figf5 ( a ) At 16 h post - transfection with GST , GST - K - α2 or GST - K - α4 along with Flag - vFLIP ( top ) or Flag - cFLIPL ( bottom ) , HEK293T cells were used for GST pulldown , followed by immunoblotting ( IB ) with anti - Flag or anti - FLIP antibody . Whole - cell lysates ( WCLs ) were used for IB with anti - GST , anti - Flag or anti - FLIP antibody . ( b ) At 16 h post - transfection with V5 - Atg3 and Flag - vFLIP with increasing amounts of GST - K - α2 or GST - K - α4 , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - V5 antibody or for immunoprecipitation ( IP ) with anti - V5 antibody , followed by IB with anti - Flag antibody . WCLs were used for IB with anti - GST , anti - Flag or anti - V5 antibody . ( c ) At 12 - 16 h post - transfection with GST - Atg3 along with Flag - vFLIP ( first ) , Flag - cFLIPS ( second ) , or HA - LC3 ( third ) with increasing amounts of the K - α2 + K - α4 peptides ( top three panels ) or C - α2 + C - α4 peptides ( bottom panel ) , HEK293T cells were used for GST pulldown , followed by IB with anti - Flag or anti - HA antibodies . WCLs were used for IB with anti - GST and anti - Flag antibody . ( d ) At 12 - 16 h post - transfection with Flag - vFLIP , HA - LC3 and V5 - Atg3 , HEK293T cells were treated with increasing amounts of the K - α2 + K - α4 peptides and used for IP anti - V5 antibody , followed by IB with anti - Flag or anti - HA antibody . WCLs were used for IB with anti - V5 , anti - HA and anti - Flag antibody .

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 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 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

figf5(a) At 16 h post-transfection with GST, GST-K-α2 or GST-K-α4 along with Flag-vFLIP (top) or Flag-cFLIPL (bottom), HEK293T cells were used for GST pulldown, followed by immunoblotting (IB) with anti-Flag or anti-FLIP antibody. Whole-cell lysates (WCLs) were used for IB with anti-GST, anti-Flag or anti-FLIP antibody.(b) At 16 h post-transfection with V5-Atg3 and Flag-vFLIP with increasing amounts of GST-K-α2 or GST-K-α4, HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-V5 antibody or for immunoprecipitation (IP) with anti-V5 antibody, followed by IB with anti-Flag antibody. WCLs were used for IB with anti-GST, anti-Flag or anti-V5 antibody.(c) At 12-16 h post-transfection with GST-Atg3 along with Flag-vFLIP (first), Flag-cFLIPS (second), or HA-LC3 (third) with increasing amounts of the K-α2+K-α4 peptides (top three panels) or C-α2+C-α4 peptides (bottom panel), HEK293T cells were used for GST pulldown, followed by IB with anti-Flag or anti-HA antibodies. WCLs were used for IB with anti-GST and anti-Flag antibody.(d) At 12-16 h post-transfection with Flag-vFLIP, HA-LC3 and V5-Atg3, HEK293T cells were treated with increasing amounts of the K-α2+K-α4 peptides and used for IP anti-V5 antibody, followed by IB with anti-Flag or anti-HA antibody. WCLs were used for IB with anti-V5, anti-HA and anti-Flag antibody.


Figure 1 ( A - J ) Confocal images of different z sections ( 0 . 2 µm of thickness ) of a neuron transfected with GFP - LC3 ( A - D ) and all sections are flattened ( E ) . Higher magnification images of the area outlined in white are shown in the inserts ( A - D ) . Different sections of images of transfected neuron with GFP - LC3 ( green ) merged with DAPI ( blue ) . Scale bar , 20 µm . ( K - O ) Three - dimensional reconstruction from serial sections using IMARIS software , cell body and process ( K ) and higher magnification of the zoomed area outlined in white ( K - I ) . Reconstructed 3D image of the same neuron using IMARIS , which evaluates AV localization in the reconstructed 3D image ( M - O ) . Enlarged areas of neuron revealed the presence of AV in soma and neurites ( N - O ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1  (A-J) Confocal images of different z sections (0.2 µm of thickness) of a neuron transfected with GFP-LC3 (A-D) and all sections are flattened (E). Higher magnification images of the area outlined in white are shown in the inserts (A-D). Different sections of images of transfected neuron with GFP-LC3 (green) merged with DAPI (blue). Scale bar, 20 µm. (K-O) Three-dimensional reconstruction from serial sections using IMARIS software, cell body and process (K) and higher magnification of the zoomed area outlined in white (K-I). Reconstructed 3D image of the same neuron using IMARIS, which evaluates AV localization in the reconstructed 3D image (M-O). Enlarged areas of neuron revealed the presence of AV in soma and neurites (N-O).


Figure 2 ( B ) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV - infected microglia supernatant for 3 or 24 hr . Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV - infected microglia supernatant ( Sup ) exposure for 24 hr . * * * P < 0 . 001 , * P < 0 . 05 for n = 6 experiments . All values are mean ± SEM . AV counting using 3D model reconstruction for neurons exposed to different treatments . ( C ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV - infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV - infected microglia supernatant . The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres . Scale bar , 20 µm .

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 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

Figure 2(B) Total AV counting results show that a significant drop in the number of AV occurs in neurons exposed to SIV-infected microglia supernatant for 3 or 24 hr. Total number of AV are significantly increased after the pretreatment with rapamycin followed by SIV-infected microglia supernatant (Sup) exposure for 24 hr. *** P<0.001, * P<0.05 for n = 6 experiments. All values are mean±SEM. AV counting using 3D model reconstruction for neurons exposed to different treatments.(C) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The five panels display a typical sample image of a neuron under control conditions as well as 3 hr and 24 hr exposure to SIV-infected microglia supernatant or pretreated with rapamycin prior the exposure to SIV-infected microglia supernatant. The right hand panels show a snap shot of a 3D outline of the neuron with AV marked as green spheres. Scale bar, 20 µm.


Figure 3 ( A ) The number of AV in neurites decreased significantly in neurons exposed to the SIV - infected microglia supernatant for 3 or 24 hr , however ; pretreatment with rapamycin blocked this effect . * * * P < 0 . 001 , for n = 6 experiments . Scale bar , 20 µm . ( B ) The AV number is unchanged in neuronal soma after exposure to the SIV - infected microglia supernatant for 3 or 24 hr . ( C and D ) Both the total length of neuronal processes ( C ) and number of brunch points ( D ) are decreased after exposure to after SIV - infected microglia supernatant treatment . * * * P < 0 . 001 , for n = 6 experiments . All values are mean ± SEM .

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 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 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

Figure 3(A) The number of AV in neurites decreased significantly in neurons exposed to the SIV-infected microglia supernatant for 3 or 24 hr, however; pretreatment with rapamycin blocked this effect. *** P<0.001, for n = 6 experiments. Scale bar, 20 µm.(B) The AV number is unchanged in neuronal soma after exposure to the SIV-infected microglia supernatant for 3 or 24 hr.(C and D) Both the total length of neuronal processes (C) and number of brunch points (D) are decreased after exposure to after SIV-infected microglia supernatant treatment. *** P<0.001, for n = 6 experiments. All values are mean±SEM.


Figure 4 ( A ) The LC3 - II level is reduced after 3 or 24 hr exposure to SIV - infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin ( 2 µM ) . The protein level of elongation complex Atg12 - Atg5 is also reduced , and again the effect is blocked in the presence of rapamycin . The protein level of p62 is increased for similar conditions with SIV - infected microglia supernatant , and the increase is blocked in the presence of rapamycin . GAPDH protein was used in these experiments as the loading control . One representative experiment of n = 4 is shown . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 . ( B , C and D ) Ratios between LC3 - II , Atg12 - Atg5 , p62 , respectively , normalized to GAPDH . Data are reported as mean ± SEM ( n = 4 ) . * * * P < 0 . 001 .

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 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 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 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

Figure 4(A) The LC3-II level is reduced after 3 or 24 hr exposure to SIV-infected microglia supernatant and this decrease effect is blocked when is pretreated with rapamycin (2 µM). The protein level of elongation complex Atg12-Atg5 is also reduced, and again the effect is blocked in the presence of rapamycin. The protein level of p62 is increased for similar conditions with SIV-infected microglia supernatant, and the increase is blocked in the presence of rapamycin. GAPDH protein was used in these experiments as the loading control. One representative experiment of n = 4 is shown.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.(B, C and D) Ratios between LC3-II, Atg12-Atg5, p62, respectively, normalized to GAPDH. Data are reported as mean±SEM (n = 4). *** P<0.001.


Figure 5 ( A ) Total AV counting results show that a significant drop occurs in neurons exposed to TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , * * P < 0 . 01 for n = 6 experiments . All values are mean ± SEM . ( B and C ) Distribution of AV number in soma and neurites respectively . ( B ) The number of AV in neurites decreased significantly in neurons exposed to both TNF - α or NMDA for 3 or 24 hr . * * * P < 0 . 001 , for n = 6 experiments . ( C ) The AV number in soma is increased in neurons after exposure to TNF - α for 3 or 24 hr , however it remained unaffected in neurons after exposure to NMDA . * * P < 0 . 01 for n = 6 experiments . ( D ) Flattened images of multi - stack confocal optical slices of neurons transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . transfected with GFP - LC3 ( green ) to label AV , and DAPI ( blue ) to label the nucleus . The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF - α or NMDA at 25 ng / ml and 35 µM respectively . Scale bar , 20 µm .

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 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 O O O O O O O O O O O O O O O O O O O O O O O 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 5(A) Total AV counting results show that a significant drop occurs in neurons exposed to TNF-α or NMDA for 3 or 24 hr. *** P<0.001, ** P<0.01 for n = 6 experiments. All values are mean±SEM. (B and C) Distribution of AV number in soma and neurites respectively.(B) The number of AV in neurites decreased significantly in neurons exposed to both TNF-α or NMDA for 3 or 24 hr. *** P<0.001, for n = 6 experiments.(C) The AV number in soma is increased in neurons after exposure to TNF-α for 3 or 24 hr, however it remained unaffected in neurons after exposure to NMDA. ** P<0.01 for n = 6 experiments.(D) Flattened images of multi-stack confocal optical slices of neurons transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. transfected with GFP-LC3 (green) to label AV, and DAPI (blue) to label the nucleus. The four panels display a typical sample image of a neuron under 3 hr and 24 hr exposure to TNF-α or NMDA at 25 ng/ml and 35 µM respectively. Scale bar, 20 µm.


Figure 6 ( A ) . Neuronal survival tested by MTT . There are significant decreases of neuronal survival after exposure to SIV - infected microglia supernatant for 3 or 24 hr , which is significantly abrogated in the presence of rapamycin . Rapamycin alone has no effect . MK - 801 ( 100 µM ) also prevented the neurotoxicity induced by SIV - infected microglia supernatant . * * * P < 0 . 001 , for n = 6 experiments . ( B ) Neuronal survival was examined with MTT in cultures exposed to SIV - infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3 - MA ( 1 mM ) or Baf ( 100 nM ) . There is no significant difference between cells exposed to SIV - infected microglia supernatant and a pretreatment with 3 - MA followed by exposure to SIV - infected microglia supernatant . However , there is a significant decrease of neuronal survival exposed to SIV - infected microglia supernatant when cells are pretreated with Baf . The SIV - infected microglia supernatant from no infected monkey ( no Sup ) doesn ' t affect neuronal survival and there is no significant difference with control samples . All values are mean ± SEM .

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

Figure 6(A). Neuronal survival tested by MTT. There are significant decreases of neuronal survival after exposure to SIV-infected microglia supernatant for 3 or 24 hr, which is significantly abrogated in the presence of rapamycin. Rapamycin alone has no effect. MK-801 (100 µM) also prevented the neurotoxicity induced by SIV-infected microglia supernatant. *** P<0.001, for n = 6 experiments.(B) Neuronal survival was examined with MTT in cultures exposed to SIV-infected microglia supernatant for 24 hr and in the presence or absence of different drugs such as 3-MA (1 mM) or Baf (100 nM). There is no significant difference between cells exposed to SIV-infected microglia supernatant and a pretreatment with 3-MA followed by exposure to SIV-infected microglia supernatant. However, there is a significant decrease of neuronal survival exposed to SIV-infected microglia supernatant when cells are pretreated with Baf. The SIV-infected microglia supernatant from no infected monkey (no Sup) doesn't affect neuronal survival and there is no significant difference with control samples. All values are mean±SEM.


Figure 7 ( A ) Relative values ( RV ) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected ( UN ) monkeys , n = 7 for SIVE , n = 12 for SIV and n = 9 for UN , P < 0 . 05 . B ) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects ( NNDS ) , n = 8 for HAD and n = 10 for NNDS , P < 0 . 0001 . ( C ) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys , as well as those with HAD in brain sections from hippocampus . p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues . Higher magnification views are shown in green insets . Black arrows indicate p62 - or ubiquitin - immunoreactive structures within the neuronal cell body or proximal processes , and red arrowheads indicate a dot - like immunoreactivity in the neuropil . Scale bar , 20 µm .

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 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

Figure 7(A) Relative values (RV) of p62 mRNA level in frontal lobe of SIVE versus SIV and uninfected (UN) monkeys, n = 7 for SIVE, n = 12 for SIV and n = 9 for UN, P<0.05.B) RV of p62 mRNA level in frontal cortex of HAD versus non neurological diseased subjects (NNDS), n = 8 for HAD and n = 10 for NNDS, P<0.0001.(C) p62 and ubiquitin immunoreactivity on serial sections in SIVE monkeys, as well as those with HAD in brain sections from hippocampus. p62 has a diffuse immunoreactivity in UN monkeys and in human NNDS and a dot profile structures in SIVE and HAD brain tissues. Higher magnification views are shown in green insets. Black arrows indicate p62- or ubiquitin-immunoreactive structures within the neuronal cell body or proximal processes, and red arrowheads indicate a dot-like immunoreactivity in the neuropil. Scale bar, 20 µm.


Figure 1Maximum projection ( 2 z - slices ) of HeLa cells immunostained for F - actin ( Phalloidin ) , Tubulin , Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase . Scale bar - 10µm Quantification of F - actin ( Phalloidin ) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in ( A ) , showing the increase in F - actin around centrosomes at early anaphase . Mean actin metaphase = 1 ± 0 . 03996 , n = 92 , mean actin anaphase = 1 . 875 ± 0 . 08895 , n = 121 , Student t - test , * * * * p < 0 . 0001 ; Time - lapse sequence from a representative Hela cell expressing Lifeact - GFP transiting from metaphase to anaphase , showing accumulation of actin in the presumptive centrosomal region , red arrows . Images represent 4z - projection . Scale bar - 10µm . Time - lapse sequence of a representative HeLa cell expressing GFP - alpha - tubulin and RFP - Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules . Images represent 4 z - projection of movies taken every 1min . Bottom - higher magnification view of the centrosomes C1 and C2 from time - lapse above showing a transient increase in actin around the centrosomes in early anaphase . Scale bar = 10µm , 1µm in zoom . T = 0 is one frame before anaphase onset . Dotted circle shows centrosome position . Integrated fluorescence intensity of GFP - alpha - tubulin and RFP - Lifeact around the centrosomes for a population of cells exiting mitosis , showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time . N = 30 centrosomes ( 15 cells ) . T = 0 is one frame before anaphase onset . Graph shows mean + / - SD , values were normalized to intensity at T = 0 . Quantification and comparison of actin ( RFP - Lifeact ) and tubulin ( a - tubulin - GFP ) fluorescence intensity around the centrosomes at metaphase ( - 4 to - 1min ) , early anaphase ( 2 - 5min ) and late anaphase ( 7 - 10min ) for a population of cells . Mean actin intensity at Metaphase = 1 . 002 ± 0 . 0239 , n = 97 , Early Anaphase = 1 . 023 ± 0 . 03047 , n = 101 , Late anaphase = 0 . 9703 ± 0 . 05004 , n = 98 . Tubulin intensity at Metaphase = 1 . 008 ± 0 . 03356 , n = 106 , Early anaphase = 0 . 9748 ± 0 . 04124 , n = 116 , Late anaphase = 0 . 9276 ± 0 . 05382 , n = 101 , One - way ANOVA .

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 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 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 O O O O 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 1Maximum projection (2 z-slices) of HeLa cells immunostained for F-actin (Phalloidin), Tubulin, Pericentrin and DAPI at metaphase and early anaphase showing actin accumulation around centrosomes in early anaphase. Scale bar- 10µm Quantification of F-actin (Phalloidin) intensity around pericentrin positive centrosomes in HeLa cells immunostained as in (A), showing the increase in F-actin around centrosomes at early anaphase. Mean actin metaphase = 1 ± 0.03996, n=92, mean actin anaphase = 1.875 ± 0.08895, n=121, Student t-test, ****p<0.0001;Time-lapse sequence from a representative Hela cell expressing Lifeact-GFP transiting from metaphase to anaphase, showing accumulation of actin in the presumptive centrosomal region, red arrows. Images represent 4z-projection. Scale bar-10µm.Time-lapse sequence of a representative HeLa cell expressing GFP-alpha-tubulin and RFP-Lifeact transiting from metaphase to anaphase showing the changes in actin and microtubules. Images represent 4 z-projection of movies taken every 1min. Bottom - higher magnification view of the centrosomes C1 and C2 from time-lapse above showing a transient increase in actin around the centrosomes in early anaphase. Scale bar = 10µm, 1µm in zoom. T=0 is one frame before anaphase onset. Dotted circle shows centrosome position.Integrated fluorescence intensity of GFP-alpha-tubulin and RFP-Lifeact around the centrosomes for a population of cells exiting mitosis, showing actin accumulation around the centrosomes and a simultaneous decrease in tubulin over time. N=30 centrosomes (15 cells). T=0 is one frame before anaphase onset. Graph shows mean +/- SD, values were normalized to intensity at T=0. Quantification and comparison of actin (RFP-Lifeact) and tubulin (a-tubulin-GFP) fluorescence intensity around the centrosomes at metaphase (-4 to -1min), early anaphase (2-5min) and late anaphase (7-10min) for a population of cells. Mean actin intensity at Metaphase =1.002 ± 0.0239, n=97, Early Anaphase = 1.023 ± 0.03047, n=101, Late anaphase = 0.9703 ± 0.05004, n=98. Tubulin intensity at Metaphase = 1.008 ± 0.03356, n=106, Early anaphase = 0.9748 ± 0.04124, n=116, Late anaphase = 0.9276 ± 0.05382, n=101, One-way ANOVA.


Figure 2Stills from time - lapse of HeLa cells expressing GFP - alpha - tubulin and RFP - lifeact arrested at prometaphase with STLC ( t = 0 ) and forced to exit mitosis with Cdk1 inhibition ( Ro3306 ) imaged every 90 seconds . Scale bar = 10µm and for zoom = 4µm . n = 21 cells from 4 independent experiments . Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in ( A ) , showing a similar accumulation of actin as observed in bipolar divisions . Graph shows mean actin accumulation normalized to t = 0 , errors bars indicate standard deviation . HeLa cells expressing GFP - centrin1 arrested at prometaphase or forced mitotic exit ( Ro3306 5 ' ) and stained with phalloidin . Scale bar - 5µm and for zoom - 2µm . The level of actin around the centrosome in ( C ) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit . STLC arrest 1 ± 0 . 01317 , n = 210 , STLC + Ro - 5mins = 1 . 251 ± 0 . 02441 , n = 144 , Unpaired t - test with Welch correction , * * * * p < 0 . 0001 . Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor . Tubulin intensity decreses during forced mitotic exit . STLC - DMSO - 1 ± 0 . 02407 , n = 180 , STLC - RO - 3306 - 0 . 6516 ± 0 . 02337 , n = 136 . P < 0 . 0001 , Welch ' s t test .

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 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 O O O O O O O O O O O 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 2Stills from time-lapse of HeLa cells expressing GFP-alpha-tubulin and RFP-lifeact arrested at prometaphase with STLC (t=0) and forced to exit mitosis with Cdk1 inhibition (Ro3306) imaged every 90 seconds. Scale bar = 10µm and for zoom = 4µm. n = 21 cells from 4 independent experiments.Quantification of actin around centrosomes when cells are forced to exit mitosis using Cdk1 inhibition as in (A), showing a similar accumulation of actin as observed in bipolar divisions. Graph shows mean actin accumulation normalized to t=0, errors bars indicate standard deviation.HeLa cells expressing GFP-centrin1 arrested at prometaphase or forced mitotic exit (Ro3306 5') and stained with phalloidin. Scale bar- 5µm and for zoom-2µm.The level of actin around the centrosome in (C) was quantified and normalised relative to metaphase and shows an increase during forced mitotic exit. STLC arrest 1 ± 0.01317, n=210, STLC+Ro-5mins=1.251 ± 0.02441, n=144, Unpaired t-test with Welch correction, ****p<0.0001. Quantification of tubulin intensity around centrosomes during prometaphase arrested cells or cells forced to exit with Cdk1 inhibitor. Tubulin intensity decreses during forced mitotic exit. STLC-DMSO- 1 ± 0.02407, n=180, STLC-RO-3306- 0.6516 ± 0.02337, n=136. P<0.0001, Welch's t test.


Figure 3Maximum projection ( 2 - z slices ) view of Hela cells pre - treated with DMSO and 0 . 2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase . Scale bar - 10µm . Quantification of actin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the reduction in actin accumulation around the centrosomes following CK666 treatment . DMSO - metaphase - 1 ± 0 . 4325 , n = 54 , CK666 - metaphase - 0 . 936 ± 0 . 4604 , n = 43 , DMSO - anaphase - 1 . 8 ± 0 . 9736 , n = 76 , CK666 - anaphase - 1 . 087 ± 0 . 4597 , n = 73 ; one - way ANOVA , p < 0 . 0001 . Date pooled from 3 independent experiments . Z - projection of HeLa cells expressing GFP - centrin1 pre - treated with DMSO or 0 . 2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F - actin . Quantification of the level of actin around the centrosome from ( C ) , which shows the reduction in actin accumulation around centrosomes following CK666 pre - treatment . DMSO - STLC = 1 ± 0 . 02768 , n = 99 , CK666 STLC = 0 . 776 ± 0 . 02186 , n = 87 , DMSO - RO - 3306 = 1 . 339 ± 0 . 03048 , n = 127 , CK666 - RO - 3306 = 0 . 7699 ± 0 . 02246 , n = 70 , one - way ANOVA , p < 0 . 0001 . Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit . DMSO - metaphase - 1 ± 0 . 1968 , n = 54 , DMSO - anaphase - 0 . 867 ± 0 . 2345 , n = 78 , CK666 - metaphase - 0 . 7995 ± 0 . 1275 , n = 43 , CK666 - anaphase - 0 . 7407 ± 0 . 1859 , n = 74 ; one - way ANOVA , p < 0 . 0001 . Data pooled from 3 independent experiments . Error bars indicated standard deviation . Quantification of tubulin around centrosomes for cells treated with DMSO or 0 . 2mM CK666 , during monopolar exit , showing the failure to reduce tubulin density around centrosomes following CK666 treatment . Data from three independent experiments . Prometaphase arrest ( p . a . ) 1 ± 0 . 34 , n = 27 ; forced mitotic exit ( f . m . e ) 0 . 78 ± 0 . 30 , n = 26 ; p . a . plus CK666 0 . 49 ± 0 . 34 , n = 30 ; f . m . e plus CK666 0 . 92 ± 0 . 37 , n = 30 . One - way ANOVA with Sidak ' s multiple comparisons test .

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

Figure 3Maximum projection (2-z slices) view of Hela cells pre-treated with DMSO and 0.2mM CK666 for 15 mins during their mitotic exit showing that treatment with CK666 leads to reduced accumulation of actin around the centrosomes during anaphase. Scale bar-10µm. Quantification of actin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the reduction in actin accumulation around the centrosomes following CK666 treatment. DMSO-metaphase-1±0.4325, n=54, CK666-metaphase-0.936±0.4604, n=43, DMSO-anaphase-1.8±0.9736, n=76, CK666-anaphase-1.087±0.4597, n=73; one-way ANOVA, p<0.0001. Date pooled from 3 independent experiments.Z-projection of HeLa cells expressing GFP-centrin1 pre-treated with DMSO or 0.2mM CK666 during prometaphase arrest and forced mitotic exit and stained with phalloidin for F-actin.Quantification of the level of actin around the centrosome from (C), which shows the reduction in actin accumulation around centrosomes following CK666 pre-treatment. DMSO-STLC= 1 ± 0.02768, n=99, CK666 STLC= 0.776 ± 0.02186, n=87, DMSO-RO-3306= 1.339 ± 0.03048, n=127, CK666-RO-3306= 0.7699 ± 0.02246, n=70, one-way ANOVA, p<0.0001.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, showing the failure to reduce tubulin density around centrosomes following CK666 treatment during bipolar exit. DMSO-metaphase-1±0.1968, n=54, DMSO-anaphase-0.867±0.2345, n=78, CK666-metaphase-0.7995±0.1275, n=43, CK666-anaphase-0.7407±0.1859, n=74; one-way ANOVA, p<0.0001. Data pooled from 3 independent experiments. Error bars indicated standard deviation.Quantification of tubulin around centrosomes for cells treated with DMSO or 0.2mM CK666, during monopolar exit, showing the failure to reduce tubulin density around centrosomes following CK666 treatment. Data from three independent experiments. Prometaphase arrest (p.a.) 1±0.34 , n=27; forced mitotic exit (f.m.e) 0.78±0.30, n=26; p.a. plus CK666 0.49±0.34, n=30 ; f.m.e plus CK666 0.92±0.37, n=30. One-way ANOVA with Sidak's multiple comparisons test.


Figure 4Z - projection of HeLa cells expressing GFP - centrin1 immunostained for p34 - Arc ( sub - domain Arp2 / 3 ) following prometaphase arrest and forced mitotic exit for 5min . N = 3 independent experiments . Scale bar = 10µm and for zoom 2µm . Levels of p34 - Arc around the centrosome ( 3um area ) were quantified relative to metaphase and increase during forced mitotic exit . STLC arrested = 1 ± 0 . 01576 , n = 153 , STLC + RO - 3306 = 1 . 268 ± 0 . 01748 , n = 191 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation . Z - projection of centrosomes isolated from cells expressing GFP - centrin1 during prometaphase arrest and forced exit , which were immunostained with p34 - Arc . N = 2 independent experiments . Scale bar = 2µm . Quantification of p34 - Arc levels around the centrosome in ( C ) , relative to metaphase , showing its increase during forced exit . STLC arrested = 1 ± 0 . 00779 , n = 93 , STLC + RO - 3306 = 1 . 249 ± 0 . 02446 , n = 134 , * * * * p < 0 . 0001 , Unpaired t - test with Welch correction . Error bars represent standard deviation . Time - lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time . Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in ( E ) over an area of 2 μm , ( white circle ) , showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit . N = 2 isolations , 5 independent experiments , n = 16 STLC and n = 19 STLC RO . Color switch experiment using green - and red - labelled actin . Red actin was used first , followed by Capping protein , then green - labelled actin . The accumulation of green in the centre indicates that actin is nucleated at the centrosome . Scale bar = 10µm . Quantification of ( G ) over the time . N = 2 isolations , 2 independent experiments , n = 19 STLC and n = 14 STLC RO . Error bars represent standard deviation . Time - lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre - treated with DMSO or CK666 . Scale bar = 10µm . Quantification of ( I ) , showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre - treated with CK666 . N = 2 isolations , 2 independent experiments , n = 17 DMSO , 26 Ro CK666 24 STLC DMSO , 23 STLC CK666 . Error bars represent standard deviation .

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

Figure 4Z-projection of HeLa cells expressing GFP-centrin1 immunostained for p34-Arc (sub-domain Arp2/3) following prometaphase arrest and forced mitotic exit for 5min. N=3 independent experiments. Scale bar = 10µm and for zoom 2µm.Levels of p34-Arc around the centrosome (3um area) were quantified relative to metaphase and increase during forced mitotic exit. STLC arrested = 1 ± 0.01576, n=153, STLC+RO-3306= 1.268 ± 0.01748, n=191, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Z-projection of centrosomes isolated from cells expressing GFP-centrin1 during prometaphase arrest and forced exit, which were immunostained with p34-Arc. N=2 independent experiments. Scale bar = 2µm.Quantification of p34-Arc levels around the centrosome in (C), relative to metaphase, showing its increase during forced exit. STLC arrested = 1 ± 0.00779, n=93, STLC+RO-3306= 1.249 ± 0.02446, n=134, ****p<0.0001, Unpaired t-test with Welch correction. Error bars represent standard deviation.Time-lapse of in vitro assay on centrosomes isolated from prometaphase arrested and cells forced to exit mitosis showing centrosomal actin nucleation over time. Scale bar = 10µm Quantification of actin nucleation from isolated centrosomes as in (E) over an area of 2 μm, (white circle), showing an increase in actin nucleation around centrosomes isolated from cells undergoing forced mitotic exit. N=2 isolations, 5 independent experiments, n=16 STLC and n=19 STLC RO.Color switch experiment using green- and red-labelled actin. Red actin was used first, followed by Capping protein, then green-labelled actin. The accumulation of green in the centre indicates that actin is nucleated at the centrosome. Scale bar = 10µm. Quantification of (G) over the time. N=2 isolations, 2 independent experiments, n=19 STLC and n=14 STLC RO. Error bars represent standard deviation.Time-lapse showing cytoplasmic actin filament formation at centrosomes isolated from cells undergoing forced exit when pre-treated with DMSO or CK666. Scale bar = 10µm. Quantification of (I), showing that centrosomal actin nucleation fails when centrosomes isolated from cells undergoing exit are pre-treated with CK666. N=2 isolations, 2 independent experiments, n = 17 DMSO, 26 Ro CK666 24 STLC DMSO, 23 STLC CK666. Error bars represent standard deviation.


Figure 5Phos - tag Western blot of MCF7 - Her2 - 18 cells synchronised into distinct phases of the cell cycle showing the phospho - shift in WASH1 during mitosis . Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment .

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Figure 5Phos-tag Western blot of MCF7-Her2-18 cells synchronised into distinct phases of the cell cycle showing the phospho-shift in WASH1 during mitosis.Blue native PAGE Western blot showing shift in the size of WASH1 protein complex isolated from STLC treated prometaphase arrested cells when compared to cells forced to exit mitosis following Ro3306 treatment.


Figure 6Time - lapse of HeLa cells expressing GFP - alpha tubulin and RFP - lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition ( t = 0 ) . Scale bar = 5µm . Quantification of actin around the centrosome in ( A ) , showing the failure to accumulate actin around the centrosome in siWASH . N = 4 experiments . Error bars represent standard deviation . Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody , showing the reduction in WASH levels following treatment of cells with siRNA against WASH . Scale bar = 5µm and 2µm in zoom . Z - projection of HeLa cells expressing GFP - centrin1 treated with control siRNA or siRNA against WASH , arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin . Scale bar - 5µm Quantification of actin around the centrosome in ( F ) , showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH . siControl - STLC = 1 ± 0 . 0189 , n = 165 , siWASH - STLC = 0 . 9138 ± 0 . 0298 , n = 118 , siControl - STLC + RO - 3306 = 1 . 911 ± 0 . 06221 , n = 147 , siWASH - STLC + RO - 3306 = 1 . 103 ± 0 . 0301 , n = 140 . Error bars represent standard deviation . P < 0 . 0001 , one - way ANOVA . Time - lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre - treated with either no antibody or anti - WASH1 antibody for 1 hour . Scale bar - 10µm Quantification of in vitro assay from ( F ) , showing the reduction in actin nucleation around centrosomes when they are pre - treated with anti - WASH1 . Error bars represent standard deviation . 2 independent experiments .

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

Figure 6Time-lapse of HeLa cells expressing GFP-alpha tubulin and RFP-lifeact treated with control siRNA or siRNA against WASH1 for 48 hours arrested at prometaphase and forced to exit mitosis with Ro3306 addition (t=0). Scale bar = 5µm.Quantification of actin around the centrosome in (A), showing the failure to accumulate actin around the centrosome in siWASH. N = 4 experiments. Error bars represent standard deviation.Western blots of lysates from cells treated with siRNA control and siRNA against WASH1 probed with WASH1 antibody, showing the reduction in WASH levels following treatment of cells with siRNA against WASH. Scale bar = 5µm and 2µm in zoom.Z-projection of HeLa cells expressing GFP-centrin1 treated with control siRNA or siRNA against WASH, arrested at prometaphase or forced to exit mitosis and immunostained with phalloidin. Scale bar-5µm Quantification of actin around the centrosome in (F), showing the failure to accumulated actin around the centrosome during forced exit in cells treated with siRNA against WASH. siControl-STLC= 1 ± 0.0189, n=165, siWASH-STLC=0.9138 ± 0.0298, n=118, siControl-STLC+RO-3306=1.911 ± 0.06221, n=147, siWASH-STLC+RO-3306=1.103 ± 0.0301, n=140. Error bars represent standard deviation. P<0.0001, one-way ANOVA.Time-lapse from in vitro assay with centrosomes isolated from cells undergoing forced exit were pre-treated with either no antibody or anti-WASH1 antibody for 1 hour. Scale bar- 10µm Quantification of in vitro assay from (F), showing the reduction in actin nucleation around centrosomes when they are pre-treated with anti-WASH1. Error bars represent standard deviation. 2 independent experiments.


Figure 1HeLa cells overexpressing the HA - tagged MICU1 WT , MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10 μmRepresentative images of the 2mt - GCaMP6m 474 / 410 ratio of ShMICU1 HeLa stable cells expressing an empty vector ( ctrl ) or the MICU1 WT , MICU1 SD , and MICU1 SA . Scale bar 10 μmResting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA control ( plko ) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 5 independent experiments ; 55 - 67cells ) Representative kinetics ( E ) and analysis ( F ) of aequorin - based [ Ca2 + ] m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2 , 5 - di - tert - butylhydroquinone ( TBHQ ) in the absence of extracellular Ca2 + ( n = 3 independent experiments ) Representative kinetics ( G ) and analysis ( H ) of aequorin - based [ Ca2 + ] m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments ) Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR / Cas9 - mediated genome editing . The results for 12 of the 36 clones that were examined are shownResting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m , in MICU1 - KO cells generated using the CRISPR / Cas9 technique and transfected with the indicated constructs ( n = 3 independent experiments ; 30 - 56 cells ) Representative kinetics ( K ) and analysis ( L ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2 + ( n = 3 independent experiments . ) Representative kinetics ( M ) and analysis ( N ) of aequorin - based [ Ca2 + ] m measurements in intact MICU1 - KO cells generated using the CRISPR / Cas9 technique , transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA ( n = 3 independent experiments ) .

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 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 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 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 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 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 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

Figure 1HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μmRepresentative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μmResting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells)Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments)Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shownResting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells)Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.)Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).


Figure 2HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated ( S473 ) Akt ( p - Akt ) or HSP60 ( mitochondrial marker ) . Merged images are indicated ( merge ) . Scale bar 10 μmAnalysis of the number of cells , expressed as a percentage , showing obvious mitochondrial staining of activated ( S473 phosphorylated ) Akt ( p - Akt ) ( n = 3 independent experiments ; 350 - 355 cells ) Hek293T cells treated with vehicle or 1 μM rapamycin ( Rapa . ) for 4 h were fractionated into Cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell homogenateMouse livers treated with vehicle or 1 μM rapamycin ( Rapa . ) for 16 h by an intraperitoneal injection were fractionated into cytosol ( Cyt . ) or mitochondrial ( Mito . ) extracts and analyzed by western blotting . Phosphorylated ( S2468 ) mTOR ( p - mTOR ) was used to assess the rapamycin activity . E - cadherin : plasma membrane marker ; β - tubulin : cytosolic marker ; VDAC : mitochondrial marker ; Homo : cell homogenateMitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , and mitochondrial proteins with known localizations . Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture . OMM : outer mitochondrial membrane ; IMS : intermembrane spaceWestern blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of vehicle - or rapamycin ( Rapa . , 1 μM for 4 h ) - treated Hek293T cell mitochondria following carbonate extraction at pH 11 . 5 . Akt , phosphorylated ( S473 ) Akt ( p - Akt ) , MICU1 , the established integral membrane proteins TIM23 and MCU , and the soluble protein cytochrome c ( Cyt . c ) was analyzedHek293T cells were transfected with the GFP - tagged wild - type ( WT ) MICU1 and then treated with 1 μM rapamycin ( Rapa . ) alone for 4 h or in combination with the Akt inhibitor triciribine ( 10 μM ) . GFP - MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) and phosphorylated ( S473 ) Akt ( p - Akt ) antibodies by western blottingHek293T cells were transfected with either the GFP - tagged wild - type ( WT ) MICU1 or MICU1 S124A - GFP mutant . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blotting .

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 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 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 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 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 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

Figure 2HeLa cells treated with vehicle or 1 μM rapamycin for 4 h were stained for phosphorylated (S473) Akt (p-Akt) or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μmAnalysis of the number of cells, expressed as a percentage, showing obvious mitochondrial staining of activated (S473 phosphorylated) Akt (p-Akt) (n=3 independent experiments; 350-355 cells)Hek293T cells treated with vehicle or 1 μM rapamycin (Rapa.) for 4 h were fractionated into Cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenateMouse livers treated with vehicle or 1 μM rapamycin (Rapa.) for 16 h by an intraperitoneal injection were fractionated into cytosol (Cyt.) or mitochondrial (Mito.) extracts and analyzed by western blotting. Phosphorylated (S2468) mTOR (p-mTOR) was used to assess the rapamycin activity. E-cadherin: plasma membrane marker; β-tubulin: cytosolic marker; VDAC: mitochondrial marker; Homo: cell homogenateMitochondria isolated from Hek293T cells were subjected to the indicated treatments and analyzed by western blotting against Akt, phosphorylated (S473) Akt (p-Akt), and mitochondrial proteins with known localizations. Osmotic swelling through the removal of sucrose from the buffer was used to induce OMM rupture. OMM: outer mitochondrial membrane; IMS: intermembrane spaceWestern blot analysis of the supernatant (S) and insoluble pellet (P) fractions of vehicle- or rapamycin (Rapa., 1 μM for 4 h)-treated Hek293T cell mitochondria following carbonate extraction at pH 11.5. Akt, phosphorylated (S473) Akt (p-Akt), MICU1, the established integral membrane proteins TIM23 and MCU, and the soluble protein cytochrome c (Cyt. c) was analyzedHek293T cells were transfected with the GFP-tagged wild-type (WT) MICU1 and then treated with 1 μM rapamycin (Rapa.) alone for 4 h or in combination with the Akt inhibitor triciribine (10 μM). GFP-MICU1 immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) and phosphorylated (S473) Akt (p-Akt) antibodies by western blottingHek293T cells were transfected with either the GFP-tagged wild-type (WT) MICU1 or MICU1 S124A-GFP mutant. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blotting.


Figure 3Mitochondrial preparations of both Hek293T ( left ) and HeLa ( right ) cells transiently transfected with an HA - tagged constitutively active Akt T308D / S473D ( Akt D / D ) construct were analyzed by western blotting . GAPDH : cytosolic marker ; TIM23 : mitochondrial markerResting mitochondrial calcium levels in the control ( ctrl ) and Akt D / D - expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 44 - 49 cells ) Schematic model of the HA - tagged constitutively active Akt form targeted to the mitochondrial intermembrane space ( mt - Akt D / D ) . Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence . TOM20 was used as a mitochondrial marker . Merged images are indicated ( merge ) . Scale bar 10 μmHek293T cells were transfected with the indicated constructs . The GFP - tagged MICU1 was immunoprecipitated from the whole - cell lysate , and the precipitated proteins were immunoblotted with the HA and phosphorylated ( S473 ) Akt ( p - Akt ) antibodiesHek293T cells were transfected with the indicated constructs . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blottingResting mitochondrial calcium levels in control ( ctrl ) and mitochondrial - targeted Akt D / D ( mt - Akt D / D ) - expressing HeLa cells , evaluated through ratiometric imaging of the mitochondrial - targeted GCaMP6m ( n = 4 independent experiments ; 43 - 47 cells ) Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m ( n = 3 independent experiments ; 38 - 50 cells ) Representative kinetics ( H ) and analysis ( I ) of aequorin - based [ Ca2 + ] m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [ Ca2 + ] ( n = 3 independent experiments ) .

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Figure 3Mitochondrial preparations of both Hek293T (left) and HeLa (right) cells transiently transfected with an HA-tagged constitutively active Akt T308D/S473D (Akt D/D) construct were analyzed by western blotting. GAPDH: cytosolic marker; TIM23: mitochondrial markerResting mitochondrial calcium levels in the control (ctrl) and Akt D/D-expressing HeLa cells evaluated by ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 44-49 cells)Schematic model of the HA-tagged constitutively active Akt form targeted to the mitochondrial intermembrane space (mt-Akt D/D). Both the expression and the mitochondrial localization of the chimera were assessed by immunofluorescence. TOM20 was used as a mitochondrial marker. Merged images are indicated (merge). Scale bar 10 μmHek293T cells were transfected with the indicated constructs. The GFP-tagged MICU1 was immunoprecipitated from the whole-cell lysate, and the precipitated proteins were immunoblotted with the HA and phosphorylated (S473) Akt (p-Akt) antibodiesHek293T cells were transfected with the indicated constructs. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blottingResting mitochondrial calcium levels in control (ctrl) and mitochondrial-targeted Akt D/D (mt-Akt D/D)-expressing HeLa cells, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m (n=4 independent experiments; 43-47 cells)Resting mitochondrial calcium levels in ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m (n=3 independent experiments; 38-50 cells)Representative kinetics (H) and analysis (I) of aequorin-based [Ca2+]m measurements in permeabilized ShRNA MICU1 HeLa stable cells transfected with the indicated constructs and challenged with 400 nM buffered [Ca2+] (n=3 independent experiments).


Figure 4Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . pre - MICU1 : precursor form of MICU1 ; m - MICU1 : mature form of MICU1Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs . Where indicated , the cells were treated with 5 μM MG132 for 4 h . pre : precursor ; int : intermediate ; m : mature formWestern blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs , following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . The cells were treated with 5 μM MG132 for 4 h before fractionationWestern blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT - HA or MICU1 SD - HA , together with MCU - flag and EMRE constructs , following carbonate extraction at pH 7 . 4 , pH 10 . 5 , pH 11 . 5 or pH 12 . 5 . The cells were treated with 5 μM MG132 for 4 h before fractionationShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) , both WT and mutants , is represented relative to the amount at time 0 . ( n = 3 independent experiments ) ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs . Cells were harvested , and total protein was extracted and subjected to western blotting analysis with the indicated antibodies . Immunoblotting was performed under reducing ( with the presence of DTT ) or non - reducing ( non - red ) conditions ( without DTT ) . Cells were treated with 5 μM MG132 for 4 h before lysisQuantification of HA levels in non - reducing condition showed in ( E ) . ( n = 3 independent experiments ) Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs ( n = 3 independent experiments ; 46 - 54 cells ) .

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Figure 4Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. pre-MICU1: precursor form of MICU1; m-MICU1: mature form of MICU1Western blot analysis of ShRNA MICU1 HeLa stable cells transfected with the indicated constructs. Where indicated, the cells were treated with 5 μM MG132 for 4 h. pre: precursor; int: intermediate; m: mature formWestern blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with the indicated constructs, following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. The cells were treated with 5 μM MG132 for 4 h before fractionationWestern blot analysis of the supernatant (S) and insoluble pellet (P) fractions of ShRNA MICU1 HeLa cell mitochondria transfected with MICU1 WT-HA or MICU1 SD-HA, together with MCU-flag and EMRE constructs, following carbonate extraction at pH 7.4, pH 10.5, pH 11.5 or pH 12.5. The cells were treated with 5 μM MG132 for 4 h before fractionationShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1), both WT and mutants, is represented relative to the amount at time 0. (n=3 independent experiments)ShRNA MICU1 HeLa stable cells were transfected with the indicated constructs. Cells were harvested, and total protein was extracted and subjected to western blotting analysis with the indicated antibodies. Immunoblotting was performed under reducing (with the presence of DTT) or non-reducing (non-red) conditions (without DTT). Cells were treated with 5 μM MG132 for 4 h before lysisQuantification of HA levels in non-reducing condition showed in (E). (n=3 independent experiments)Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=3 independent experiments; 46-54 cells).


Figure 5Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and / or IMMP2L . int : intermediate ; m : mature formWestern blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and / or IMMP2L . int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before lysisWestern blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markersWestern blot analysis of the supernatant ( S ) and insoluble pellet ( P ) fractions of control or IMMP1L - silenced HeLa cell mitochondria transfected with HA - tagged MICU1 WT following carbonate extraction at pH 10 . The established integral membrane protein TIM23 and the soluble protein cytochrome c ( Cyt . c ) were analyzed as markers . pre : precursor ; int : intermediate ; m : mature form . Cells were treated with 5 μM MG132 for 4 h before lysisControl or IMMP1L - silenced HeLa cells were transfected with HA - tagged MICU1 WT . Cells were incubated with 50 μg / ml cycloheximide ( CHX ) for the indicated times , collected and subjected to western blotting analysis as indicated . In the graph , the amount of HA ( MICU1 ) is represented relative to the amount at time 0 . ( n = 3 independent experiments ) Representative kinetics ( F ) and analysis ( G ) of aequorin - based [ Ca2 + ] m measurements in control or IMMP1L - silenced HeLa cells challenged with 10 μM cyclopiazonic acid ( CPA ) in the presence of 100 μM EGTA ( n = 3 independent experiments ) Resting mitochondrial calcium levels , evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m , in control or IMMP1L - silenced HeLa cells ( n = 3 independent experiments ; 37 - 55 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 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 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 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 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

Figure 5Western blot analysis of endogenous MICU1 levels in HeLa cells upon silencing of IMMP1L and/or IMMP2L. int: intermediate; m: mature formWestern blot analysis of HeLa cells transfected with the indicated constructs and silenced with IMMP1L and/or IMMP2L. int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysisWestern blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markersWestern blot analysis of the supernatant (S) and insoluble pellet (P) fractions of control or IMMP1L-silenced HeLa cell mitochondria transfected with HA-tagged MICU1 WT following carbonate extraction at pH 10. The established integral membrane protein TIM23 and the soluble protein cytochrome c (Cyt. c) were analyzed as markers. pre: precursor; int: intermediate; m: mature form. Cells were treated with 5 μM MG132 for 4 h before lysisControl or IMMP1L-silenced HeLa cells were transfected with HA-tagged MICU1 WT. Cells were incubated with 50 μg/ml cycloheximide (CHX) for the indicated times, collected and subjected to western blotting analysis as indicated. In the graph, the amount of HA (MICU1) is represented relative to the amount at time 0. (n=3 independent experiments)Representative kinetics (F) and analysis (G) of aequorin-based [Ca2+]m measurements in control or IMMP1L-silenced HeLa cells challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments)Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in control or IMMP1L-silenced HeLa cells (n=3 independent experiments; 37-55 cells).


Figure 6Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activityLung cancer cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS ( phospho - Akt substrate ) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activityMelanoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activityGlioblastoma cells were transfected with the GFP - tagged WT MICU1 . MICU1 - GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS ( phospho - Akt substrate ) antibody by western blottingNumber of soft agar colonies ( n = 3 independent experiments formed by Rat2 cells stably expressing the indicated constructsxenograft tumor volumes ( H ) and tumor weights at day 21 ( n = 8 mice for each group ) ( I ) formed by Rat2 cells stably expressing the indicated constructsRepresentative images ( J ) and analysis ( K ) of MitoSOX - based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) ( n = 3 independent experiments ) . Scale bar 10 μm . f . a . u . : fluorescence arbitrary unitShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D ( SD ) or MICU1 S124A ( SA ) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated . l . e . : long exposure .

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Figure 6Western blot analysis of MICU1 and MICU2 levels in lung cancer cells with low or high Akt activityLung cancer cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with a PAS (phospho-Akt substrate) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in melanoma cells with low or high Akt activityMelanoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with a GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blottingWestern blot analysis of MICU1 and MICU2 levels in glioblastoma cells with low or high Akt activityGlioblastoma cells were transfected with the GFP-tagged WT MICU1. MICU1-GFP immunocomplexes were precipitated with GFP antibody and analyzed with PAS (phospho-Akt substrate) antibody by western blottingNumber of soft agar colonies (n=3 independent experiments formed by Rat2 cells stably expressing the indicated constructsxenograft tumor volumes (H) and tumor weights at day 21 (n=8 mice for each group) (I) formed by Rat2 cells stably expressing the indicated constructsRepresentative images (J) and analysis (K) of MitoSOX-based ROS measurements in ShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) (n=3 independent experiments). Scale bar 10 μm. f.a.u.: fluorescence arbitrary unitShRNA MICU1 HeLa stable cells stably expressing either MICU1 S124D (SD) or MICU1 S124A (SA) were treated with 500 μM H2O2 alone or in combination with the ROS scavenger Tempol and analyzed by western blotting as indicated. l. e.: long exposure.


Figure 1B Plot of the whole - cell current ( Vhold - 65 mV ) in control conditions ( ctrl ) and after application of NASPM ( NSP , p < 0 . 0001 , n = 18 ) .

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Figure 1B Plot of the whole-cell current (Vhold -65 mV) in control conditions (ctrl) and after application of NASPM (NSP, p<0.0001, n=18).


Figure 2B Current - voltage relationship of the tonic Ba2 + - sensitive ( 100 µM ) outward GIRK current demonstrating reversal near expected EK and inward rectification ( n = 6 biological replicates ) .

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Figure 2B Current-voltage relationship of the tonic Ba2+-sensitive (100 µM) outward GIRK current demonstrating reversal near expected EK and inward rectification (n=6 biological replicates).


Figure 3A With GDPβS - containing internal solution , noradrenaline - induced GluD1R current ( INA ) was diminished by > 20 minutes post - dialysis , shown in a representative trace . C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl - , GDPβS - Li3 - , and GTP - containing internal solution , displaying no significant difference between the groups ( p = 0 . 46 , n = 9 , 13 , 8 biological replicates respectively ) .

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Figure 3A With GDPβS-containing internal solution, noradrenaline-induced GluD1R current (INA) was diminished by >20 minutes post-dialysis, shown in a representative trace.C Plot of the magnitude of tonic GluD1R current measured after dialysis with GTP + LiCl-, GDPβS-Li3-, and GTP-containing internal solution, displaying no significant difference between the groups (p=0.46, n=9, 13, 8 biological replicates respectively).


Figure 4B - C Plot of the amplitude of the α1 - AR - EPSC in control conditions ( ctrl ) and in glycine ( 1 mM , p = 0 . 43 , n = 10 biological replicates , B ) , or D - serine ( 1 mM , p = 0 . 13 , n = 9 biological replicates , C ) . E Glycine had no significant effect on the amplitude of the α1 - AR - EPSC ( arrow ) or whole - cell current , shown in a representative trace .

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Figure 4B-C Plot of the amplitude of the α1-AR-EPSC in control conditions (ctrl) and in glycine (1 mM, p=0.43, n=10 biological replicates, B), or D-serine (1 mM, p=0.13, n=9 biological replicates, C).E Glycine had no significant effect on the amplitude of the α1-AR-EPSC (arrow) or whole-cell current, shown in a representative trace.


Figure 5A Increasing extracellular calcium to 4 . 8 mM reduced the amplitude of the α1 - AR - EPSC , shown in a time - course plot ( n = 18 and 26 biological replicates for 2 . 4 and 4 . 8 mM respectively ) . F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions ( 1 . 2 mM ) , 2 . 4 mM and 4 . 8 mM calcium . The magnitude of tonic GluD1R current in 2 . 4 and 4 . 8 mM calcium was reduced relative to control conditions ( 1 . 2 mM : n = 18 ; 2 . 4 mM : p = 0 . 03 , n = 17 ; 4 . 8 mM : p = 0 . 005 , n = 22 biological replicates ) . J Elevating extracellular calcium to 4 . 8 mM reduces the change in whole - cell current to NASPM and accelerates recovery of the α1 - AR - EPSC from NASPM - block in control conditions ( 1 . 2 mM extracellular calcium ) , shown in a representative whole - cell voltage - clamp recording .

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Figure 5A Increasing extracellular calcium to 4.8 mM reduced the amplitude of the α1-AR-EPSC, shown in a time-course plot (n=18 and 26 biological replicates for 2.4 and 4.8 mM respectively).F Plot of the magnitude of tonic GluD1R current measured by application of NASPM in control conditions (1.2 mM), 2.4 mM and 4.8 mM calcium. The magnitude of tonic GluD1R current in 2.4 and 4.8 mM calcium was reduced relative to control conditions (1.2 mM: n=18; 2.4 mM: p=0.03, n=17; 4.8 mM: p=0.005, n=22 biological replicates).J Elevating extracellular calcium to 4.8 mM reduces the change in whole-cell current to NASPM and accelerates recovery of the α1-AR-EPSC from NASPM-block in control conditions (1.2 mM extracellular calcium), shown in a representative whole-cell voltage-clamp recording.


Figure 6A Representative traces of whole - cell current clamp recordings of membrane potential and AP firing evoked by current injection ( 1 . 5 s ) demonstrating hyperpolarization by NASPM . Dashed line is at - 80 mV . C Plot of the membrane potential ( Vm ) versus injected current relative to rheobase ( rb ) in control and NASPM , demonstrating that NASPM produced a hyperpolarization at subthreshold potentials ( n = 10 biological replicates ) .

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Figure 6A Representative traces of whole-cell current clamp recordings of membrane potential and AP firing evoked by current injection (1.5 s) demonstrating hyperpolarization by NASPM. Dashed line is at -80 mV.C Plot of the membrane potential (Vm) versus injected current relative to rheobase (rb) in control and NASPM, demonstrating that NASPM produced a hyperpolarization at subthreshold potentials (n=10 biological replicates).


Figure 1 ( A ) The T cell clone 20 - 4 / A4 recognizes an epitope derived from the NeoR protein in the context of HLA ‐ DP3 . MHC class II expression on RCC1 . 24 ‐ NeoR cells following IFN ‐ γ treatment confers recognition by 20 - 4 / A4 . This recognition is almost completely blocked by the HLA ‐ DP ‐ specific antibody B7 / 21 . EBV ‐ B cells transfected with pINCO ‐ NeoR are able to present endogenous NeoR on MHC class II . As exemplified by the cell lines EBV ‐ B1 . 11 ( HLA ‐ DP3 ) and EBV ‐ B1 . 25 ( HLA ‐ DP4 ) , recognition by 20 - 4 / A4 is dependent on HLA ‐ DP3 expression . Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216 - 229 served as controls . ( B ) Cross - presentation experiments to exclude release of NeoR and re ‐ uptake as exogenous protein . EBV ‐ B1 . 25 cells transfected with the neoR expression plasmid pINCO ‐ NeoR , were co ‐ cultured for 24 h with the same number of untransfected EBV ‐ B1 . 11 cells , or vice versa . Then , T cells were added and co ‐ cultured with the cells for additional 24 h . To test for NeoR release by neoR ‐ transfected EBV ‐ B1 . 11 cells , total supernatant ( 200 μl ) of 1 × 105 EBV ‐ B1 . 11 cells was harvested 48 h post transfection by centrifugation , and was used as culture media for 1 × 105 untransfected EBV ‐ B1 . 11 cells . After 48 h , 1 × 105 20 - 4 / A4 T cells were added and GM ‐ CSF concentration determined after additional 24 h .

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Figure 1(A) The T cell clone 20-4/A4 recognizes an epitope derived from the NeoR protein in the context of HLA‐DP3. MHC class II expression on RCC1.24‐NeoR cells following IFN‐γ treatment confers recognition by 20-4/A4. This recognition is almost completely blocked by the HLA‐DP‐specific antibody B7/21. EBV‐B cells transfected with pINCO‐NeoR are able to present endogenous NeoR on MHC class II. As exemplified by the cell lines EBV‐B1.11 (HLA‐DP3) and EBV‐B1.25 (HLA‐DP4), recognition by 20-4/A4 is dependent on HLA‐DP3 expression. Bacterially expressed NeoR protein and a synthetic peptide encompassing amino acids NeoR216-229 served as controls.(B) Cross-presentation experiments to exclude release of NeoR and re‐uptake as exogenous protein. EBV‐B1.25 cells transfected with the neoR expression plasmid pINCO‐NeoR, were co‐cultured for 24 h with the same number of untransfected EBV‐B1.11 cells, or vice versa. Then, T cells were added and co‐cultured with the cells for additional 24 h. To test for NeoR release by neoR‐transfected EBV‐B1.11 cells, total supernatant (200 μl) of 1×105 EBV‐B1.11 cells was harvested 48 h post transfection by centrifugation, and was used as culture media for 1×105 untransfected EBV‐B1.11 cells. After 48 h, 1×105 20-4/A4 T cells were added and GM‐CSF concentration determined after additional 24 h.


Figure 2 ( A ) , pINCO ‐ NeoR transfected EBV ‐ B1 . 11 cells ( B ) , and as control pINCO ‐ Tyrosinase ‐ transfected EBV ‐ B1 . 11 cells ( C ) , were either left untreated or treated with 5 μM lactacystin or 50 μM AAF ‐ CMK for 20 h . Following fixation with 0 . 5 % paraformaldehyde , cells were incubated with T cells , and 24 h later GM ‐ CSF release was determined by ELISA . ( D ) FACS analysis of EBV ‐ B1 . 11 cells ( open histograms ) and EBV ‐ B1 . 11 cells stably transfected with the ICP47 gene ( shaded histograms ) . Inhibition of TAP by ICP47 down ‐ regulates HLA class I but not HLA ‐ DP expression . An isotype ‐ matched antibody was used as control ( thin ‐ lined histogram ) . EBV ‐ B1 . 11 ‐ ICP47 cells were transfected with pINCO ‐ NeoR ( E ) , pINCO ‐ Tyrosinase ( F ) , or mock transfected ( pINCO ) and MHC ‐ restricted presentation monitored with T cells .

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Figure 2(A), pINCO‐NeoR transfected EBV‐B1.11 cells (B), and as control pINCO‐Tyrosinase‐transfected EBV‐B1.11 cells (C), were either left untreated or treated with 5 μM lactacystin or 50 μM AAF‐CMK for 20 h. Following fixation with 0.5% paraformaldehyde, cells were incubated with T cells, and 24 h later GM‐CSF release was determined by ELISA.(D) FACS analysis of EBV‐B1.11 cells (open histograms) and EBV‐B1.11 cells stably transfected with the ICP47 gene (shaded histograms). Inhibition of TAP by ICP47 down‐regulates HLA class I but not HLA‐DP expression. An isotype‐matched antibody was used as control (thin‐lined histogram). EBV‐B1.11‐ICP47 cells were transfected with pINCO‐NeoR (E), pINCO‐Tyrosinase (F), or mock transfected (pINCO) and MHC‐restricted presentation monitored with T cells.


Figure 3 ( A ) RCC1 . 24 ‐ NeoR incubated with IFN ‐ γ were either left untreated ( w / o ) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing . Subsequently cells were fixed with 0 . 5 % paraformaldehyde and tested with 20 - 4 / A4 in a GM ‐ CSF release assay . EBV ‐ B1 . 11 cells transfected with pINCO ‐ NeoR ( B ) or pINCO ‐ Tyrosinase ( C ) were tested likewise . Both substances abrogated recognition of neoR ‐ transfected cells , while HLA ‐ A2 ‐ restricted presentation of the tyrosinase peptide was not affected ( D ) ) EBV ‐ B1 . 11 cells were incubated with 200 ng / ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in ( A ) . Presentation of exogenous NeoR on HLA ‐ DP3 is blocked by both substances as well , indicating that NeoR presentation is dependent on lysosomal processing .

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Figure 3(A) RCC1.24‐NeoR incubated with IFN‐γ were either left untreated (w/o) or treated with chloroquine or leupeptin for 24 h to block lysosomal processing. Subsequently cells were fixed with 0.5% paraformaldehyde and tested with 20-4/A4 in a GM‐CSF release assay. EBV‐B1.11 cells transfected with pINCO‐NeoR (B) or pINCO‐Tyrosinase (C) were tested likewise. Both substances abrogated recognition of neoR‐transfected cells, while HLA‐A2‐restricted presentation of the tyrosinase peptide was not affected(D) ) EBV‐B1.11 cells were incubated with 200 ng/ml of recombinant NeoR protein and treated with leupeptin or chloroquine as described in (A). Presentation of exogenous NeoR on HLA‐DP3 is blocked by both substances as well, indicating that NeoR presentation is dependent on lysosomal processing.


Figure 4 ( A ) 293T cells transfected with pINCO ‐ NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot . In addition , supernatant of NeoR ‐ pINCO transfected 293T cells ( 96 h post transfection ) was included to test for release of NeoR into the culture supernatant . At 6 h post transfection , NeoR protein becomes detectable in the cytoplasm , while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal / lysosomal fraction . For further characterization of the various fractions , blots were stripped and re ‐ probed with antibodies specific for the cytoplasmic protein LDH , the nuclear protein c ‐ myc , early endosomes ( Rab ‐ 5 ) , late endosomes and lysosomes ( Lamp ‐ 1 ) and mitochondria ( cytochrome c ) , which co ‐ purify with endosomes / lysosomes . ( B ) Endosomal / lysosomal fractions were prepared from pINCO ‐ NeoR ‐ transfected cells 96 h post transfection as in ( A ) . To prevent lysosomal degradation of NeoR , half of the cells were treated with leupeptin for the last 48 h . These fractions were separated on a 27 % Percoll gradient and eight different fractions collected . Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp ‐ 1 , Rab ‐ 5 and cytochrome c . Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal / lysosomal fractions 7 and 8 . ( C ) To determine where NeoR gains access to the vacuolar system , 293T cells were transfected with pINCO ‐ NeoR , treated with leupeptin to prevent NeoR degradation , and the endosomal / lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation . Starting at 16 h post transfection , NeoR becomes detectable in the Rab ‐ 5 ‐ positive fraction 5 , and then spreads into late endosomal / lysosomal fractions . At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA .

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Figure 4(A) 293T cells transfected with pINCO‐NeoR were harvested 6 and 96 h post transfection and NeoR localization within various cell fractions determined by Western blot. In addition, supernatant of NeoR‐pINCO transfected 293T cells (96 h post transfection) was included to test for release of NeoR into the culture supernatant. At 6 h post transfection, NeoR protein becomes detectable in the cytoplasm, while at 96 h post transfection a substantial amount of NeoR protein is also localized in the endosomal/lysosomal fraction. For further characterization of the various fractions, blots were stripped and re‐probed with antibodies specific for the cytoplasmic protein LDH, the nuclear protein c‐myc, early endosomes (Rab‐5), late endosomes and lysosomes (Lamp‐1) and mitochondria (cytochrome c), which co‐purify with endosomes/lysosomes.(B) Endosomal/lysosomal fractions were prepared from pINCO‐NeoR‐transfected cells 96 h post transfection as in (A). To prevent lysosomal degradation of NeoR, half of the cells were treated with leupeptin for the last 48 h. These fractions were separated on a 27% Percoll gradient and eight different fractions collected. Successful separation into fractions with increasing density was verified by Western blot analysis with antibodies directed against Lamp‐1, Rab‐5 and cytochrome c. Inhibition of NeoR degradation by leupeptin causes accumulation of NeoR protein in the late endosomal/lysosomal fractions 7 and 8.(C) To determine where NeoR gains access to the vacuolar system, 293T cells were transfected with pINCO‐NeoR, treated with leupeptin to prevent NeoR degradation, and the endosomal/lysosomal fractions prepared at various time points post transfection and separated by Percoll gradient centrifugation. Starting at 16 h post transfection, NeoR becomes detectable in the Rab‐5‐positive fraction 5, and then spreads into late endosomal/lysosomal fractions. At 26 h post transfection an almost identical NeoR distribution pattern is observed in cells treated with or without BfA.


Figure 5 ( A ) RCC were transfected with the NeoR ‐ GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence . As compared to untreated cells , cells treated with 3 ‐ MA show a much brighter UV fluorescence . ( B ) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR ‐ GFP fusion construct . Compared to untreated cells , cells treated for 24 h with 7 . 5 mM 3 ‐ MA show an about threefold increase in mean fluorescence intensity . ( C ) Western blot analysis of neoR ‐ transfected cells . Treatment of cells with 7 . 5 mM 3 ‐ MA causes a dramatic increase in NeoR protein levels in only 12 h . The ER ‐ resident protein BiP was used as gel loading control . ( D ) This increase in total NeoR protein in 3 ‐ MA ‐ treated cells is associated with an increase in cytoplasmic , and a decrease in endosomal / lysosomal NeoR protein levels . ( E ) Western blot analysis of whole cell lysates prepared from neoR ‐ transfected cells . Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels . Simultaneous treatment of cells with 3 ‐ MA prevents NeoR degradation . BiP was again used as a gel loading control .

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Figure 5(A) RCC were transfected with the NeoR‐GFP expression construct and subcellular localization of the fusion protein monitored by UV fluorescence. As compared to untreated cells, cells treated with 3‐MA show a much brighter UV fluorescence.(B) Semiquantitative analysis of fluorescence intensity of cells transfected with the NeoR‐GFP fusion construct. Compared to untreated cells, cells treated for 24 h with 7.5 mM 3‐MA show an about threefold increase in mean fluorescence intensity.(C) Western blot analysis of neoR‐transfected cells. Treatment of cells with 7.5 mM 3‐MA causes a dramatic increase in NeoR protein levels in only 12 h. The ER‐resident protein BiP was used as gel loading control.(D) This increase in total NeoR protein in 3‐MA‐treated cells is associated with an increase in cytoplasmic, and a decrease in endosomal/lysosomal NeoR protein levels.(E) Western blot analysis of whole cell lysates prepared from neoR‐transfected cells. Inhibition of protein synthesis by cycloheximide treatment leads to a rapid decrease in NeoR protein levels. Simultaneous treatment of cells with 3‐MA prevents NeoR degradation. BiP was again used as a gel loading control.