019 - nCoV pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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HEK293T cells were transfected with 200 nM siRNAs per well. After 24 h, the cells were infected with HIV-luc/2019-nCoV pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.


assay . B , Teicoplanin inhibit the entry of 2019 - nCoV and SARS - nCoV . Chemical structure of teicoplanin ( Left ) . A549 cells were seeded in a 96 - well plate , and 24 h later , the cells were infected with HIV - luc / 2019 - nCoV , HIV - luc / SARS - nCoV or HIV - luc / VSVG pseudoviruses . After washing and incubation with fresh medium for 48 h , the intracellular luciferase activity was measured . Ordinary one - way ANOVA test was used for this analysis . The results are shown as the mean and SEM . * P < 0 . 05 .

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assay.B, Teicoplanin inhibit the entry of 2019-nCoV and SARS-nCoV. Chemical structure of teicoplanin (Left). A549 cells were seeded in a 96-well plate, and 24 h later, the cells were infected with HIV-luc/2019-nCoV, HIV-luc/SARS-nCoV or HIV-luc/VSVG pseudoviruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. Ordinary one-way ANOVA test was used for this analysis. The results are shown as the mean and SEM. *P< 0.05.C


, A549 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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, A549 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.


types . A , HEK293T cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

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types.A, HEK293T cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.B


software . B , Huh7 cells were infected with HIV - luc / 2019 - nCoV pseudoviruses and then incubated with teicoplanin at various concentrations . The intracellular luciferase activity was measured at 48 h post - infection . The IC50 was calculated using GraphPad Prism software .

software.B, Huh7 cells were infected with HIV-luc/2019-nCoV pseudoviruses and then incubated with teicoplanin at various concentrations. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software.C


A549 cells were incubated with dalbavancin ( D ) at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis .

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A549 cells were incubated with dalbavancin (D) at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. dalbavancin


D , A549 cells were incubated with vancomycin at various concentrations and infected with HIV - luc / 2019 - nCoV pseudoviruses . The intracellular luciferase activity was measured at 48 h post - infection . Ordinary one - way ANOVA test was used for this analysis . Left : Chemical structure of

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D, A549 cells were incubated with vancomycin at various concentrations and infected with HIV-luc/2019-nCoV pseudoviruses. The intracellular luciferase activity was measured at 48 h post-infection. Ordinary one-way ANOVA test was used for this analysis. Left: Chemical structure of vancomycin


Isosurface of the subtomogram average ( in pink ) with an applied C5 symmetry and 0 . 69 nm tomographic slices ( A - E ) at the indicated heights . Scale bars 10 nm . The average is shown in side view , whereas the slices represent perpendicular slices . The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base ( base not visible at the used threshold ) . The division of the structure into subparts was adapted from ( Durand et al . , 2015 ) . Subvolumes were extracted from cryotomograms of ghost cells and FIB - milled intact E . coli BL21 cells heterologously expressing TssJLM Slice ( 0 . 69 nm ) through the non - symmetrized average . Note that the C5 symmetry was

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Isosurface of the subtomogram average (in pink) with an applied C5 symmetry and 0.69 nm tomographic slices (A-E) at the indicated heights. Scale bars 10 nm. The average is shown in side view, whereas the slices represent perpendicular slices. The 5 pairs of pillars formed a narrow central channel and separated into 10 arches towards the base (base not visible at the used threshold). The division of the structure into subparts was adapted from (Durand et al., 2015). Subvolumes were extracted from cryotomograms of ghost cells and FIB-milled intact E. coli BL21 cells heterologously expressing TssJLM Slice (0.69 nm) through the non-symmetrized average. Note that the C5 symmetry was visible


Slice ( 9 . 7 nm ) through a cryotomogram of a FIB - milled E . coli BL21 cell expressing TssJLM . The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average . The zoomed - in area highlights the position of the TssJLM particles within the inner and the outer membrane . Some particles were found in cytoplasmic membrane invaginations , as indicated by white arrows . Scale bar 100

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Slice (9.7 nm) through a cryotomogram of a FIB-milled E. coli BL21 cell expressing TssJLM. The average shown in Figure 1 was placed back at the positions and orientations of the individual subvolumes that were used to generate the final average. The zoomed-in area highlights the position of the TssJLM particles within the inner and the outer membrane. Some particles were found in cytoplasmic membrane invaginations, as indicated by white arrows. Scale bar 100 nm


Cryotomographic slices ( 9 . 7 nm ) showing side views of TssJLM . In these examples , the basal parts of TssJLM ( red arrows ) could be seen extending into the cytoplasm . Scale bar 10

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Cryotomographic slices (9.7 nm) showing side views of TssJLM. In these examples, the basal parts of TssJLM (red arrows) could be seen extending into the cytoplasm. Scale bar 10 nm


Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4 h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar .

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Fluorescence of prey cells (green) indicating their survival against attacker cells after 4 h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.


Time - lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental ( WT ) and TssJ mutated strains ( TssJ D97K ) . Individual images were taken every 60 seconds . The corresponding time of each micrograph is indicated in the lower right part of the image . Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads . Scale bars , 1 µm .

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Time-lapse fluorescence microscopy recordings showing localization and dynamics of the sfGFPTssM fusion protein in the parental (WT) and TssJ mutated strains (TssJ D97K). Individual images were taken every 60 seconds. The corresponding time of each micrograph is indicated in the lower right part of the image. Stable foci in the WT strain and less stable foci in the TssJ D97K strain are respectively indicated by white and blue arrowheads. Scale bars, 1 µm.


Fluorescence of prey cells ( green ) indicating their survival against attacker cells after 4h . The image of a representative bacterial competition spot is shown on the upper part . The relative fluorescent level ( in AU ) and the number of recovered E . coli recipient cells ( in log10 of colony forming units ( cfu ) ) are indicated in the lower graphs . The assays were performed from at least three independent cultures , with technical triplicates and a representative technical triplicate is shown . Error bars in the left graph represent ± standard deviation ( SD ) from technical triplicate . The circles in the right graph indicate values from technical triplicate , and the average is indicated by the bar .

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Fluorescence of prey cells (green) indicating their survival against attacker cells after 4h. The image of a representative bacterial competition spot is shown on the upper part. The relative fluorescent level (in AU) and the number of recovered E. coli recipient cells (in log10 of colony forming units (cfu)) are indicated in the lower graphs. The assays were performed from at least three independent cultures, with technical triplicates and a representative technical triplicate is shown. Error bars in the left graph represent ± standard deviation (SD) from technical triplicate. The circles in the right graph indicate values from technical triplicate, and the average is indicated by the bar.


Fluorescence microscopy recordings showing sfGFPTssM foci in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . TssM foci containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 1 µm .

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Fluorescence microscopy recordings showing sfGFPTssM foci in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). TssM foci containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.


Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental ( WT ) and TssM mutated strains ( TssM Q779C / N780C , TssM ∆ 777 - 783 ) . Fluorescent sheath containing cells are indicated by arrowheads . Microscopy analyses were performed independently three times , each in technical triplicate , and a representative experiment is shown . Scale bars , 1 µm .

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Fluorescence microscopy recordings showing TssBsfGFP sheath in the parental (WT) and TssM mutated strains (TssM Q779C/N780C, TssM ∆777-783). Fluorescent sheath containing cells are indicated by arrowheads. Microscopy analyses were performed independently three times, each in technical triplicate, and a representative experiment is shown. Scale bars, 1 µm.


( B ) TLC analysis of ganglioside species in human serum . ( C ) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum . Data expressed as mean ± SD , n = 6 .

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(B) TLC analysis of ganglioside species in human serum. (C) Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum. Data expressed as mean ± SD, n = 6.


( B ) TLC analysis of ganglioside species in human serum .

(B) TLC analysis of ganglioside species in human serum.


( E ) Quantification by LC - MS / MS analysis of serum GM3 species with differing acyl chain structures . Total abundance of 10 species was defined as 1 . Data expressed as mean ± SD , n = 6 .

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(E) Quantification by LC-MS/MS analysis of serum GM3 species with differing acyl chain structures. Total abundance of 10 species was defined as 1. Data expressed as mean ± SD, n = 6.


( A ) Heat map analysis of serum GM3 species in various pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Colors indicate fold change average of each species relative to control ( defined as 1 ) , as shown in key at right . Order of pathological phases corresponds to increments of HOMA - IR and serum CRP .

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(A) Heat map analysis of serum GM3 species in various pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP.


( B - D ) Properties of various GM3 species as a function of pathological phases : control ( n = 24 ) , VFA ( n = 38 ) , lipidemia ( n = 28 ) , glycemia ( n = 15 ) , and lipidemia + glycemia ( n = 17 ) . Data shown are relative abundances of total LCFA species ( 16 : 0 , 18 : 0 , 20 : 0 ) ( B ) , total VLCFA species ( 22 : 0 , 23 : 0 , 24 : 0 , h24 : 0 ) ( C ) , and total unsaturated VLCFA species ( 22 : 1 , 24 : 1 , h24 : 1 ) ( D ) relative to total of ten major GM3 species ( defined as 1 ) in each subject .

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(B-D) Properties of various GM3 species as a function of pathological phases: control (n=24), VFA (n=38), lipidemia (n=28), glycemia (n=15), and lipidemia + glycemia (n=17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject.


( E - H ) Properties of various GM3 species as a function of BMI : LCFA - GM3 ( E ) , VLCFA - GM3 ( F ) , unsaturated VLCFA - GM3 ( G ) , and α - hydroxyVLCFA - GM3 ( h24 : 0 ) ( H ) . Colors indicate disease severity : light blue , no abnormal scores ( n = 25 ) ; orange , early - phase obesity ( n = 74 ) ; purple , severe obesity ( n = 23 ) .

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(E-H) Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxyVLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n=25); orange, early-phase obesity (n=74); purple, severe obesity (n=23).


( I , J ) Spearman ' s correlations for GM3 h24 : 0 vs . ALT ( I ) and vs . HOMA - IR ( J ) .

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(I, J) Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J).


( K ) Plots of α - hydroxylation rate ( h24 : 0 / 24 : 0 ) vs . serum CRP . Colors indicate range of CRP value ( mg / dL ) : light blue , 0 . 01 - 0 . 02 ( n = 21 ) ; orange , 0 . 03 - 0 . 09 ( n = 56 ) ; gray , 0 . 10 - 0 . 29 ( n = 29 ) ; red , 0 . 3 - 1 . 0 ( diagnostically abnormal ; n = 15 ) .

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(K) Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dL): light blue, 0.01-0.02 (n=21); orange, 0.03-0.09 (n=56); gray, 0.10-0.29 (n=29); red, 0.3-1.0 (diagnostically abnormal; n=15).


( B , C ) GM3 - mediated enhancement and inhibition of proinflammatory cytokine production from LPS - stimulated monocytes ( LPS : 0 . 06 , 0 . 13 , 0 . 25 ng / mL ) . TNF - α ( B ) and IL - 6 ( C ) production in culture supernatant was measured by ELISA .

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(B, C) GM3-mediated enhancement and inhibition of proinflammatory cytokine production from LPS-stimulated monocytes (LPS: 0.06, 0.13, 0.25 ng/mL). TNF-α (B) and IL-6 (C) production in culture supernatant was measured by ELISA.


( D , E ) Co - stimulation of monocytes by LPS plus GM3 species or complex ganglioside species ( 1 . 5 , 3 . 0 , 4 . 5 µM ) . TNF - α ( D ) and IL - 6 ( E ) production were shown in heat maps .

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(D, E) Co-stimulation of monocytes by LPS plus GM3 species or complex ganglioside species (1.5, 3.0, 4.5 µM). TNF-α (D) and IL-6 (E) production were shown in heat maps.


( F ) Co - stimulation of monocytes by LPS plus GM3 species or precursor GSL species . TNF - α , IL - 6 , and IL - 12 / 23 production were shown in heat maps .

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(F) Co-stimulation of monocytes by LPS plus GM3 species or precursor GSL species. TNF-α, IL-6, and IL-12/23 production were shown in heat maps.


( G ) Inhibitory effect of LCFA and unsaturated VLCFA - GM3 on LVCFA - GM3 species . IL - 6 production in culture supernatant was measured by ELISA .

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(G) Inhibitory effect of LCFA and unsaturated VLCFA-GM3 on LVCFA-GM3 species. IL-6 production in culture supernatant was measured by ELISA.


( A ) Co - stimulation of human monocytes by GM3 species plus various TLR ligands : LPS ( 0 . 13 ng / mL ) , TLR4 / MD - 2 , Pam3CSK4 ( 0 . 5 µg / mL ) , TLR1 / 2 , Flagellin ( 50 ng / mL ) , TLR5 , R848 ( 0 . 5 µg / mL ) , TLR7 / 8 , MALP - 2 ( 1 . 0 ng / mL ) , TLR2 / 6 . IL - 6 production in culture supernatant was quantified by ELISA ( shown in a heat map ) .

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(A) Co-stimulation of human monocytes by GM3 species plus various TLR ligands: LPS (0.13 ng/mL), TLR4/MD-2, Pam3CSK4 (0.5 µg/mL), TLR1/2, Flagellin (50 ng/mL), TLR5, R848 (0.5 µg/mL), TLR7/8, MALP-2 (1.0 ng/mL), TLR2/6. IL-6 production in culture supernatant was quantified by ELISA (shown in a heat map).


( B ) Co - stimulation of monocytes by GM3 species ( 16 : 0 , 24 : 0 , 24 : 1 ) plus synthetic TLR4 ligands LA506 ( 15 ng / mL ) , LA505 ( 150 ng / mL ) , or LA504 ( 150 ng / mL ) .

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(B) Co-stimulation of monocytes by GM3 species (16:0, 24:0, 24:1) plus synthetic TLR4 ligands LA506 (15 ng/mL), LA505 (150 ng/mL), or LA504 (150 ng/mL).


( C , D ) Production of IL - 6 ( C ) and TNF - α ( D ) in culture supernatant following co - stimulation of monocytes by GM3 species plus human HMGB1 .

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(C, D) Production of IL-6 (C) and TNF-α (D) in culture supernatant following co-stimulation of monocytes by GM3 species plus human HMGB1.


( E ) Overexpression of hTLR4 , hTLR4 / hMD - 2 , hTLR4 ( P714H ) / MD - 2 in HEK293T cells , and co - stimulation by GM3 22 : 0 with LPS ( 5 ng / mL ) . TLR4 activation was monitored by NF - κB luciferase reporter assay ( termed " Relative Luc Activity " on Y - axis ) .

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(E) Overexpression of hTLR4, hTLR4/hMD-2, hTLR4 (P714H) /MD-2 in HEK293T cells, and co-stimulation by GM3 22:0 with LPS (5 ng/mL). TLR4 activation was monitored by NF-κB luciferase reporter assay (termed "Relative Luc Activity" on Y-axis).


( F , Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL )

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

(F, Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL)


G ) Co - stimulation of hTLR4 / hMD - 2 by GM3 species plus LPS ( 5 ng / mL ) and further addition of soluble human CD14 ( 1 µg / mL )

O O O O O O O O O O O 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) Co-stimulation of hTLR4/hMD-2 by GM3 species plus LPS (5 ng/mL) and further addition of soluble human CD14 (1 µg/mL)


( H ) Stimulation of Mal - overexpressing HEK293T cells by GM3 species .

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(H) Stimulation of Mal-overexpressing HEK293T cells by GM3 species.


( I ) Co - stimulation of hTLR4 / hMD - 2 by LPS ( 5 ng / mL ) plus various mixtures of GM3 species .

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

(I) Co-stimulation of hTLR4/hMD-2 by LPS (5 ng/mL) plus various mixtures of GM3 species.


( A ) Co - stimulation of RAW macrophages by GM3 species plus various TLR ligands : LPS ( 0 . 5 , 1 . 0 ng / mL ) , bovine thymus HMGB1 ( 0 . 25 µg / mL ) , Pam3CSK4 ( 50 ng / mL ) , Poly I : C ( 10 µg / mL ) , R848 ( 4 ng / mL ) , and CpG type - B ( 20 nM ) . TNF - α production in culture supernatant was quantified by ELISA .

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(A) Co-stimulation of RAW macrophages by GM3 species plus various TLR ligands: LPS (0.5, 1.0 ng/mL), bovine thymus HMGB1 (0.25 µg/mL), Pam3CSK4 (50 ng/mL), Poly I:C (10 µg/mL), R848 (4 ng/mL), and CpG type-B (20 nM). TNF-α production in culture supernatant was quantified by ELISA.


( B ) Co - stimulation of RAW macrophages by low - and high - dose LPS ( 0 , 5 , 50 ng / mL ) plus GM3 22 : 0 ( 5 µM ) . Time course of TNF - α production in culture supernatant was quantified by ELISA .

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(B) Co-stimulation of RAW macrophages by low- and high-dose LPS (0, 5, 50 ng/mL) plus GM3 22:0 (5 µM). Time course of TNF-α production in culture supernatant was quantified by ELISA.


( C ) Co - stimulation of BMDMs from C3H / HeN or C3H / HeJ mice by GM3 species plus LPS ( 0 . 5 ng / mL ) , and TNF - α production in culture supernatant .

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(C) Co-stimulation of BMDMs from C3H/HeN or C3H/HeJ mice by GM3 species plus LPS (0.5 ng/mL), and TNF-α production in culture supernatant.


( d ) Co - stimulation of mTLR4 / mMD - 2 - expressing HEK293T cells by GM3 species plus LPS ( 2 . 5 ng / mL ) , and further addition of soluble mouse CD14 - Fc fusion protein ( 1 µg / mL ) .

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(d) Co-stimulation of mTLR4/mMD-2-expressing HEK293T cells by GM3 species plus LPS (2.5 ng/mL), and further addition of soluble mouse CD14-Fc fusion protein (1 µg/mL).


( E Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and complex ganglioside species ( E ; 2 . 5 , 5 . 0 , 10 µM ) , ( shown in heat maps ) .

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(E Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and complex ganglioside species (E; 2.5, 5.0, 10 µM), (shown in heat maps).


F ) Co - stimulation of BMDMs from C3H / HeN mice by LPS plus GM3 species and precursor GSL species ( F ; 2 . 5 , 5 . 0 , 10 µM ) ( shown in heat maps ) .

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F) Co-stimulation of BMDMs from C3H/HeN mice by LPS plus GM3 species and precursor GSL species (F; 2.5, 5.0, 10 µM) (shown in heat maps).


( A ) GM3 molecular species of 6 - wk - old control C57 / BL6 mice and ob / ob mice were analyzed respectively by LC - MS / MS ( n = 3 ) .

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(A) GM3 molecular species of 6-wk-old control C57/BL6 mice and ob/ob mice were analyzed respectively by LC-MS/MS (n=3).


( B ) GM3 molecular species of normal - diet ( ND ) and high - fat - diet ( HFD ) C57 / BL6 mice were analyzed by LC - MS / MS ( n = 4 ) .

O O O O O O O O O O O O O O O O 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) GM3 molecular species of normal-diet (ND) and high-fat-diet (HFD) C57/BL6 mice were analyzed by LC-MS/MS (n=4).


( C ) TLC analysis of acidic GSL fraction ( equivalent to 0 . 1 mg protein ) from epididymal fat pads of C3H / HeN ( A ) and C3H / HeJ mice ( B ) on ND or HFD for 8 wk .

O O O O O O O O O O O O O O O O O O O O O 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) TLC analysis of acidic GSL fraction (equivalent to 0.1 mg protein) from epididymal fat pads of C3H/HeN (A) and C3H/HeJ mice (B) on ND or HFD for 8 wk.


( D ) GM3 molecular species of C3H / HeN mice ( ND , HFD ) and C3H / HeJ mice ( ND , HFD ) were analyzed by LC - MS / MS ( n = 4 ) .

(D) GM3 molecular species of C3H/HeN mice (ND, HFD) and C3H/HeJ mice (ND, HFD) were analyzed by LC-MS/MS (n=4).


( C , D ) Alanine scanning for basic residues involved in signal transduction via VLCFA - GM3 ( n = 5 ) ( C ) , and combinations of effective mutations ( n = 6 ) ( D ) . Signal transduction was monitored by NF - κB reporter assay .

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(C, D) Alanine scanning for basic residues involved in signal transduction via VLCFA-GM3 (n=5) (C), and combinations of effective mutations (n=6) (D). Signal transduction was monitored by NF-κB reporter assay.


( E - G ) Comparative inhibitory effects of GM3 16 : 0 on mTLR4 / mMD - 2 ( E ) , mTLR4 / hMD - 2 ( domain - swapped complex ) ( F ) , and hTLR4 / hMD - 2 ( G ) ( n = 3 ) .

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

(E-G) Comparative inhibitory effects of GM3 16:0 on mTLR4/mMD-2 (E), mTLR4/hMD-2 (domain-swapped complex) (F), and hTLR4/hMD-2 (G) (n=3).


( H ) Cross - linked SDS - PAGE analysis of recombinant mTLR4 ( extracellular domain ) / mMD - 2 complexed with GM3 18 : 0 , GM3 species enhancing mTLR4 activation .

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(H) Cross-linked SDS-PAGE analysis of recombinant mTLR4 (extracellular domain)/mMD-2 complexed with GM3 18:0, GM3 species enhancing mTLR4 activation.


A . Representative spinning disk confocal live - cell imaging time series of U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , treated with control and KIF4A siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

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A. Representative spinning disk confocal live-cell imaging time series of U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), treated with control and KIF4A siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.


B . Representative spinning disk confocal live - cell imaging time series images of S - trityl - L - cysteine ( STLC ) - treated U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) . Note that MT - flux is abrogated in presence of taxol ( lower panel ) . Scale bars , 10 µm . Time , min : sec .

O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

B. Representative spinning disk confocal live-cell imaging time series images of S-trityl-L-cysteine (STLC)-treated U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red). Note that MT-flux is abrogated in presence of taxol (lower panel). Scale bars, 10 µm. Time, min:sec.


C . Illustration of 405nm laser - photoactivated regions in monopolar spindles ( blue circles , left ) . The effect of photoactivation and region selected for kymograph generation ( dashed white rectangle , right ) . Scale bar , 10 µm . D . Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates ( red dotted lines highlight MT - flux slopes ) . Scale bars , 30 sec .

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C. Illustration of 405nm laser-photoactivated regions in monopolar spindles (blue circles, left). The effect of photoactivation and region selected for kymograph generation (dashed white rectangle, right). Scale bar, 10 µm. D. Corresponding kymograph profiles of the photoactivated regions in bipolar and monopolar spindles used for quantification of the flux rates (red dotted lines highlight MT-flux slopes). Scale bars, 30 sec.


Quantification of MT - flux in bipolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) : - bipolar spindles : siControl ( 49 , 5 ) , STLC ( 31 , 3 ) , siKIF15 ( 39 , 3 ) , siKID ( 32 , 3 ) , siKIF4A ( 28 , 3 ) , siKIF2A ( 36 , 3 ) , siCLASPs ( 21 , 2 ) , GSK923295 ( 44 , 3 )

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

Quantification of MT-flux in bipolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments): - bipolar spindles: siControl (49, 5), STLC (31, 3), siKIF15 (39, 3), siKID (32, 3), siKIF4A (28, 3), siKIF2A (36, 3), siCLASPs (21, 2), GSK923295 (44, 3)


Quantification of MT - flux in monopolar spindles subjected to indicated treatments . Graphs represent MT flux of individual cells with mean ± SD . N ( number of cells , number of independent experiments ) monopolar spindles : siControl ( 35 , 3 ) , siKIF15 ( 44 , 3 ) , siKID ( 38 , 3 ) , siKIF4A ( 44 , 3 ) , siKIF2A ( 38 , 3 ) , siCLASPs ( 12 , 2 ) , GSK923295 ( 33 , 3 ) .

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

Quantification of MT-flux in monopolar spindles subjected to indicated treatments. Graphs represent MT flux of individual cells with mean ± SD. N (number of cells, number of independent experiments) monopolar spindles: siControl (35, 3), siKIF15 (44, 3), siKID (38, 3), siKIF4A (44, 3), siKIF2A (38, 3), siCLASPs (12, 2), GSK923295 (33, 3).


A . Representative spinning disk confocal live - cell imaging time series images of U2OS PA - GFP - α - tubulin cells conditionally co - expressing KIF4A shRNA and RNAi - resistant mCherry - Kif4A variants induced using doxycycline . Chromosomes were stained using SiR - DNA . White arrowheads highlight poleward flux of the photoactivated regions . Scale bar , 10 µm . Time , min : sec .

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A. Representative spinning disk confocal live-cell imaging time series images of U2OS PA-GFP-α-tubulin cells conditionally co-expressing KIF4A shRNA and RNAi-resistant mCherry-Kif4A variants induced using doxycycline. Chromosomes were stained using SiR-DNA. White arrowheads highlight poleward flux of the photoactivated regions. Scale bar, 10 µm. Time, min:sec.


B . Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi - resistant mCherry - KIF4A variants . The anti - KIF4A antibody was used , together with anti - vinculin antibody as a loading control .

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B. Representative immunoblot of cell lysates obtained before and after doxycycline induction to validate the efficiency of KIF4A shRNA construct and expression of the RNAi-resistant mCherry-KIF4A variants. The anti-KIF4A antibody was used, together with anti-vinculin antibody as a loading control.


C . Quantification of MT - flux upon shRNA - mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi - resistant mCherry - KIF4A variants . The bars in graph represent mean ± SD . N ( number of cells , number of independent experiments ) : uninduced shKIF4A ( 48 , 4 ) , shKIF4A ( 60 , 4 ) , uninduced KIF4A WT ( 36 , 5 ) , shKIF4A + KIF4A WT ( 43 , 5 ) , uninduced KIF4A K94A ( 42 , 7 ) , shKIF4A + KIF4A K94A ( 38 , 6 ) , uninduced KIF4A ΔZip1 ( 32 , 4 ) , shKIF4A + ΔZip1 ( 30 , 4 ) .

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C. Quantification of MT-flux upon shRNA-mediated depletion of KIF4A alone or in combination with conditional expression of the RNAi-resistant mCherry-KIF4A variants. The bars in graph represent mean ± SD. N (number of cells, number of independent experiments): uninduced shKIF4A (48, 4), shKIF4A (60, 4), uninduced KIF4A WT (36, 5), shKIF4A + KIF4A WT (43, 5), uninduced KIF4A K94A (42, 7), shKIF4A + KIF4A K94A (38, 6), uninduced KIF4A ΔZip1 (32, 4), shKIF4A + ΔZip1 (30, 4).


A . Representative spinning disk confocal time series of MT - flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes ( MUGs ) . U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) , labeled for chromosomes with SiR - DNA ( grey ) are shown , with 5 mM caffeine used as a control . Scale bar , 10 µm . Time , min : sec . B . Quantification of the MT - flux rates from indicated conditions . MT - flux values with mean ± SD are plotted . N ( number of cells , number of independent experiments ) : Caffeine - only control ( 30 , 3 ) and MUGs ( 34 , 3 ) .

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A. Representative spinning disk confocal time series of MT-flux in bipolar spindles in control cells and cells undergoing mitosis with unreplicated genomes (MUGs). U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red), labeled for chromosomes with SiR-DNA (grey) are shown, with 5 mM caffeine used as a control. Scale bar, 10 µm. Time, min:sec. B. Quantification of the MT-flux rates from indicated conditions. MT-flux values with mean ± SD are plotted. N (number of cells, number of independent experiments): Caffeine-only control (30, 3) and MUGs (34, 3).


C . Representative point - scanning confocal maximum - intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions , immunostained with antibodies against KIF4A and α - tubulin . DNA was counterstained with DAPI . KIF4A in cyan and α - tubulin in red in merged image . Scale bar , 10 µm .

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C. Representative point-scanning confocal maximum-intensity projected images of mitotic spindles in U2OS cells subjected to indicated conditions, immunostained with antibodies against KIF4A and α-tubulin. DNA was counterstained with DAPI. KIF4A in cyan and α-tubulin in red in merged image. Scale bar, 10 µm.


A . Representative spinning disk confocal live - cell image series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated siRNAs . White arrowheads highlight poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_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

A. Representative spinning disk confocal live-cell image series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated siRNAs. White arrowheads highlight poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.


B . Quantification of the impact of the MT - crosslinking proteins on MT - flux in U2OS PA - GFP / mCherry - α - tubulin cells transfected with respective siRNAs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siPRC1 ( 45 , 3 ) , siNuMA ( 40 , 3 ) , siHSET ( 37 , 3 ) , siHSET + siNuMA ( 39 , 3 ) .

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B. Quantification of the impact of the MT-crosslinking proteins on MT-flux in U2OS PA-GFP/mCherry-α-tubulin cells transfected with respective siRNAs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siPRC1 (45, 3), siNuMA (40, 3), siHSET (37, 3), siHSET + siNuMA (39, 3).


C . Quantification of the impact of the motor activity of HSET on MT - flux in U2OS cells stably expressing mEOS - α - tubulin treated with control or HSET 3 ′ UTR siRNAs in presence or absence of the respective RNAi - resistant GFP - HSET constructs . Graph represent MT - flux values with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 23 , 3 ) , siHSET 3 ′ UTR ( 33 , 3 ) , siHSET 3 ′ UTR + HSET WT ( 29 , 5 ) , siHSET 3 ′ UTR + HSET N593K ( 30 , 4 ) .

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C. Quantification of the impact of the motor activity of HSET on MT-flux in U2OS cells stably expressing mEOS-α-tubulin treated with control or HSET 3′UTR siRNAs in presence or absence of the respective RNAi-resistant GFP-HSET constructs. Graph represent MT-flux values with mean ± SD. N (number of cells, number of independent experiments): siControl (23, 3), siHSET 3′UTR (33, 3), siHSET 3′UTR + HSET WT (29, 5), siHSET 3′UTR + HSET N593K (30, 4).


D . Representative kymographs of the photoactivated spindles displaying asynchronous flux motion ( split of the two red dashed lines towards individual poles ) upon RNAi - mediated depletion of MT - crosslinkers . E . Percentage of cells with asynchronous flux

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D. Representative kymographs of the photoactivated spindles displaying asynchronous flux motion (split of the two red dashed lines towards individual poles) upon RNAi-mediated depletion of MT-crosslinkers. E. Percentage of cells with asynchronous flux movements


A . Representative spinning disk confocal live - cell time series of MT - flux in U2OS cells stably co - expressing PA - GFP - α - tubulin ( cyan ) and mCherry - α - tubulin ( red ) treated with indicated conditions . White arrowheads follow the poleward motion of the photoactivated regions due to MT - flux . Scale bars , 10 µm . Time , min : sec .

O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Representative spinning disk confocal live-cell time series of MT-flux in U2OS cells stably co-expressing PA-GFP-α-tubulin (cyan) and mCherry-α-tubulin (red) treated with indicated conditions. White arrowheads follow the poleward motion of the photoactivated regions due to MT-flux. Scale bars, 10 µm. Time, min:sec.


B . Quantification of the impact of stable end - on KT - MT attachments on MT - flux in NDC80 - depleted cells under indicated conditions and in early prometaphase cells with and without the CENP - E inhibitor GSK923295 . MT - flux values are plotted with mean ± SD . N ( number of cells , number of independent experiments ) : siControl ( 49 , 5 ) , siNDC80 ( 59 , 6 ) , siNDC80 + siKIF4A ( 41 , 3 ) , siNDC80 + STLC ( bipol . ) ( 28 , 3 ) , siNDC80 + siKIF15 ( 36 , 4 ) , siNDC80 + siCLASPs ( 33 , 3 ) , siNDC80 + siKIF2A ( 38 , 4 ) , siNDC80 + GSK923295 ( 38 , 3 ) , early prometaphase ( 23 , 3 ) , early prometaphase + GSK923295 ( 28 , 3 ) .

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 B-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Quantification of the impact of stable end-on KT-MT attachments on MT-flux in NDC80-depleted cells under indicated conditions and in early prometaphase cells with and without the CENP-E inhibitor GSK923295. MT-flux values are plotted with mean ± SD. N (number of cells, number of independent experiments): siControl (49, 5), siNDC80 (59, 6), siNDC80 + siKIF4A (41, 3), siNDC80 + STLC (bipol.) (28, 3), siNDC80 + siKIF15 (36, 4), siNDC80 + siCLASPs (33, 3), siNDC80 + siKIF2A (38, 4), siNDC80 + GSK923295 (38, 3), early prometaphase (23, 3), early prometaphase + GSK923295 (28, 3).


C . Kymograph profiles ( left ) highlighting that the photoactivated regions in prometaphase ( upper panel ) split and move towards both poles , whereas metaphase spindles ( lower panel ) flux uniformly towards individual poles ( indicated by red dotted lines ) . Illustration of the photoactivated regions ( right ) in prometaphase and metaphase spindles , highlighting that interpolar MTs overlap over a broader region in early mitosis , becoming focused at the spindle equator in metaphase . Scale bar , 30 sec . D . Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase . N ( number of cells , number of independent experiments ) : prometaphase ( 23 , 3 ) , metaphase ( 22 , 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Kymograph profiles (left) highlighting that the photoactivated regions in prometaphase (upper panel) split and move towards both poles, whereas metaphase spindles (lower panel) flux uniformly towards individual poles (indicated by red dotted lines). Illustration of the photoactivated regions (right) in prometaphase and metaphase spindles, highlighting that interpolar MTs overlap over a broader region in early mitosis, becoming focused at the spindle equator in metaphase. Scale bar, 30 sec. D. Percentage of cells with parallel and antiparallel flux movements during prometaphase and metaphase. N (number of cells, number of independent experiments): prometaphase (23, 3), metaphase (22, 3).