D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . By 48 h infection , cells were treated with MG132 or DMSO for 6 h . Cell lysates were then analyzed by Western blot analysis with the indicated antibodies .

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D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. By 48 h infection, cells were treated with MG132 or DMSO for 6 h. Cell lysates were then analyzed by Western blot analysis with the indicated antibodies.


E . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , total RNA was subjected to real - time RT - PCR analysis . Data shown are mean ± SD ( n = 3 ; * * P < 0 . 01 , two - tailed t - test ) .

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E. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, total RNA was subjected to real-time RT-PCR analysis. Data shown are mean ± SD (n = 3; **P < 0.01, two-tailed t-test).


A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , acidification of the culture medium was evaluated by visually inspecting the color of the medium . Yellow medium indicates the presence of more lactate .

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A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, acidification of the culture medium was evaluated by visually inspecting the color of the medium. Yellow medium indicates the presence of more lactate.


B . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF , control shRNA , or lncRNA - MIF shRNA as indicated . Forty - eight hours after infection , cell lysates were analyzed by Western blotting with the indicated antibodies .

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B. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF, control shRNA, or lncRNA-MIF shRNA as indicated. Forty-eight hours after infection, cell lysates were analyzed by Western blotting with the indicated antibodies.


C . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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C. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


D . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or negative control mimics ( NC mimics ) as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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D. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or negative control mimics (NC mimics) as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


E . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , levels of lactate in the culture medium were measured and normalized to cell number . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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E. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, levels of lactate in the culture medium were measured and normalized to cell number. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


F . HeLa cells expressing either lncRNA - MIF or control shRNA were transfected with miR - 586 inhibitor or NC inhibitor as indicated . Twenty - four hours after transfection , intracellular glucose levels were measured and normalized based on protein concentration . Data shown are mean ± SD ( n = 3 ; * P < 0 . 05 , two - tailed t - test ) .

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F. HeLa cells expressing either lncRNA-MIF or control shRNA were transfected with miR-586 inhibitor or NC inhibitor as indicated. Twenty-four hours after transfection, intracellular glucose levels were measured and normalized based on protein concentration. Data shown are mean ± SD (n = 3; *P < 0.05, two-tailed t-test).


A . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

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A. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


B . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , growth curves were measured for the indicated periods of time . Data shown are mean ± SD ( n = 3 ) .

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B. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, growth curves were measured for the indicated periods of time. Data shown are mean ± SD (n = 3).


C . HeLa cells were infected with lentiviruses expressing control RNA , lncRNA - MIF or lncRNA - MIF - AS as indicated . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle .

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C. HeLa cells were infected with lentiviruses expressing control RNA, lncRNA-MIF or lncRNA-MIF-AS as indicated. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


D . HeLa cells were infected with lentiviruses expressing control shRNA , lncRNA - MIF shRNA - 1 , - 2 or - 3 . Forty - eight hours after infection , cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle .

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D. HeLa cells were infected with lentiviruses expressing control shRNA, lncRNA-MIF shRNA-1, -2 or -3. Forty-eight hours after infection, cells were stained with PI and analyzed by flow cytometry to identify phases of cell cycle.


E . The percentage numbers of cells ( Fig 7C ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

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E. The percentage numbers of cells (Fig 7C) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


F . The percentage numbers of cells ( Fig 7D ) in G1 , S or G2 / M phase were analyzed by FlowJo 7 . 6 software . Data shown are mean ± SD ( n = 3 ) .

F. The percentage numbers of cells (Fig 7D) in G1, S or G2/M phase were analyzed by FlowJo 7.6 software. Data shown are mean ± SD (n = 3).


A . HeLa cells expressing either lncRNA - MIF or control RNA were transfected with miR - 586 mimics or NC mimics as indicated . Colonies were stained with crystal violet and counted after 14 days incubation .

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A. HeLa cells expressing either lncRNA-MIF or control RNA were transfected with miR-586 mimics or NC mimics as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


B . HeLa cells expressing either lncRNA - MIF shRNA or control shRNA were transfected with miR - 586 inhibitors or NC inhibitors as indicated . Colonies were stained with crystal violet and counted after 14 days incubation .

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B. HeLa cells expressing either lncRNA-MIF shRNA or control shRNA were transfected with miR-586 inhibitors or NC inhibitors as indicated. Colonies were stained with crystal violet and counted after 14 days incubation.


C . 2 × 106 HeLa cells expressing either lncRNA - MIF , lncRNA - MIF control RNA , lncRNA - MIF - shRNA or lncRNA - MIF control shRNA were individually injected subcutaneously into flanks of nude mice ( n = 7 for each group ) . Representative photographs of xenografttumors in situ were taken 3 weeks after injection . D . Three weeks post graft , mice were euthanized , tumors were excised and weighed , and the mean tumor weight was plotted on the graph . Data shown are mean ± SD ( n = 7 ; * * P < 0 . 01 , two - tailed t - test ) .

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C. 2×106 HeLa cells expressing either lncRNA-MIF, lncRNA-MIF control RNA, lncRNA-MIF-shRNA or lncRNA-MIF control shRNA were individually injected subcutaneously into flanks of nude mice (n = 7 for each group). Representative photographs of xenografttumors in situ were taken 3 weeks after injection.D. Three weeks post graft, mice were euthanized, tumors were excised and weighed, and the mean tumor weight was plotted on the graph. Data shown are mean ± SD (n = 7; **P < 0.01, two-tailed t-test).


D ) The heart , tibialis anterior ( TA ) and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT - PCR using primers represented by the arrows . Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction .

D) The heart, tibialis anterior (TA) and triceps muscle were isolated and analyzed to identify the presence of the duplication from exons 18 to 30 via RT-PCR using primers represented by the arrows. Sanger sequencing confirmed correct splicing of exons 30 and 18 at the duplication junction.


A ) 8 - µm cross section of 15 - week - old WT and Dup18 - 30 TA and triceps were analyzed for dystrophin localization by immunofluorescence . Asterisk indicates clusters of revertant fibers . Scale bars , 100 μm . B ) The muscle architecture of the same muscles was investigated by H & E staining . A representative image is shown . Scale bars , 100 μm . Asterisk indicates areas with necrotic fibers and fibrosis . Arrows indicate fibers with central nuclei .

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A) 8-µm cross section of 15-week-old WT and Dup18-30 TA and triceps were analyzed for dystrophin localization by immunofluorescence. Asterisk indicates clusters of revertant fibers. Scale bars, 100 μm. B) The muscle architecture of the same muscles was investigated by H&E staining. A representative image is shown. Scale bars, 100 μm. Asterisk indicates areas with necrotic fibers and fibrosis. Arrows indicate fibers with central nuclei.


G , H ) Mice were tested in an open - field chamber in which total distance travelled ( G ) and vertical activity ( H ) were assessed . WT , n = 9 ; Dup18 - 30 n = 12 .

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G, H) Mice were tested in an open-field chamber in which total distance travelled (G) and vertical activity (H) were assessed. WT, n = 9; Dup18-30 n = 12.


F , G ) Western blotting detected restoration of dystrophin expression in ( F ) heart and ( G ) TA in the Dup18 - 30 mice . 25 % and 50 % of the WT proteins compared to Dup18 - 30 mice have been loaded on the gel . Calnexin was used as a loading control .

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F, G ) Western blotting detected restoration of dystrophin expression in (F) heart and (G) TA in the Dup18-30 mice. 25% and 50% of the WT proteins compared to Dup18-30 mice have been loaded on the gel. Calnexin was used as a loading control.


H ) Immunostaining showed restoration of dystrophin expression in the heart and TA . A representative sample is shown . Scale bars , 100 μm .

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H) Immunostaining showed restoration of dystrophin expression in the heart and TA. A representative sample is shown. Scale bars, 100 μm.


A ) Immunofluorescence staining for alpha - syntrophin and beta - sarcoglycan in the TA and triceps muscles of the Dup18 - 30 mice . A representative image is shown . Scale bars , 100 μm .

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A) Immunofluorescence staining for alpha-syntrophin and beta-sarcoglycan in the TA and triceps muscles of the Dup18-30 mice. A representative image is shown. Scale bars, 100 μm.


B ) TA , triceps and diaphragm muscle architecture was analyzed by H & E staining . Scale bars , 50 μm .

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B) TA, triceps and diaphragm muscle architecture was analyzed by H&E staining. Scale bars, 50 μm.


A ) Fibrosis was analyzed in the diaphragm via Masson Trichrome staining . Scale bars , 100 μm .

A) Fibrosis was analyzed in the diaphragm via Masson Trichrome staining. Scale bars, 100 μm.


WT , Dup18 - 30 untreated and Dup18 - 30 treated mice were tested 7 weeks after treatment by measuring ( C ) forelimbs grip

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WT, Dup18-30 untreated and Dup18-30 treated mice were tested 7 weeks after treatment by measuring (C) forelimbs grip strength


A A representative photograph showing the co - cultured DRG and DH neurons . The DRG neurons were expressed with Spy - pHluorin for imaging the synaptic transmission . Scale bar , 10 μm . B Images of a presynaptic bouton marked in ( A ) showing the Spy - pHluorin fluorescence at 20 s before ( - 20 s , pre - stimulus ) , 20 s , 40 s , and 120 s after electrical stimulation ( post - stimulus ) in 0Ca2 + ( upper panel ) or 2Ca2 + ( lower panel ) solution . C Averaged fluorescence changes ( ΔF / F0 ) of Spy - pHluorin in 0Ca2 + ( left ) or 2Ca2 + solution ( right ) in response to the same electrical stimulation ( 20 Hz , 20 s ) ( n = 45 puncta from 6 cells for 0Ca2 + and 57 puncta from 6 cells for 2Ca2 + ) . The shadow in the traces represents the error bars ( s . e . m ) of each point .

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A A representative photograph showing the co-cultured DRG and DH neurons. The DRG neurons were expressed with Spy-pHluorin for imaging the synaptic transmission. Scale bar, 10 μm. B Images of a presynaptic bouton marked in (A) showing the Spy-pHluorin fluorescence at 20 s before (-20 s, pre-stimulus), 20 s, 40 s, and 120 s after electrical stimulation (post-stimulus) in 0Ca2+ (upper panel) or 2Ca2+ (lower panel) solution. C Averaged fluorescence changes (ΔF/F0) of Spy-pHluorin in 0Ca2+ (left) or 2Ca2+ solution (right) in response to the same electrical stimulation (20 Hz, 20 s) (n = 45 puncta from 6 cells for 0Ca2+ and 57 puncta from 6 cells for 2Ca2+). The shadow in the traces represents the error bars (s.e.m) of each point.


D - F The same as in ( A - C ) , but the experiments were performed in cultured hippocampal neurons ( n = 70 puncta from 3 cells for 0Ca2 + and 72 puncta from 3 cells for 2Ca2 + ) .

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D-F The same as in (A-C), but the experiments were performed in cultured hippocampal neurons (n = 70 puncta from 3 cells for 0Ca2+ and 72 puncta from 3 cells for 2Ca2+).


B Representative western blots ( upper ) and analysis ( lower ) of the expression levels of Syb2 in control ( Ctrl ) and Syb2 - cKO DRG neurons ( n = 3 per group ) . Control DRG neurons is from homozygous floxed Syb2 - null mice infected with AAV5 - CAG - EGFP virus .

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B Representative western blots (upper) and analysis (lower) of the expression levels of Syb2 in control (Ctrl) and Syb2-cKO DRG neurons (n = 3 per group). Control DRG neurons is from homozygous floxed Syb2-null mice infected with AAV5-CAG-EGFP virus.


C Evoked EPSCs and statistics from DH neurons co - cultured with control ( Ctrl ) or Syb2 - cKO DRG cells ( cKO ) in 0Ca2 + solution ( n = 13 cells for Ctrl and 11 for cKO ) .

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C Evoked EPSCs and statistics from DH neurons co-cultured with control (Ctrl) or Syb2-cKO DRG cells (cKO) in 0Ca2+ solution (n = 13 cells for Ctrl and 11 for cKO).


D Evoked EPSCs and statistics from DH neurons co - cultured with DRG neurons before ( black ) and after ( red ) applying 1 μM ω - conotoxin - GVIA ( GVIA ) in 0Ca2 + bath ( n = 9 cells ) .

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D Evoked EPSCs and statistics from DH neurons co-cultured with DRG neurons before (black) and after (red) applying 1 μM ω-conotoxin-GVIA (GVIA) in 0Ca2+ bath (n = 9 cells).


F Left panels , evoked EPSCs from DH neurons co - cultured with scrambled shRNA control ( Ctrl ) or Cav2 . 2 - KD ( sh - 1 / sh - 2 ) DRG neurons in 0Ca2 + solution . Right panel , quantification of evoked EPSCs ( n = 17 cells for Ctrl , 12 for sh - 1 , and 19 for sh - 2 ) . EPSCs were evoked by local electrical stimulation ( Estim , at arrows ) .

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F Left panels, evoked EPSCs from DH neurons co-cultured with scrambled shRNA control (Ctrl) or Cav2.2-KD (sh-1/sh-2) DRG neurons in 0Ca2+ solution. Right panel, quantification of evoked EPSCs (n = 17 cells for Ctrl, 12 for sh-1, and 19 for sh-2). EPSCs were evoked by local electrical stimulation (Estim, at arrows).


( A ) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system ( Voltage 2 . 5 K ) . Error bars represent mean ± SEM , n = 4 . Slopes compared by ANCOVA test , p < 0 . 0001 .

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(A) Cell proliferation for PC3 cells transfected with siSCR or siCLU monitored in real time for 60h with SP Dynamic Xcelligence system (Voltage 2.5 K). Error bars represent mean ± SEM, n=4. Slopes compared by ANCOVA test, p<0.0001.


( B ) Left panel : Cell cycle profile of PC3 cells transfected with siSCR or siCLU . Right panel : Percentage quantification of variation in G1 , S and G2 / M phases . Error bars represent mean ± SEM , n = 3 , * * p < 0 . 01 by paired Student ' s t - test . ( G2 / M population p = 0 . 0058 ; G1 p = 0 . 008 . ) . Inset : Western blot with CLU antibody in PC3 cells after CLU silencing . Vinculin was used as loading control .

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(B) Left panel: Cell cycle profile of PC3 cells transfected with siSCR or siCLU. Right panel: Percentage quantification of variation in G1, S and G2/M phases. Error bars represent mean ± SEM, n=3, **p<0.01 by paired Student's t-test. (G2/M population p= 0.0058; G1 p=0.008.). Inset: Western blot with CLU antibody in PC3 cells after CLU silencing. Vinculin was used as loading control.


( C ) Left panel : RNA - Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP - transfected with siCLU versus siSCR . Target genes expression is shown as fold change relative to siSCR , p ≤ 0 . 05 by an unpaired t - test for all the genes listed . Central panel : Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis . The comparison of these 2 experiments shows modification of 4 mitosis regulators . Right panel : Western blot validating the expression levels of target proteins .

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(C) Left panel: RNA-Microarray analysis comparing PC3 cells transfected with siCLU versus siSCR and Kinexus phosphokinome microarray analysis comparing LNCaP-transfected with siCLU versus siSCR. Target genes expression is shown as fold change relative to siSCR, p ≤0.05 by an unpaired t-test for all the genes listed. Central panel: Venn diagram to graphically illustrate the overlap of mRNA and proteins involved in the regulation of mitosis in both transcriptome and phosphokinome analysis. The comparison of these 2 experiments shows modification of 4 mitosis regulators. Right panel: Western blot validating the expression levels of target proteins.


( A ) Real - time PCR for CLU and Cdc25C in three different prostate cancer cell lines . Target genes expression was calculated relative to GAPDH and normalized to siSCR . Error bars represent mean ± SD , n = 3 .

(A) Real-time PCR for CLU and Cdc25C in three different prostate cancer cell lines. Target genes expression was calculated relative to GAPDH and normalized to siSCR. Error bars represent mean ± SD, n=3.


( B ) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A . Vinculin was used as loading control .

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(B) Western blot for CLU and Cdc25C after siSCR or si CLU transfection in the same prostate cancer cell lines as in A. Vinculin was used as loading control.


( C ) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h . Vinculin was used as loading

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(C) Western blot for CLU and Cdc25C in PC3 cells after transfection with siSCR or siCLU followed by transfection with CLU or empty expression plasmid for additional 48h. Vinculin was used as loading control


( A ) Upper panel : Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX - 011 ( n = 8 ) or SCR ASO control ( n = 6 ) . Vinculin was used as loading control . Lower panel : Quantification of the protein levels , relative to the loading control , reported as dot plots . * p < 0 . 05 by t - test followed by Welch ' s correction ( CLU p = 0 . 049 , Cdc25C p = 0 . 024 ) .

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(A) Upper panel: Western blot for CLU and Cdc25C from LNCaP xenografts harvested from mice treated with OGX-011 (n=8) or SCR ASO control (n=6). Vinculin was used as loading control. Lower panel: Quantification of the protein levels, relative to the loading control, reported as dot plots. *p<0.05 by t-test followed by Welch's correction (CLU p= 0.049, Cdc25C p= 0.024).


( B ) Western blot with the correspondent quantification of protein levels ( left ) and real time PCR ( right ) from untreated LNCaP xenografts harvested at endpoint . The negative correlation between CLU - RNA and Cdc25C - RNA ΔΔCT values was calculated using a spearman test ( rho = - 0 . 86 ; p = 0 . 0107 ) .

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(B) Western blot with the correspondent quantification of protein levels (left) and real time PCR (right) from untreated LNCaP xenografts harvested at endpoint. The negative correlation between CLU-RNA and Cdc25C-RNA ΔΔCT values was calculated using a spearman test (rho=-0.86; p=0.0107).


( C ) Immunoistochemistry staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy ( n = 5 ) or neoadjuvant hormone therapy , ( NHT ) ( n = 5 ) or the combination of NHT and OGX - 011 ( n = 9 ) for 3 months prior surgery . Average score staining from the 3 groups ( left ) and representative sections for CLU and Cdc25C ( right ) . Error bars represent mean ± SEM . CLU : * p < 0 . 05 ; untreated versus 3mNHT ( p = 0 . 0213 ) ; 3mNHT versus 3mNHT + OGX - 011 ( p = 0 . 012 ) by ANOVA followed by Bonferroni post hoc analysis . All cases were normalized by clinical stage , Gleason score and serum PSA . For each patient , the pathologist selected the area with the highest Gleason score . Scale bar represents 100

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(C) Immunoistochemistry staining for CLU and Cdc25C of radical prostatectomy samples from historical control specimens with no neoadjuvant therapy (n=5) or neoadjuvant hormone therapy, (NHT) (n=5) or the combination of NHT and OGX-011 (n=9) for 3 months prior surgery. Average score staining from the 3 groups (left) and representative sections for CLU and Cdc25C (right). Error bars represent mean ± SEM. CLU: *p<0.05; untreated versus 3mNHT (p=0.0213); 3mNHT versus 3mNHT +OGX-011 (p=0.012) by ANOVA followed by Bonferroni post hoc analysis. All cases were normalized by clinical stage, Gleason score and serum PSA. For each patient, the pathologist selected the area with the highest Gleason score. Scale bar represents 100 µm


( A ) Co - immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors . Proteins were immunoprecipitated with specific anti - Cdc25C ( left ) and CLU ( right ) antibodies and analyzed by immunoblotting .

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(A) Co-immunoprecipitation from parental PC3 cells transfected with CLU and Cdc25C plasmid expression vectors. Proteins were immunoprecipitated with specific anti-Cdc25C (left) and CLU (right) antibodies and analyzed by immunoblotting.


( B ) Dual immunofluorescence staining with Cdc25C ( green ) and CLU ( red ) antibodies in PC3 cells . Confocal microscopy was used to identify the interaction ( white arrows ) . DNA was counterstained with ( DAPI ) ( blue ) . Scale bar represent 10 µm .

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(B) Dual immunofluorescence staining with Cdc25C (green) and CLU (red) antibodies in PC3 cells. Confocal microscopy was used to identify the interaction (white arrows). DNA was counterstained with (DAPI) (blue). Scale bar represent 10 µm.


( C ) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells . Confocal microscopy was used to detect the interaction ( red dots ) . DNA was counterstained with DAPI ( blue ) . PC3 cells transfected with siCLU were used as a negative control . Scale bar represent 10 µm .

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(C) Duolink proximity ligation assay between Cdc25C and CLU in PC3 cells. Confocal microscopy was used to detect the interaction (red dots). DNA was counterstained with DAPI (blue). PC3 cells transfected with siCLU were used as a negative control. Scale bar represent 10 µm.


( A ) Left panel : Western blot for CLU , Cdc25C , Cdc25C - T48 , Cdc25C S216 and Cdc25C S198 in PC - 3 cells after siSCR or siCLU transfection , synchronization and nocodazole release at indicated time points . Vinculin was used as loading control . Right panel : Immunofluorescence microscopy for Cdc25C - T48 in PC3 cells after siSCR or siCLU transfection . DNA was counterstained with ( DAPI ) ( blue ) . Scale bar represents 10 µm .

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(A) Left panel: Western blot for CLU, Cdc25C, Cdc25C-T48, Cdc25C S216 and Cdc25C S198 in PC-3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Vinculin was used as loading control. Right panel: Immunofluorescence microscopy for Cdc25C-T48 in PC3 cells after siSCR or siCLU transfection. DNA was counterstained with (DAPI) (blue). Scale bar represents 10 µm.


( B ) Western blot for CLU , Cdc25C - T48 , Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid ( OA ) ( 100 nM ) for 12 hours . Proteins were extracted 15 min after nocodazole release . Vinculin was used as loading control .

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(B) Western blot for CLU, Cdc25C-T48, Cdk1 and PP2A in PC3 cells after siSCR or siCLU transfection and after synchronization followed by treatment with or without okadaic acid (OA) (100 nM) for 12 hours. Proteins were extracted 15 min after nocodazole release. Vinculin was used as loading control.


( C ) Western blot for PP2A , CLU , Cdc25C - T48 , Cdc25C , Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points . Vinculin was used as loading control .

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(C) Western blot for PP2A, CLU, Cdc25C-T48, Cdc25C, Wee1 and Cdk1 in PC3 cells transfected with siSCR or siPP2A synchronized and nocodazole released at indicated time points. Vinculin was used as loading control.


( D ) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection , synchronization and nocodazole release at indicated time points . Error bar represent mean ± SEM , n = 3 , * * p < 0 . 01 ( p = 0 . 0025 ) by ANOVA followed by Bonferroni ' s post hoc comparisons . Cells treated with no peptide were used as a negative control .

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(D) PP2A phosphatase activity measurement in PC3 cells after siSCR or siCLU transfection, synchronization and nocodazole release at indicated time points. Error bar represent mean ± SEM, n=3, ** p<0.01 (p=0.0025) by ANOVA followed by Bonferroni's post hoc comparisons. Cells treated with no peptide were used as a negative control.


( A ) Western blot for CLU , Cdk1 Y15 , Cdk1 and Wee1 in PC - 3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points . Vinculin was used as loading

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(A) Western blot for CLU, Cdk1 Y15, Cdk1 and Wee1 in PC-3 cells after siSCR or siCLU transfection followed by synchronization and nocodazole release at indicated time points. Vinculin was used as loading control


( B ) Wee1 expression level was evaluated in PC3 cells after CLU silencing by real - time PCR . Error bar represent mean ± SEM , n = 3 , * * * p < 0 . 001 ( p = 0 . 0001 ) by unpaired t - test followed by Welch ' s correction .

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(B) Wee1 expression level was evaluated in PC3 cells after CLU silencing by real-time PCR. Error bar represent mean ± SEM, n=3, *** p<0.001 (p=0.0001) by unpaired t-test followed by Welch's correction.


( C ) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor , MK - 1775 . Error bar represent mean ± SEM , n = 3 , * * * * p < 0 . 0001 by Mann -

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(C) FACS analysis showing percentage of the PC3 cells positive for phosphohistone H3 after transfection with siSCR or siCLU in presence and absence of Wee1 inhibitor, MK-1775. Error bar represent mean ± SEM, n=3, ****p<0.0001 by Mann-Whitney


( D ) Western blot for CLU , Wee1 , Cdk1 Y15 , Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK - 1775 , a specific Wee1 inhibitor , for 12 hours . Cells were harvested at different time points after nocodazole release . Vinculin was used as loading control .

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(D) Western blot for CLU, Wee1, Cdk1 Y15, Cdk1 and Cyclin B1 in PC3 cells after transfection with siSCR or siCLU followed by synchronization and treatment with or without MK-1775, a specific Wee1 inhibitor, for 12 hours. Cells were harvested at different time points after nocodazole release. Vinculin was used as loading control.


( A ) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU ( left ) , and SCR or OGX - 011 ( right ) followed by 72h of cabazitaxel treatment . Percentage of surviving cells was calculated relative to control . Error bar represent mean ± SEM , n = 3 . Inset : Western blot was performed to verify CLU knockdown in cells .

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(A) Cabazitaxel IC50 calculation using WST1 assay in PC3 cells after transfection with siSCR or siCLU (left), and SCR or OGX-011 (right) followed by 72h of cabazitaxel treatment. Percentage of surviving cells was calculated relative to control. Error bar represent mean ± SEM, n=3. Inset: Western blot was performed to verify CLU knockdown in cells.


( B ) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC - 3 cells after SCR or OGX - 011 transfection followed by different concentration of cabazitaxel as indicated ( > 1000 counts ) Error bar represent mean ± SEM . , * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 by Student ' s t - test . ( caba 1 . 5 nM p = 0 . 00115 ; 3 nM p = 0 . 0105 ; 4 . 5 nM p = 1 . 93x10 - 7 , ; 6 . 25 nM p = 8 . 9x10 - 9 ; 12 . 5 nM p = 6 . 8x10 - 9 ; 25 nM p = 1 . 2x10 - 9 ) .

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(B) Mitotic cells and cells in mitotic catastrophe were counted using DAPI in PC-3 cells after SCR or OGX-011 transfection followed by different concentration of cabazitaxel as indicated (>1000 counts) Error bar represent mean ± SEM.,*p<0.05, ***p<0.001, ****p<0.0001 by Student's t-test.( caba 1.5 nM p=0.00115 ; 3 nM p=0.0105 ; 4.5 nM p=1.93x10-7, ; 6.25 nM p=8.9x10-9; 12.5 nM p=6.8x10-9; 25 nM p=1.2x10-9).


( C ) Clonogenic assay in PC - 3 cells after SCR or OGX - 011 transfection followed by treatment with different concentrations of cabazitaxel as indicated . The number of survival clones was assessed after 15 days using crystal violet assay . Error bars represent mean ± SEM n = 3 , * p < 0 . 05 , * * p < 0 . 01 by Mann - Whitney ( 0 nM caba p = 0 . 039 ; 0 . 5 nM p = 0 . 0079 ; 2 nM p = 0 . 0079 )

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(C) Clonogenic assay in PC-3 cells after SCR or OGX-011 transfection followed by treatment with different concentrations of cabazitaxel as indicated. The number of survival clones was assessed after 15 days using crystal violet assay. Error bars represent mean ± SEM n=3,*p<0.05, **p<0.01 by Mann-Whitney (0 nM caba p=0.039; 0.5 nM p=0.0079; 2 nM p=0.0079)


( A ) Cell cycle distribution of syncronized PC3 and PC3 - R - caba cells 60 min after nocodazole release at indicated time points .

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(A) Cell cycle distribution of syncronized PC3 and PC3-R-caba cells 60 min after nocodazole release at indicated time points.


( B ) Western blot for CLU , Cdc25C , Cdc25C - T48 , Cdk1 , Cdk1 - Y15 and Wee1 in IGRCaP - 1 and PC3 parental ( WT ) or cabazitaxel - resistant ( R - caba ) cells as well as for PC3 - R - caba cells transfected with siSCR or siCLU . Vinculin was used as loading control .

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(B) Western blot for CLU, Cdc25C, Cdc25C-T48, Cdk1, Cdk1-Y15 and Wee1 in IGRCaP-1 and PC3 parental (WT) or cabazitaxel-resistant (R-caba) cells as well as for PC3-R-caba cells transfected with siSCR or siCLU. Vinculin was used as loading control.


( C ) Cell survival measured using WST1 in PC3 - R - caba cells after SCR or OGX - 011 transfection and treatment with 10 nM cabazitaxel for 48h . Error bar represent mean ± SEM , n = 3 , * * * p < 0 . 001 by one way ANOVA followed by Bonferroni ' s post hoc correction .

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(C) Cell survival measured using WST1 in PC3-R-caba cells after SCR or OGX-011 transfection and treatment with 10 nM cabazitaxel for 48h. Error bar represent mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.


( D ) Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel ( 10 nM ; C10 ) , MK - 1775 ( 10 nM ; MK10 ) or both for 72 hours . . Error bar represent the mean ± SEM , n = 3 , * * * p < 0 . 001 by one way ANOVA followed by Bonferroni ' s post hoc correction .

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(D) Cell survival measured using WST1 in PC3 resistant cells after transfection with siSCR or siCLU followed by treatment with cabazitaxel (10 nM; C10), MK-1775 (10 nM; MK10) or both for 72 hours. .Error bar represent the mean ± SEM, n=3, *** p<0.001 by one way ANOVA followed by Bonferroni's post hoc correction.


( E ) PARP cleavage was assessed by western blot in cells treated as in D and its relative expression was quantified by densitometry using ImageJ . Vinculin was used as loading

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(E) PARP cleavage was assessed by western blot in cells treated as in D and its relative expression was quantified by densitometry using ImageJ. Vinculin was used as loading control


( F ) Left panel : Quantification of tumour volume over 52 days in nude male mice xenotransplanted with PC3 - docetaxel resistant cells and treated with either MK - 1775 ( M , black line ) , MK - 1775 and micellar taxane ( MT , blue line ) , micellar taxane and OGX - 011 ( TO , red line ) or MK - 1775 , micellar taxane and OGX - 011 ( MTO , green line ) . The error bars indicate mean ± SEM , n = 8 . Right panel : Slopes of tumor growth between groups , were compared by ANOVA followed by Dunnett ' s post hoc analysis . . * p < 0 . 05 , * * * p < 0 . 001 . p = 0 . 0488 ( M to TO ) ; p = 0 . 0007 ( M to MTO ) .

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(F) Left panel: Quantification of tumour volume over 52 days in nude male mice xenotransplanted with PC3-docetaxel resistant cells and treated with either MK-1775 (M, black line), MK-1775 and micellar taxane (MT, blue line), micellar taxane and OGX-011 (TO, red line) or MK-1775, micellar taxane and OGX-011 (MTO, green line). The error bars indicate mean ± SEM, n=8. Right panel: Slopes of tumor growth between groups, were compared by ANOVA followed by Dunnett's post hoc analysis..*p<0.05, ***p<0.001 .p=0.0488 (M to TO); p=0.0007 (M to MTO).


A Immunohistochemical positive staining for NALCN in human prostate adenocarcinoma tissues , but not in normal or hyperplastic prostate tissues ( tissue microarray : PR484 , US Biomax , Inc ) . T2N0M0 - tumor invaded submucosa without any regional or distant metastases ( stage II ) ; T3aN0M0 - tumor had broken through the capsule of the prostate gland and invaded through muscularis propria ( stage III ) ; T3N1M0 - tumor invaded through muscularis propria with 1 to 3 regional lymph node metastases ( stage IV ) . Data information : The images : scale bar is 100 µm .

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A Immunohistochemical positive staining for NALCN in human prostate adenocarcinoma tissues, but not in normal or hyperplastic prostate tissues (tissue microarray: PR484, US Biomax, Inc). T2N0M0 - tumor invaded submucosa without any regional or distant metastases (stage II); T3aN0M0 - tumor had broken through the capsule of the prostate gland and invaded through muscularis propria (stage III); T3N1M0 - tumor invaded through muscularis propria with 1 to 3 regional lymph node metastases (stage IV). Data information: The images: scale bar is 100 µm.


C Immunohistochemistry : significant correlation ( P < 0 . 0001 ) between positive staining of NALCN and Src in clinically localized prostate cancer , but not in non - cancerous prostate . Data information : The images : scale bar is 100 µm .

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C Immunohistochemistry: significant correlation (P<0.0001) between positive staining of NALCN and Src in clinically localized prostate cancer, but not in non-cancerous prostate. Data information: The images: scale bar is 100 µm.


D Immunohistochemistry : positive staining of NALCN in bone metastases produced by breast and colon cancers , but not in corresponding normal tissues . Data information : The images : scale bar is 100 µm .

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D Immunohistochemistry: positive staining of NALCN in bone metastases produced by breast and colon cancers, but not in corresponding normal tissues. Data information: The images: scale bar is 100 µm.


F NALCN knockdown suppresses invadopodia formation reported by Cy3 - fluorescent gelatin degradation ( arrows ) . Confocal images ( left ) of Cy3 ( red ) and DAPI ( blue ; nuclei ) fluorescence in control ( shCTL ) and shNALCN 48 hours after plating the cells on gelatin . Bar diagram plot : NALCN suppression causes significant decrease of the number of gelatin degradation puncta counted within 48 h of incubation . Data information : The images : scale bar is 100 µm . Bar diagram plots show the degradation puncta per cell ( F ) mean ± S . E . M . for N = 5 ( F ) biological replicates per condition . * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - tailed Student ' s t - test .

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F NALCN knockdown suppresses invadopodia formation reported by Cy3-fluorescent gelatin degradation (arrows). Confocal images (left) of Cy3 (red) and DAPI (blue; nuclei) fluorescence in control (shCTL) and shNALCN 48 hours after plating the cells on gelatin. Bar diagram plot: NALCN suppression causes significant decrease of the number of gelatin degradation puncta counted within 48 h of incubation. Data information: The images: scale bar is 100 µm. Bar diagram plots show the degradation puncta per cell (F) mean±S.E.M. for N=5 (F) biological replicates per condition. **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.


A Co - localisation of NALCN with invadopodia markers - Dynamin , Src and Tks : confocal images ( left ) , 2D intensity histograms ( middle ) and statistical analysis ( right ) . Scale bar : 5 µm . . Data information : The colocalization intensity ( A ) was measured using Pearson ' s correlation coefficient with Fiji ImageJ software . Bar diagram plots show mean ± S . E . M . : ( A ) Pearson ' s correlation coefficient as a measure of co - localization with NALCN of Dynamin ( n = 4 ) , Src ( n = 14 ) and Tks ( n = 4 ) , n = number of cell * * * P < 0 . 001 , unpaired two - tailed Student ' s t - test .

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A Co-localisation of NALCN with invadopodia markers - Dynamin, Src and Tks: confocal images (left), 2D intensity histograms (middle) and statistical analysis (right). Scale bar: 5 µm.. Data information: The colocalization intensity (A) was measured using Pearson's correlation coefficient with Fiji ImageJ software. Bar diagram plots show mean±S.E.M.: (A) Pearson's correlation coefficient as a measure of co-localization with NALCN of Dynamin (n=4), Src (n=14) and Tks (n=4), n= number of cell ***P<0.001, unpaired two-tailed Student's t-test.


C Top : temporal profiles of self - normalised fluorescence intensity ( ∆ F / F0 ) of fluo - 4 ( green ) , averaged within outlined region ( inset : left , green outline ) , and mCherry ( red ) , averaged within 14 circles ( inset : right , red outlines ) . Middle : galleries show confocal images of fluo - 4 and mCherry fluorescence captured during two periods of interests , POIs ( blue bars ) : for POI1 - every image , for POI2 - every 100th image . Bottom left : confocal images of Dynamin2 - mCherry fluorescence captured before and 3 min after stimulation with 10 % FBS . Bottom middle and right : plots relate the dynamics of [ Ca2 + ] c changes ( Fluo - 4 ) to formation of dynamin puncta ( Dynamin2 - mCherry ) at two regions of interests ( ROIs ) ( insets ) ; the galleries ( below ) show every 40th image ( after 90 ° rotation ) during the POIs ( plots : blue bars ) .

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C Top: temporal profiles of self-normalised fluorescence intensity (∆F/F0) of fluo-4 (green), averaged within outlined region (inset: left, green outline), and mCherry (red), averaged within 14 circles (inset: right, red outlines). Middle: galleries show confocal images of fluo-4 and mCherry fluorescence captured during two periods of interests, POIs (blue bars): for POI1- every image, for POI2 - every 100th image. Bottom left: confocal images of Dynamin2-mCherry fluorescence captured before and 3 min after stimulation with 10% FBS. Bottom middle and right: plots relate the dynamics of [Ca2+]c changes (Fluo-4) to formation of dynamin puncta (Dynamin2-mCherry) at two regions of interests (ROIs) (insets); the galleries (below) show every 40th image (after 90° rotation) during the POIs (plots: blue bars).


D Top the overlay of NALCN - GFP and Cal - 590 images is related to the gallery of self - normalized rainbow - coded Cal - 590 images showing [ Ca2 + ] c response to 10 % FBS . Bottom : 3 rainbow - coded Cal - 590 images are superimposed on grey - coded NALCN - GFP image and rotated by 90 ° . Arrows : NALCN - enriched structure .

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D Top the overlay of NALCN-GFP and Cal-590 images is related to the gallery of self-normalized rainbow-coded Cal-590 images showing [Ca2+]c response to 10% FBS. Bottom: 3 rainbow-coded Cal-590 images are superimposed on grey-coded NALCN-GFP image and rotated by 90°. Arrows: NALCN-enriched structure.


A FBS - induced transient [ Ca2 + ] c elevations ( Fluo - 4 , top left ) are associated with transient decreases of Ca2 + concentration in endoplasmic reticulum ( [ Ca2 + ] ER ) ( Mag - Fluo - 4 , bottom left ) . Corresponding galleries ( right ) show every 100th image during the highlighted POIs ( I and II , respectively ) . Visualization of the ER with pDsRed2 - ER confirms the ER origin of Mag - Fluo - 4 signal ( inset bottom left : confocal images of cell fragment ) .

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A FBS-induced transient [Ca2+]c elevations (Fluo-4, top left) are associated with transient decreases of Ca2+ concentration in endoplasmic reticulum ([Ca2+]ER) (Mag-Fluo-4, bottom left). Corresponding galleries (right) show every 100th image during the highlighted POIs (I and II, respectively). Visualization of the ER with pDsRed2-ER confirms the ER origin of Mag-Fluo-4 signal (inset bottom left: confocal images of cell fragment).


C - D [ Ca2 + ] c oscillations reported by Fluo - 4 fluorescence in FBS - exposed PC - 3 cells pre - treated with siCTL ( C ) or siNALCN ( D ) for 72 hours . Top : sample traces from 8 different cells . Middle : POIs ( top : grey background ) are presented on enlarged plots to emphasize that NALCN suppression abolishes " pacemaker events " observed in control ( fitted red curves ) . Bottom : the galleries show every 3rd image during the POIs ( middle : orange background ) . Scale bars : 10 μm .

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C-D [Ca2+]c oscillations reported by Fluo-4 fluorescence in FBS-exposed PC-3 cells pre-treated with siCTL (C) or siNALCN (D) for 72 hours. Top: sample traces from 8 different cells. Middle: POIs (top: grey background) are presented on enlarged plots to emphasize that NALCN suppression abolishes "pacemaker events" observed in control (fitted red curves). Bottom: the galleries show every 3rd image during the POIs (middle: orange background). Scale bars: 10 μm.


A - D Cytosolic Na + concentration ( [ Na + ] c ) changes reported by ratiometric Na + indicator SBFI following ( A - C ) switch of extracellular Na + concentration ( [ Na + ] o ) from 0 to 130 mM or ( D ) application of 1 µM thapsigargin ( TG ) . The effects of : ( A ) NALCN knockdown , ( B ) extracellular Ca2 + concentration ( [ Ca2 + ] o ) and ( C ) Ca2 + store depletion . Data information : Data show mean ± S . E . M . The cartoons highlight experimental design and the plots show mean ± S . E . M . traces ( n = 45 - 501 ) of SBFI ( olive ) and Fura - 2 ( wine ) fluorescence with the axes ( ∆ R / R0 ) presented in corresponding colour .

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A-D Cytosolic Na+ concentration ([Na+]c) changes reported by ratiometric Na+ indicator SBFI following (A-C) switch of extracellular Na+ concentration ([Na+]o) from 0 to 130 mM or (D) application of 1 µM thapsigargin (TG). The effects of: (A) NALCN knockdown, (B) extracellular Ca2+ concentration ([Ca2+]o) and (C) Ca2+ store depletion. Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.


E - H Store - operated Ca2 + entry ( SOCE ) - induced changes of [ Na + ] c ( SBFI ) and [ Ca2 + ] c ( Fura - 2 ) . The effects of : NALCN knockdown ( E ) , the reverse - mode plasmalemmal Na + / Ca2 + exchanger ( RM - NCX ) inhibitor KB - R7943 ( 1 µM ) either at [ Na + ] o = 130 mM ( F ) or upon [ Na + ] o switch from 0 to 130 mM following SOCE activation ( G ) , and knockdown of the mitochondrial Na + / Ca2 + exchanger , siNCLX ( H ) . Data information : Data show mean ± S . E . M . The cartoons highlight experimental design and the plots show mean ± S . E . M . traces ( n = 45 - 501 ) of SBFI ( olive ) and Fura - 2 ( wine ) fluorescence with the axes ( ∆ R / R0 ) presented in corresponding colour .

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E-H Store-operated Ca2+ entry (SOCE)-induced changes of [Na+]c (SBFI) and [Ca2+]c (Fura-2). The effects of: NALCN knockdown (E), the reverse-mode plasmalemmal Na+/Ca2+ exchanger (RM-NCX) inhibitor KB-R7943 (1 µM) either at [Na+]o=130 mM (F) or upon [Na+]o switch from 0 to 130 mM following SOCE activation (G), and knockdown of the mitochondrial Na+/Ca2+ exchanger, siNCLX (H). Data information: Data show mean±S.E.M. The cartoons highlight experimental design and the plots show mean±S.E.M. traces (n=45-501) of SBFI (olive) and Fura-2 (wine) fluorescence with the axes (∆R/R0) presented in corresponding colour.


I SOCE augments NALCN - mediated Na + influx ( top ) by addressing NALCN to plasma membrane ( bottom ) . Data information : Plots ( I , top ) compare mean rates of Na + influx induced by [ Na + ] o - switch ( n = 283 - 360 ) and SOCE ( n = 180 - 216 ) , and their shNALCN - sensitive fractions . Immunoblotting ( I , bottom ) compares NALCN expression in total cell lysates and biotinylated fractions before and after SOCE induction . Numbers show ( in corresponding colour ) mean values ( n = 3 ) of NALCN protein levels ( band intensity ) normalized to N - Cadherin , Calnexin and β - actin .

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I SOCE augments NALCN-mediated Na+ influx (top) by addressing NALCN to plasma membrane (bottom). Data information: Plots (I, top) compare mean rates of Na+ influx induced by [Na+]o-switch (n=283-360) and SOCE (n=180-216), and their shNALCN-sensitive fractions. Immunoblotting (I, bottom) compares NALCN expression in total cell lysates and biotinylated fractions before and after SOCE induction. Numbers show (in corresponding colour) mean values (n=3) of NALCN protein levels (band intensity) normalized to N-Cadherin, Calnexin and β-actin.


J - K The effect of KB - R7943 ( 1 µM ) on FBS - induced [ Ca2 + ] c responses ( Fura - 2 , ∆ R / R0 ) and inward current ( perforated patch , Vh = - 80 mV ) , respectively . Data information : The bar diagram plots show mean signal temporal densities ( J , n = 20 ) and mean current densities ( K , n = 6 - 8 ) during initial transient ( cyan ) and oscillations ( orange ) . n = number of cell * * P < 0 . 01 , * * * P < 0 . 001 , unpaired two - tailed Student ' s t - test .

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J-K The effect of KB-R7943 (1 µM) on FBS-induced [Ca2+]c responses (Fura-2, ∆R/R0) and inward current (perforated patch, Vh=-80 mV), respectively. Data information: The bar diagram plots show mean signal temporal densities (J, n=20) and mean current densities (K, n=6-8) during initial transient (cyan) and oscillations (orange). n= number of cell **P<0.01, ***P<0.001, unpaired two-tailed Student's t-test.


A Top : visualisation of mitochondria with MitoTracker Green confirms mitochondrial origin of Rhod - 2 signal . Middle : FBS - induced [ Ca2 + ] c oscillations ( Fluo - 4 ) are associated with [ Ca2 + ] mito oscillations ( Rhod - 2 ) . Bottom : every 2nd image from the highlighted ( orange background ) period . B Top : FBS - induced [ Ca2 + ] c oscillations ( Fluo - 4 ) are associated with oscillations in O2 - production by mitochondria ( MitoSOX Red ) . Bottom : every 3rd image from the highlighted ( orange background ) period . Data information : Cartoons ( left ) highlight experimental design and assessed signaling pathways .

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A Top: visualisation of mitochondria with MitoTracker Green confirms mitochondrial origin of Rhod-2 signal. Middle: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with [Ca2+]mito oscillations (Rhod-2). Bottom: every 2nd image from the highlighted (orange background) period. B Top: FBS-induced [Ca2+]c oscillations (Fluo-4) are associated with oscillations in O2- production by mitochondria (MitoSOX Red). Bottom: every 3rd image from the highlighted (orange background) period. Data information: Cartoons (left) highlight experimental design and assessed signaling pathways.


C O2 - metabolite , H2O2 , suppresses FBS - induced [ Ca2 + ] c oscillations ( Fluo - 4 ) . Data information : In C , mean signal temporal density during POI2 was normalized to that during POI1 in control ( n = 58 ) and in 50 µM of H2O2 ( n = 74 ) , and compared . Bar diagram plots : mean ± S . E . M . n = number of cell , * * * P < 0 . 001 , unpaired two - tailed Student ' s t - test . Cartoons ( left ) highlight experimental design and assessed signaling pathways .

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C O2- metabolite, H2O2, suppresses FBS-induced [Ca2+]c oscillations (Fluo-4). Data information: In C, mean signal temporal density during POI2 was normalized to that during POI1 in control (n=58) and in 50 µM of H2O2 (n=74), and compared. Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways.


D Suppression of NCLX with siNCLX attenuates [ Ca2 + ] c oscillations ( Fluo - 4 ) . Data information : In D , mean signal temporal densities during highlighted periods were compared in control ( n = 18 ) and following 48 h pre - treatment with siNCLX ( n = 17 ) . Bar diagram plots : mean ± S . E . M . n = number of cell , * * * P < 0 . 001 , unpaired two - tailed Student ' s t - test . Cartoons ( left ) highlight experimental design and assessed signaling pathways . Note that Na + delivered to the cytosol by NALCN , on the one hand , activates RM - NCX , while on the other hand , is exchanged by NCLX to Ca2 + thus restricting elevation of [ Ca2 + ] mito and , hence , production of superoxide ( O2 - ) and H2O2 , which is known to inhibit SERCA , RM - NCX and SOCE elements .

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D Suppression of NCLX with siNCLX attenuates [Ca2+]c oscillations (Fluo-4). Data information: In D, mean signal temporal densities during highlighted periods were compared in control (n=18) and following 48 h pre-treatment with siNCLX (n=17). Bar diagram plots: mean±S.E.M. n= number of cell, ***P<0.001, unpaired two-tailed Student's t-test. Cartoons (left) highlight experimental design and assessed signaling pathways. Note that Na+ delivered to the cytosol by NALCN, on the one hand, activates RM-NCX, while on the other hand, is exchanged by NCLX to Ca2+ thus restricting elevation of [Ca2+]mito and, hence, production of superoxide (O2-) and H2O2, which is known to inhibit SERCA, RM-NCX and SOCE elements.


D Corresponding traces of relative changes in total z - stack fluorescence , as indicated .

D Corresponding traces of relative changes in total z-stack fluorescence, as indicated.


E Plot : mean rates of secretion calculated as signal mass ( cyan , left ) per second . Data information : Data ( E ) : mean ± S . E . M . for siCTL ( n = 35 ) , siNALCN ( n = 40 ) , Control ( n = 45 ) , EGTA - AM ( n = 35 ) and BAPTA - AM ( n = 62 ) . n = number of cell , * * * P < 0 . 001 ; and n . s . , not significant , unpaired two - tailed Student ' s t - test .

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E Plot: mean rates of secretion calculated as signal mass (cyan, left) per second. Data information: Data (E): mean±S.E.M. for siCTL (n=35), siNALCN (n=40), Control (n=45), EGTA-AM (n=35) and BAPTA-AM (n=62). n= number of cell, ***P<0.001; and n.s., not significant, unpaired two-tailed Student's t-test.


D Left : Dorsal ( top ) and ventral ( bottom ) images of bioluminescence from mice 31 days after intra - tibial injection of control ( shCTL : PC - 3 Luc - shCTL ) or NALCN depleted cells ( shCTL : PC - 3 Luc - shNALCN ) . Right : statistical comparison of total photon fluxes between the two groups of 10 mice ( c . p . s . : count of photons per second ± SD ) . Data information : * P < 0 . 05 , * * P < 0 . 01 , and n . s . , not significant , two - tailed Mann - Whitney U test .

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D Left: Dorsal (top) and ventral (bottom) images of bioluminescence from mice 31 days after intra-tibial injection of control (shCTL: PC-3 Luc-shCTL) or NALCN depleted cells (shCTL: PC-3 Luc-shNALCN). Right: statistical comparison of total photon fluxes between the two groups of 10 mice (c.p.s.: count of photons per second±SD). Data information: *P<0.05, **P<0.01, and n.s., not significant, two-tailed Mann-Whitney U test.


E Left : x - ray 3D images obtained from mice 8 weeks after intracardiac injections with control ( PC - 3 Luc ) and cells stably overexpressing NALCN ( + hNALCN ) . Right : box plots compare the incidence and total volume of bone lesions between the two groups of 11 mice : central band shows the mean , the boxes show lower and upper quartiles and whiskers show maximum data values . Data information : Red numbers and / or arrows in the images depict tumour take incidence / bone lesions . * P < 0 . 05 , * * P < 0 . 01 , and n . s . , not significant , two - tailed Mann - Whitney U test .

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E Left: x-ray 3D images obtained from mice 8 weeks after intracardiac injections with control (PC-3 Luc) and cells stably overexpressing NALCN (+hNALCN). Right: box plots compare the incidence and total volume of bone lesions between the two groups of 11 mice: central band shows the mean, the boxes show lower and upper quartiles and whiskers show maximum data values. Data information: Red numbers and/or arrows in the images depict tumour take incidence/bone lesions. *P<0.05, **P<0.01, and n.s., not significant, two-tailed Mann-Whitney U test.


( B ) Expression of S protein on the plasma membrane of Jurkat - S cells assessed by surface biotinylation and followed by immunoprecipitation with anti - T2A , SDS - PAGE under reducing conditions and western blot with streptavidin - peroxidase . Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control . The arrow indicates the position of the reduced S protein . ( C ) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat - S and Jurkat cells followed by immunoprecipitation with anti - T2A followed by SDS - PAGE under non - denaturing conditions ( i . e . without reducing agents and without boiling ) . The nitrocellulose membrane was blotted with streptavidin - peroxidase . Arrows point at different oligomerization forms of the S protein .

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(B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein. (C) Expression of the S protein in native form was assessed by surface biotinylation of Jurkat-S and Jurkat cells followed by immunoprecipitation with anti-T2A followed by SDS-PAGE under non-denaturing conditions (i.e. without reducing agents and without boiling). The nitrocellulose membrane was blotted with streptavidin-peroxidase. Arrows point at different oligomerization forms of the S protein.


( B ) Expression of S protein on the plasma membrane of Jurkat - S cells assessed by surface biotinylation and followed by immunoprecipitation with anti - T2A , SDS - PAGE under reducing conditions and western blot with streptavidin - peroxidase . Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control . The arrow indicates the position of the reduced S protein .

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(B) Expression of S protein on the plasma membrane of Jurkat-S cells assessed by surface biotinylation and followed by immunoprecipitation with anti-T2A, SDS-PAGE under reducing conditions and western blot with streptavidin-peroxidase. Biotinylation of the parental Jurkat cells was carried out in parallel as a negative control. The arrow indicates the position of the reduced S protein.


( D ) Formation of syncytia between the CTFR - labelled ACE2 + human cell line and the CFSE - labelled Jurkat - S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers . The Bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat . Parental Jurkat cells ( not expressing S protein ) are considered negative controls . Data represent the mean ± SD of triplicated datasets .

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(D) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells was measured by flow cytometry by analyzing the percentage of cells that become double positive for CTFR and CFSE markers. The Bar plot to the right shows the effect of different doses of HepG2 cells on the formation of syncytia with a fixed number of Jurkat. Parental Jurkat cells (not expressing S protein) are considered negative controls. Data represent the mean±SD of triplicated datasets.


( E ) Formation of syncytia between the CTFR - labelled ACE2 + human cell line and the CFSE - labelled Jurkat - S cells , in an experiment as in D , was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE . Nuclei were identified by DAPI staining . Red arrowhead in the DAPI image indicates a nucleus of Hep - G2 origin ; the green arrowhead the nucleus of Jurkat - S origin .

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(E) Formation of syncytia between the CTFR-labelled ACE2+ human cell line and the CFSE-labelled Jurkat-S cells, in an experiment as in D, was confirmed by confocal microscopy after sorting cells double positive for CTFR and CFSE. Nuclei were identified by DAPI staining. Red arrowhead in the DAPI image indicates a nucleus of Hep-G2 origin; the green arrowhead the nucleus of Jurkat-S origin.


( F ) Overlay plot of Jurkat - S cells that were incubated with anti - EGFR mAb conjugated to Bv421 and serum from either donor # 15 or from a pre - COVID - 19 donor and followed by a secondary anti - human IgG1 antibody conjugated to PE .

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(F) Overlay plot of Jurkat-S cells that were incubated with anti-EGFR mAb conjugated to Bv421 and serum from either donor #15 or from a pre-COVID-19 donor and followed by a secondary anti-human IgG1 antibody conjugated to PE.


( A ) Overlay histograms of Jurkat - S staining with different human sera diluted 1 : 50 . A pre - COVID19 serum sample is taken as negative control ( grey histogram ) .

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(A) Overlay histograms of Jurkat-S staining with different human sera diluted 1:50. A pre-COVID19 serum sample is taken as negative control (grey histogram).


( B ) Bar plots of flow cytometry data generated with Jurkat - S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests : green , positive for the test ; orange , weak or unclear ; magenta , negative samples . Flow cytometry data is expressed as the ratio between the MFI of the antibody anti - S and the MFI for EGFR . Negative values for the flow cytometry test are those with a S / EGFR MFI ratio lower than 0 . 5 . This ratio was set in order to fit most of the data negative for the other serological tests ( pink triangles ) under that threshold . Data represent the mean ± s . e . m . All datapoints are shown .

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(B) Bar plots of flow cytometry data generated with Jurkat-S cells and the panel of serum samples of Table EV1 classified according to their result in the indicated commercial tests: green, positive for the test; orange, weak or unclear; magenta, negative samples. Flow cytometry data is expressed as the ratio between the MFI of the antibody anti-S and the MFI for EGFR. Negative values for the flow cytometry test are those with a S/EGFR MFI ratio lower than 0.5. This ratio was set in order to fit most of the data negative for the other serological tests (pink triangles) under that threshold.Data represent the mean±s.e.m. All datapoints are shown.


( A ) Box and whiskers plot of MFI and absorbance data in human sera according to their clinical classification of COVID - 19 symptoms . Clinical classification was done according to the following parameters : Asymptomatic , no symptoms ; Mild , 3 or more of the following symptoms : non - productive cough , hyperthermia , headache , odynophagia , dyspnea , asthenia , myalgia , ageusia , anosmia , cutaneous involvement ; Moderate , 3 or more of the above symptoms plus gastrointestinal symptoms , or more than 3 of the above for 7 or more days ; Moderate - Severe , 3 or more of the above symptoms plus pneumonia . Box and whiskers are shown to represent the minimum and maximum values as well as the median . All datapoints are shown .

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(A) Box and whiskers plot of MFI and absorbance data in human sera according to their clinical classification of COVID-19 symptoms. Clinical classification was done according to the following parameters: Asymptomatic, no symptoms; Mild, 3 or more of the following symptoms: non-productive cough, hyperthermia, headache, odynophagia, dyspnea, asthenia , myalgia, ageusia, anosmia, cutaneous involvement; Moderate, 3 or more of the above symptoms plus gastrointestinal symptoms, or more than 3 of the above for 7 or more days; Moderate-Severe, 3 or more of the above symptoms plus pneumonia. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown.


( B ) Comparison of MFI versus absorbance data generated by flow cytometry and ELISA for all human sera . A lineal regression curve was adjusted , with a 95 % confidence interval , to all data with the R2 values indicated in the plots . # 5Selected samples of outliers ( # 4 , # 5 , # 8 , # 31 , # 46 , # 48 , # 49 and # 58 ) as well as samples close to a diagonal ( # 36 , # 52 and # 15 ) are indicated .

(B) Comparison of MFI versus absorbance data generated by flow cytometry and ELISA for all human sera. A lineal regression curve was adjusted, with a 95% confidence interval, to all data with the R2 values indicated in the plots. #5Selected samples of outliers (#4, #5, #8, #31, #46, #48, #49 and #58) as well as samples close to a diagonal (#36, #52 and #15) are indicated.


( C ) Titration of selected human sera by ELISA using the S1 protein and by flow cytometry with Jurkat - S cells . Samples with antibodies detected by flow cytometry and not by ELISA are indicated ( # 8 , # 46 , # 48 , # 49 ) . Data represent the mean ± SD of duplicates . An unpaired two - tailed t - test was carried out to compare the results of 1 : 50 , 1 : 150 and 1 : 450 dilutions of sera # 8 , # 46 , # 48 and # 49 by FC to the pre - COVID - 1 sample as a negative control . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0005 .

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(C) Titration of selected human sera by ELISA using the S1 protein and by flow cytometry with Jurkat-S cells. Samples with antibodies detected by flow cytometry and not by ELISA are indicated (#8, #46, #48, #49). Data represent the mean±SD of duplicates. An unpaired two-tailed t-test was carried out to compare the results of 1:50, 1:150 and 1:450 dilutions of sera #8, #46, #48 and #49 by FC to the pre-COVID-1 sample as a negative control. *, p<0.05; **, p<0.005; ***, p<0.0005.


( B ) Validation of the neutralization method was carried out with cell HEK - 293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein . The supernatants were mixed with sera from donor # 66 at the indicated dilutions or from a pre - COVID sample as a control before addition onto HEK - 293T - ACE2 + cell cultures . No serum , refers to a control containing no human sera . Data represent the mean ± SD of triplicates .

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(B) Validation of the neutralization method was carried out with cell HEK-293T cell culture supernatants containing lentiviral particles pseudotyped with either the S protein or with the VSV G protein. The supernatants were mixed with sera from donor #66 at the indicated dilutions or from a pre-COVID sample as a control before addition onto HEK-293T-ACE2+ cell cultures. No serum, refers to a control containing no human sera. Data represent the mean±SD of triplicates.


( C ) The presence of neutralizing antibodies in sera from donors # 46 and # 48 , found negative by ELISA and positive by flow cytometry ( Fig . 3C ) was demonstrated as in panel B . Data represent the mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample ) .

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(C) The presence of neutralizing antibodies in sera from donors #46 and #48, found negative by ELISA and positive by flow cytometry (Fig. 3C) was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( D ) Bar plot showing the S / EGFR MFI ratio determined by flow cytometry analysis with Jurkat - S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS - CoV - 2 at the Ramón y Cajal Hospital of Madrid . A negative result is considered for a ratio lower than 0 . 5 . Two clear cases of sera positive for anti - S IgG1 are indicated ( RyC52 and RyC65 ) . A borderline sample just above the threshold line ( RyC58 ) is also indicated . Data show the mean ± s . e . m . All datapoints are shown .

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(D) Bar plot showing the S/EGFR MFI ratio determined by flow cytometry analysis with Jurkat-S of serum samples from 30 sanitary personnel repeatedly tested as PCR negative for SARS-CoV-2 at the Ramón y Cajal Hospital of Madrid. A negative result is considered for a ratio lower than 0.5. Two clear cases of sera positive for anti-S IgG1 are indicated (RyC52 and RyC65). A borderline sample just above the threshold line (RyC58) is also indicated. Data show the mean±s.e.m. All datapoints are shown.


( F ) Plot of the S / EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S / EGFR and shape ( see Materials and Methods and Fig . EV1 ) . The green line indicates the threshold point ( 0 . 024 ) for the Score above which a serum is considered positive .

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(F) Plot of the S/EGFR MFI ratio for the 30 RyC samples versus a Score generated according to the slope of S/EGFR and shape (see Materials and Methods and Fig. EV1). The green line indicates the threshold point (0.024) for the Score above which a serum is considered positive.


( G ) The presence of neutralizing antibodies in sera from donors RyC46 , RyC52 , RyC56 and RyC58 diluted 1 / 15 was demonstrated as in panel B . Data represent the mean ± SD of triplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ( Unpaired two - tailed t - test comparing each serum to the No serum sample )

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(G) The presence of neutralizing antibodies in sera from donors RyC46, RyC52, RyC56 and RyC58 diluted 1/15 was demonstrated as in panel B. Data represent the mean±SD of triplicates. *, p<0.05; **, p<0.005; ****, p<0.00005 (Unpaired two-tailed t-test comparing each serum to the No serum sample)


( A ) A total of 52 serum samples from Hospital Universitario La Princesa ( HUP ) and 52 samples of pre - COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S / EGFR - based Score . Of the 52 HUP samples , a total of 40 were from donors testing positive by PCR and by an ELISA test , 6 were from donors testing negative by PCR but positive by ELISA , 1 from an individual testing negative by ELISA and positive by PCR , and 5 were not tested by PCR . Box and whiskers are shown to represent the minimum and maximum values as well as the median . All datapoints are shown . The broken line indicates the threshold for a Score value of 0 . 024 .

(A) A total of 52 serum samples from Hospital Universitario La Princesa (HUP) and 52 samples of pre-COVID donors stored at the CBMSO were tested by the flow cytometry method and classified according to the S/EGFR-based Score. Of the 52 HUP samples, a total of 40 were from donors testing positive by PCR and by an ELISA test, 6 were from donors testing negative by PCR but positive by ELISA, 1 from an individual testing negative by ELISA and positive by PCR, and 5 were not tested by PCR. Box and whiskers are shown to represent the minimum and maximum values as well as the median. All datapoints are shown. The broken line indicates the threshold for a Score value of 0.024.


C ) Four HUP serum samples giving discrepant results by flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus ( Fig . 4A ) . Data represent the mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * * , p < 0 . 00005 ; ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample )

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C) Four HUP serum samples giving discrepant results by flow cytometry method were tested in the neutralization assay of pseudotyped lentivirus (Fig. 4A). Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ****, p<0.00005; (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample)


( A ) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat - S cells at the indicated dilutions . Flow cytometry data are shown as the S / EGFR MFI ratio . Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer .

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(A) A collection of serum samples from volunteers working at the CBMSO were taken in the months of June and October 2020 and tested by flow cytometry using Jurkat-S cells at the indicated dilutions. Flow cytometry data are shown as the S/EGFR MFI ratio. Red arrows indicate a loss in titer of antibodies between June and October whereas blue arrows indicate an increase in antibody titer.


( B ) A neutralization assay with S protein - pseudotyped lentivirus was carried out to resolve the discrepancies . Data represent the mean ± SD of duplicates . * , p < 0 . 05 ; * * , p < 0 . 005 ; * * * , p < 0 . 0005 ; * * * * * , p < 0 . 000005 ( Paired two - tailed t - test comparing all serum dilutions to the pre - COVID sample ) .

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(B) A neutralization assay with S protein-pseudotyped lentivirus was carried out to resolve the discrepancies. Data represent the mean±SD of duplicates. *, p<0.05; **, p<0.005; ***, p<0.0005; *****, p<0.000005 (Paired two-tailed t-test comparing all serum dilutions to the pre-COVID sample).


( C ) Classification of the CBMSO cohort according to the Score generated from S / EGFR data . The Broken line indicates the 0 . 024 threshold above which samples are considered positive .

(C) Classification of the CBMSO cohort according to the Score generated from S/EGFR data. The Broken line indicates the 0.024 threshold above which samples are considered positive.


( b ) Effects of C18 - Pyr - Cer ( C18PC , lanes 2 and 4 ) on the lipidation of LC3B ( LC3B - II ) were examined by western blotting in the absence or presence of a pan - caspase inhibitor Z - VAD ( lanes 3 and 4 ) and compared to vehicle - treated controls ( lanes 1 and 3 ) . Full blots can be found in Supplementary Figure 13 .

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(b) Effects of C18-Pyr-Cer (C18PC, lanes 2 and 4) on the lipidation of LC3B (LC3B-II) were examined by western blotting in the absence or presence of a pan-caspase inhibitor Z-VAD (lanes 3 and 4) and compared to vehicle-treated controls (lanes 1 and 3). Full blots can be found in Supplementary Figure 13.


( c ) Formation of double - membrane autophagosomal vesicles ( black arrows ) in the absence ( control ) or presence of C18PC was visualized by TEM ( left and right , respectively ) . Higher magnification of TEM visualization is shown in lower panels . Scale bars , 10 μm ( top ) and 500 nm ( bottom ) .

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(c) Formation of double-membrane autophagosomal vesicles (black arrows) in the absence (control) or presence of C18PC was visualized by TEM (left and right, respectively). Higher magnification of TEM visualization is shown in lower panels. Scale bars, 10 μm (top) and 500 nm (bottom).


( d ) Effects of siRNA - mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs ( controls ) using Trypan blue exclusion assay .

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(d) Effects of siRNA-mediated knockdown of Atg3 or Atg7 on cell death in the absence or presence of C18PC were determined and compared to effects of transfection with Scr siRNAs (controls) using Trypan blue exclusion assay.


( e ) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild - type ( ATG5 + / + ) and ATG5 − / − knockout mice with C18PC . Control cells were treated with vehicle .

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(e) Roles of C18PC in the regulation of lethal autophagy were assessed after treatment of MEFs isolated from wild-type (ATG5+/+) and ATG5−/− knockout mice with C18PC. Control cells were treated with vehicle.


( f ) Effects of C18PC on caspase - dependent or caspase - independent cell death were determined in MEFs isolated from Bax − / − Bak − / − or Casp3 − / − Casp7 − / − mice and from wild - type ( Bax + / + Bak + / + ) or Casp3 + / − Casp7 + / − mice , used as controls . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(f) Effects of C18PC on caspase-dependent or caspase-independent cell death were determined in MEFs isolated from Bax−/− Bak−/− or Casp3−/− Casp7−/− mice and from wild-type (Bax+/+ Bak+/+) or Casp3+/− Casp7+/− mice, used as controls. Data shown are an average of at least three experiments ± s.d. (*P 0.05).


( a ) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18 - Pyr - Cer ( C18PC ) at 2 h in UM - SCC22A ( 22A ) cells was examined by visualizing the colocalization of MTG ( green ) and LTR ( red ) using live - cell imaging and confocal microscopy . Scale bars , 10 μm . Enlarged images are shown in boxed areas .

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(a) Targeting of autophagolysosomes to mitochondria in the absence or presence of C18-Pyr-Cer (C18PC) at 2 h in UM-SCC22A (22A) cells was examined by visualizing the colocalization of MTG (green) and LTR (red) using live-cell imaging and confocal microscopy. Scale bars, 10 μm. Enlarged images are shown in boxed areas.


( b ) Effects of C18 - dihydro - Cer - 14 - piperidine ( dihydro - C18PC ) on OCR measured using the SeaHorse alongside those of C18PC - and vehicle - treated controls . Data shown are an average of at least two independent experiments performed in duplicate ± s . d . ( * P 0 . 05 ) .

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(b) Effects of C18-dihydro-Cer-14-piperidine (dihydro-C18PC) on OCR measured using the SeaHorse alongside those of C18PC- and vehicle-treated controls. Data shown are an average of at least two independent experiments performed in duplicate ± s.d. (*P 0.05).


( a ) Effects of expression of wild - type ( WT ) CerS1 and its catalytically inactive V5 - tagged mutant ( Mut ) , which cannot generate C18 - ceramide , on the formation of LC3B - II were determined by western blotting ( + / − tet ) . Successful induction of wild - type and mutant CerS1 expression was confirmed using anti - V5 . β - actin was used as a loading control . Full blots can be found in Supplementary Figure 13 .

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(a) Effects of expression of wild-type (WT) CerS1 and its catalytically inactive V5-tagged mutant (Mut), which cannot generate C18-ceramide, on the formation of LC3B-II were determined by western blotting (+/− tet). Successful induction of wild-type and mutant CerS1 expression was confirmed using anti-V5. β-actin was used as a loading control. Full blots can be found in Supplementary Figure 13.


( b ) Generation of endogenous C18 - and C18 : 1 - ceramides in response to wild - type CerS1 and mutant CerS1 induction was measured by LC / MS / MS .

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(b) Generation of endogenous C18- and C18:1-ceramides in response to wild-type CerS1 and mutant CerS1 induction was measured by LC/MS/MS.


( c ) Effects of wild - type CerS1 and C18 - ceramide induction ( + tet ) on GFP and LC3B - GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls ( − tet ) . Scale bars , 10

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(c) Effects of wild-type CerS1 and C18-ceramide induction (+ tet) on GFP and LC3B-GFP lipidation were visualized using confocal microscopy and compared to effects of noninduced controls (− tet). Scale bars, 10 μm


( d ) The effects of induction of wild - type CerS1 and C18 - ceramide and catalytically inactive mutant CerS1 ( + tet ) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls ( − tet ) .

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(d) The effects of induction of wild-type CerS1 and C18-ceramide and catalytically inactive mutant CerS1 (+ tet) in the regulation of mitochondrial function were assessed by measuring OCR using the SeaHorse and compared to roles of noninduced controls (− tet).


( e ) Targeting mitochondria with autophagolysosomes in the absence ( − tet ) or presence ( + tet ) of wild - type CerS1 and C18 - ceramide was visualized by colocalization of MTG and LTR using confocal microscopy . Scale bars , 10 μm .

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(e) Targeting mitochondria with autophagolysosomes in the absence (− tet) or presence (+ tet) of wild-type CerS1 and C18-ceramide was visualized by colocalization of MTG and LTR using confocal microscopy. Scale bars, 10 μm.


( f ) Effects of wild - type and mutant CerS1 ( − / + tet ) on ATP generation .

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(f) Effects of wild-type and mutant CerS1 (−/+ tet) on ATP generation.


( g ) Effects of wild - type CerS1 ( − / + tet ) on ATP generation in the absence ( control shRNA ) or presence of shRNA - mediated knockdown of LC3B . Data shown are an average of at least three experiments ± s . d . ( * P 0 . 05 ) .

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(g) Effects of wild-type CerS1 (−/+ tet) on ATP generation in the absence (control shRNA) or presence of shRNA-mediated knockdown of LC3B. Data shown are an average of at least three experiments ± s.d. (*P 0.05).