C ) Representative traces of IKr steady state activation in WT hiPSC - CMs in Tyrode ( left ) , in the presence of 5 µM LUF7346 ( middle ) and after the application of 5 µM E4031 to selectively block IKr ( right ) . Inset : voltage clamp protocol .

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C) Representative traces of IKr steady state activation in WT hiPSC-CMs in Tyrode (left), in the presence of 5 µM LUF7346 (middle) and after the application of 5 µM E4031 to selectively block IKr (right). Inset: voltage clamp protocol.


D ) Average I / V relationships ( left ) and steady - state activation curves with superimposed Boltzmann ' s fittings ( right ) under baseline conditions ( black ) and in the presence of 5 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 19 .

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D) Average I/V relationships (left) and steady-state activation curves with superimposed Boltzmann's fittings (right) under baseline conditions (black) and in the presence of 5 µM LUF7346 (red). * = p<0.05. N: 19.


E ) Plot of the time constants ( τ ) of deactivation derived from biexponential fits ( left ) and representative examples ( right ) under baseline conditions ( black ) and in the presence of 3 µM LUF7346 ( red ) . * = p < 0 . 05 . N : 17 - 21 .

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E) Plot of the time constants (τ) of deactivation derived from biexponential fits (left) and representative examples (right) under baseline conditions (black) and in the presence of 3 µM LUF7346 (red). * = p<0.05. N: 17-21.


A ) Representative MEA trace ( left ) showing the effect of increasing concentrations of LUF7346 on FP contour , measured in LQT2N996I - CMs . Average data ( right ) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs baseline . Colour in the heatmap defines the magnitude of QT shortening ( blue ) after treatment with increasing concentrations of LUF7346 . N : 7 - 14 .

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A) Representative MEA trace (left) showing the effect of increasing concentrations of LUF7346 on FP contour, measured in LQT2N996I-CMs. Average data (right) showing the effect of LUF7346 on QT interval duration relative to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs baseline. Colour in the heatmap defines the magnitude of QT shortening (blue) after treatment with increasing concentrations of LUF7346. N: 7-14.


B ) Representative AP from LQT2corr - , LQT2N996I - , LQT1corr - , and LQT1R190Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of increasing concentrations of LUF7346 ( colour - code is shown ) .

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B) Representative AP from LQT2corr-, LQT2N996I-, LQT1corr-, and LQT1R190Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of increasing concentrations of LUF7346 (colour-code is shown).


C ) Effect of LUF7346 on APD90 , APD70 , and APD50 , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , LQT1R190Q - ( dark blue ) paced at 1 Hz ; * = p < 0 . 05 vs baseline . The colour of the asterisk indicates comparisons and respective statistical significance . ° = p < 0 . 05 vs the genetically - matched corrected control . # = p < 0 . 05 LQT1corr vs LQT2corr . N : 12 - 15 .

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C) Effect of LUF7346 on APD90, APD70, and APD50, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), LQT1R190Q- (dark blue) paced at 1 Hz; * = p<0.05 vs baseline. The colour of the asterisk indicates comparisons and respective statistical significance. ° = p<0.05 vs the genetically-matched corrected control. # = p<0.05 LQT1corr vs LQT2corr. N: 12-15.


D ) Effect of LUF7346 on APA and Ediast , measured in LQT2corr - ( blue ) , LQT2N996I - ( green ) , LQT1corr - ( purple ) , and LQT1R190Q - CMs ( dark blue ) paced at 1 Hz . N : 12 - 15 .

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D) Effect of LUF7346 on APA and Ediast, measured in LQT2corr- (blue), LQT2N996I- (green), LQT1corr- (purple), and LQT1R190Q-CMs (dark blue) paced at 1 Hz. N: 12-15.


A ) Representative MEA trace ( left ) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour , measured in LQT1R594Q - CMs . Average data ( right ) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study . * = p < 0 . 05 vs respective baseline . ° = p < 0 . 05 vs AST . Colour in the heatmap defines the magnitude of QT prolongation ( red ) and QT shortening ( blue ) , respectively , after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST . N : 5 - 10 .

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A) Representative MEA trace (left) showing the effect of 100 nM AST and increasing concentrations of LUF7346 in the presence of AST on FP contour, measured in LQT1R594Q-CMs. Average data (right) of the effect of AST and LUF7346 in the presence of AST on QT interval duration compared to baseline in CMs derived from all the hiPSC lines used in this study. * = p<0.05 vs respective baseline. ° = p<0.05 vs AST. Colour in the heatmap defines the magnitude of QT prolongation (red) and QT shortening (blue), respectively, after treatment with AST and with increasing concentrations of LUF7346 in the presence of AST. N: 5-10.


B ) Representative AP from WT - , LQT1R594Q - and JLNSR594Q - CMs , paced at 1 Hz , under baseline conditions ( black ) , after application of 100 nM AST ( red ) , and after addition of increasing concentrations of LUF7346 in the presence of AST ( colour - code is shown ) .

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B) Representative AP from WT-, LQT1R594Q- and JLNSR594Q-CMs, paced at 1 Hz, under baseline conditions (black), after application of 100 nM AST (red), and after addition of increasing concentrations of LUF7346 in the presence of AST (colour-code is shown).


C ) Effect of AST and LUF7346 in the presence of AST on APD90 , APD70 , and APD50 in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . * = p < 0 . 05 vs respective baseline ; ° = p < 0 . 05 vs AST . N : 11 - 16 .

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C) Effect of AST and LUF7346 in the presence of AST on APD90, APD70, and APD50 in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. * = p<0.05 vs respective baseline; ° = p<0.05 vs AST. N: 11-16.


D ) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT - ( grey ) , LQT1R594Q - ( red ) and JLNSR594Q - CMs ( black ) paced at 1 Hz . N : 11 - 16 .

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D) Effect of AST and LUF7346 in the presence of AST on APA and Ediast in WT- (grey), LQT1R594Q- (red) and JLNSR594Q-CMs (black) paced at 1 Hz. N: 11-16.


A : Representative AP traces measured in JLNSR594Q - CMs paced at 1 Hz , under baseline conditions ( black ) and after application of either AST alone ( red ) , AST + LUF7346 3 µM ( green ) and AST + LUF7346 5 µM ( blue ) .

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A: Representative AP traces measured in JLNSR594Q-CMs paced at 1 Hz, under baseline conditions (black) and after application of either AST alone (red), AST+LUF7346 3 µM (green) and AST+LUF7346 5 µM (blue).


B : STV values calculated on spontaneously beating hiPSC - CM clusters over 30 consecutive beats as measured with MEA . N = 7 - 24 . * = p < 0 . 05 vs respective isogenic pair .

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B: STV values calculated on spontaneously beating hiPSC-CM clusters over 30 consecutive beats as measured with MEA. N = 7-24. * = p<0.05 vs respective isogenic pair.


C : STV changes ( left ) and representative examples of QT interval oscillations plotted over 30 consecutive beats ( right ) in the presence of AST and AST + LUF7346 20 µM . * = p < 0 . 05 vs Baseline . ° = p < 0 . 05 vs AST . N = 7 - 24 .

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C: STV changes (left) and representative examples of QT interval oscillations plotted over 30 consecutive beats (right) in the presence of AST and AST+LUF7346 20 µM. * = p<0.05 vs Baseline. ° = p<0.05 vs AST. N = 7-24.


Dose - response curves representing 72 - h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib . Ranges of estimated IC50 and Emax for the selected lines are shown . Data are represented as mean ± SD .

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Dose-response curves representing 72-h viability measurements for six selected BRAFV600E melanoma cell lines after treatment with vemurafenib. Ranges of estimated IC50 and Emax for the selected lines are shown. Data are represented as mean ± SD.


Pairwise distribution and correlation of estimated IC50 and Emax values for responses ( 72 - h viability ) of 8 cell lines to four RAF inhibitors and one MEK inhibitor . Each dot represents one drug / cell line combination . Sensitive cell lines are arbitrarily defined based on their IC50s ( the most widely used metric for evaluation of drug response ; log10 [ IC50 ] < − 6 . 5 ) .

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Pairwise distribution and correlation of estimated IC50 and Emax values for responses (72-h viability) of 8 cell lines to four RAF inhibitors and one MEK inhibitor. Each dot represents one drug/cell line combination. Sensitive cell lines are arbitrarily defined based on their IC50s (the most widely used metric for evaluation of drug response; log10[IC50] < −6.5).


c - Jun and p - c - Jun ( Ser63 ) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h ( or 5 h in the case of WM115cell line ) and 48 h .

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c-Jun and p-c-Jun(Ser63) changes as measured by RPPA in six melanoma cell lines in response to different doses of RAF and MEK inhibitors for 10 h (or 5 h in the case of WM115cell line) and 48 h.


B , C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( B ) Dose - response profiles for apoptosis induction with vemurafenib and JNK - IN - 8 combination .

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B, C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (B) Dose-response profiles for apoptosis induction with vemurafenib and JNK-IN-8 combination.


C Synergistic apoptosis induction in four cell lines ( WM115 , WM1552C , LOXIMVI , and COLO858 ) treated for 72 h with combinations of vemurafenib and JNK - IN - 8 . ( C ) Excess over the predicted Bliss independence ( EOBI ) calculated for different combined doses of vemurafenib and JNK - IN - 8 .

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C Synergistic apoptosis induction in four cell lines (WM115, WM1552C, LOXIMVI, and COLO858) treated for 72 h with combinations of vemurafenib and JNK-IN-8. (C) Excess over the predicted Bliss independence (EOBI) calculated for different combined doses of vemurafenib and JNK-IN-8.


D c - Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non - targeting controls quantified in triplicate 48 h after transfection .

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D c-Jun expression in WM115 cells transfected with JUN siRNA relative to no RNA and non-targeting controls quantified in triplicate 48 h after transfection.


E , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of

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E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of vemurafenib


E , F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96 - h treatment with increasing doses of selumetinib ( F )

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E, F Apoptosis in WM115 cells with or without 48 h JUN knockdown after 96-h treatment with increasing doses of selumetinib (F)


Time - dose - response plots indicating changes in RPPA measurements for p27 versus p - histone H3 for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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Time-dose-response plots indicating changes in RPPA measurements for p27 versus p-histone H3 for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


B - D Time - dose - response plots indicating changes in RPPA measurements for pERK ( Thr202 / Tyr204 ) versus pAKT ( Ser473 ) for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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B-D Time-dose-response plots indicating changes in RPPA measurements for pERK(Thr202/Tyr204) versus pAKT(Ser473) for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


Time - dose - response plots indicating changes in RPPA measurements for pS6 ( Ser235 / 236 ) versus total c - Jun levels for two selected cell lines ( C32 and WM115 ) after exposure to PLX4720 . Mean values of four biological replicates are shown . Protein levels represent log2 fold change of each signal ( at a specific dose and time ) relative to a DMSO - treated control .

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Time-dose-response plots indicating changes in RPPA measurements for pS6(Ser235/236) versus total c-Jun levels for two selected cell lines (C32 and WM115) after exposure to PLX4720. Mean values of four biological replicates are shown. Protein levels represent log2 fold change of each signal (at a specific dose and time) relative to a DMSO-treated control.


Covariate single - cell analysis of Ki - 67 ( left ) and pRb ( Ser807 / 811 ) ( right ) versus p - cJun ( Ser73 ) in WM1552C cells before and 24 h after exposure to 0 . 8 μM vemurafenib . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The vertical lines were used to gate p - cJunHigh versus p - cJunLow cells . The horizontal lines were used to gate Ki - 67High versus Ki - 67Low cells , and pRbHigh versus pRbLow cells .

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Covariate single-cell analysis of Ki-67 (left) and pRb(Ser807/811) (right) versus p-cJun(Ser73) in WM1552C cells before and 24 h after exposure to 0.8 μM vemurafenib. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The vertical lines were used to gate p-cJunHigh versus p-cJunLow cells. The horizontal lines were used to gate Ki-67High versus Ki-67Low cells, and pRbHigh versus pRbLow cells.


Analysis of drug dose - dependent changes in proportion of pRbLow / p - cJunHigh and pRbHigh / p - cJunHigh subpopulations in four melanoma cell lines ( WM115 , WM1552C , LOXIMVI , COLO858 ) after exposure to vemurafenib for 24 h . These subpopulations were gated as shown in ( A ) . Data are represented as mean ± SD for two

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Analysis of drug dose-dependent changes in proportion of pRbLow/p-cJunHigh and pRbHigh/p-cJunHigh subpopulations in four melanoma cell lines (WM115, WM1552C, LOXIMVI, COLO858) after exposure to vemurafenib for 24 h. These subpopulations were gated as shown in (A). Data are represented as mean ± SD for two replicates


Covariate single - cell analysis of pRb ( Ser807 / 811 ) versus pS6 ( Ser235 / 236 ) following 24 - h treatment of WM1552C cells with different doses of vemurafenib alone ( top ) and vemurafenib and JNK - IN - 8 together ( bottom ) . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy . The horizontal and vertical lines were used to gate pS6High versus pS6Low cells , and pRbHigh versus pRbLow cells , respectively .

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Covariate single-cell analysis of pRb(Ser807/811) versus pS6(Ser235/236) following 24-h treatment of WM1552C cells with different doses of vemurafenib alone (top) and vemurafenib and JNK-IN-8 together (bottom). Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. Density scatter plots were generated using signal intensities for individual cells as measured by immunofluorescence microscopy. The horizontal and vertical lines were used to gate pS6High versus pS6Low cells, and pRbHigh versus pRbLow cells, respectively.


Selected immunofluorescence images of pS6 ( Ser235 / 236 ) and Hoechst staining in WM1552C cells in a DMSO - treated control and 24 h after exposure to 5 μM of vemurafenib , JNK - IN - 8 , and their combination .

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Selected immunofluorescence images of pS6(Ser235/236) and Hoechst staining in WM1552C cells in a DMSO-treated control and 24 h after exposure to 5 μM of vemurafenib, JNK-IN-8, and their combination.


Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines ( LOXIMVI , WM115 and WM1552C ) as a function of drug concentration for single - drug vemurafenib and JNK - IN - 8 treatments and their combination treatment . Drugs were added at a 1 : 1 ratio , each at indicated concentrations when used in combination . pS6High population of cells was gated as indicated in ( C ) . Data are represented as mean ± SD for two replicates . Data comparison between vemurafenib treatment and vemurafenib / JNK - IN - 8 combined treatment was made by using two - way analysis of variance ( ANOVA ) .

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Analysis of the changes in proportion of pS6Highcell population in three melanoma cell lines (LOXIMVI, WM115 and WM1552C) as a function of drug concentration for single-drug vemurafenib and JNK-IN-8 treatments and their combination treatment. Drugs were added at a 1:1 ratio, each at indicated concentrations when used in combination. pS6High population of cells was gated as indicated in (C). Data are represented as mean ± SD for two replicates. Data comparison between vemurafenib treatment and vemurafenib/JNK-IN-8 combined treatment was made by using two-way analysis of variance (ANOVA).


Fraction of c - JunHighcells ( as measured by single - cell ( immunofluorescence microscopy ) after 48 h JUN knockdown followed by 24 - h treatment with vemurafenib . Fold changes are shown relative to control - treated cells . Data are presented as mean ± SD .

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Fraction of c-JunHighcells (as measured by single-cell (immunofluorescence microscopy) after 48 h JUN knockdown followed by 24-h treatment with vemurafenib. Fold changes are shown relative to control-treated cells. Data are presented as mean ± SD.


Single - cell pS6 ( Ser235 / 236 ) levels in the c - JunHigh and c - JunLow fractions of cells ( as measured by single - cell multiplex immunofluorescence microscopy ) after 48 h of JUN knockdown and 24 - h treatment with 0 . 32 μM vemurafenib . Single - cell pS6 data are presented as box - and - whisker plots with median signal intensities and interquartile ranges ; bars extending to 1 . 5 × the interquartile range are shown for each condition as a measure of variance . P - values were calculated using a two - sided nonparametric Mann - Whitney U - test .

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Single-cell pS6(Ser235/236) levels in the c-JunHigh and c-JunLow fractions of cells (as measured by single-cell multiplex immunofluorescence microscopy) after 48 h of JUN knockdown and 24-h treatment with 0.32 μM vemurafenib. Single-cell pS6 data are presented as box-and-whisker plots with median signal intensities and interquartile ranges; bars extending to 1.5× the interquartile range are shown for each condition as a measure of variance. P-values were calculated using a two-sided nonparametric Mann-Whitney U-test.


Pairwise Spearman ' s correlation between the 1 - h changes in pERK ( Thr202 / Tyr204 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the seven dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study .

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Pairwise Spearman's correlation between the 1-h changes in pERK(Thr202/Tyr204) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the seven dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.


Comparison of the level of 1 - h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720 .

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Comparison of the level of 1-h pERK inhibition for 8 cell lines treated with SB590885 versus PLX4720.


Comparison of the 72 - h measured relative viability AUC for 8 / 10 cell lines treated with SB590885 versus

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Comparison of the 72-h measured relative viability AUC for 8/10 cell lines treated with SB590885 versus PLX4720


Comparison of the level of 1 - h pERK inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor .

Comparison of the level of 1-h pERK inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.


Comparison of the 72 - h measured relative viability AUC between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK inhibitor .

Comparison of the 72-h measured relative viability AUC between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor.


Pairwise Spearman ' s correlation between the 24 - h changes in pS6 ( Ser235 / 236 ) levels as measured by immunofluorescence microscopy and 72 - h relative viability both represented by the z - score - scaled AUC of the dose - response curves for four RAF inhibitors and one MEK inhibitor across 8 / 10 cell lines investigated in this study .

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Pairwise Spearman's correlation between the 24-h changes in pS6(Ser235/236) levels as measured by immunofluorescence microscopy and 72-h relative viability both represented by the z-score-scaled AUC of the dose-response curves for four RAF inhibitors and one MEK inhibitor across 8/10 cell lines investigated in this study.


Pairwise Spearman ' s correlation between the 24 - h p - cJun levels as measured by immunofluorescence microscopy and 72 - h relative viability after treatment with PLX4720 for 8 cell lines . p - cJun levels are averaged over five doses ( 0 . 1 - 5 μM ) , and viability is represented by the AUC of the dose - response curves .

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Pairwise Spearman's correlation between the 24-h p-cJun levels as measured by immunofluorescence microscopy and 72-h relative viability after treatment with PLX4720 for 8 cell lines. p-cJun levels are averaged over five doses (0.1-5 μM), and viability is represented by the AUC of the dose-response curves.


Comparison of the level of 24 - h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720 .

Comparison of the level of 24-h pS6 inhibition for 8 cell lines treated with SB590885 versus PLX4720.


Comparison of the level of 24 - h pS6 inhibition between two cell lines ( C32 and WM1552C ) treated with four RAF inhibitors and one MEK

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Comparison of the level of 24-h pS6 inhibition between two cell lines (C32 and WM1552C) treated with four RAF inhibitors and one MEK inhibitor


DRiP labelling in GFP - NCL HeLa Kyoto cells that were either left untreated or treated with OP - puro ( 25 µM ) for 2 h . Where indicated cells were allowed to recover in drug - free medium ( control ) or in presence of MG132 ( 10 µM ) for 5 h . Scale bars : 5 µm . GFP - NCL HeLa Kyoto cells were treated as described in A , with the exception that a lower concentration of OP - puro was used ( 5 µM ) . The distribution of DRiPs and GFP - NCL was analyzed by STED super - resolution microscopy . Scale bars : 5 µm .

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DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with OP-puro (25 µM) for 2 h. Where indicated cells were allowed to recover in drug-free medium (control) or in presence of MG132 (10 µM) for 5 h. Scale bars: 5 µm. GFP-NCL HeLa Kyoto cells were treated as described in A, with the exception that a lower concentration of OP-puro was used (5 µM). The distribution of DRiPs and GFP-NCL was analyzed by STED super-resolution microscopy. Scale bars: 5 µm.


Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin ( 25 µM ) for 2 h . Puromycylated proteins were detected by immunoblotting ( right panel ) . The left panel shows the purity of isolated nucleoli by western blotting , where fibrillarin was used as nucleolar marker , FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker .

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Isolation of nucleoli from HeLa Kyoto cells untreated or treated with puromycin (25 µM) for 2 h. Puromycylated proteins were detected by immunoblotting (right panel). The left panel shows the purity of isolated nucleoli by western blotting, where fibrillarin was used as nucleolar marker, FUS as nucleoplasmic marker and TUBA4A as cytoplasmic marker.


HeLa cells were left untreated or treated with heat shock ( HS ) at 42 ˚ C for 3 h , alone or with cycloheximide ( CHX ; 50 μg / ml ) , or Actinomycin D ( Act . D ; 3 µM ) followed by staining with the dye Amylo - glo .

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HeLa cells were left untreated or treated with heat shock (HS) at 42˚C for 3 h, alone or with cycloheximide (CHX; 50 μg/ml), or Actinomycin D (Act. D; 3 µM) followed by staining with the dye Amylo-glo.


DRiP labelling in GFP - NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42 ˚ C and OP - puro ( 25 µM ) for 2 h . Where indicated , cells were cotreated with CHX ( 50 μg / ml ) .

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DRiP labelling in GFP-NCL HeLa Kyoto cells that were either left untreated or treated with HS at 42˚C and OP-puro (25 µM) for 2 h. Where indicated, cells were cotreated with CHX (50 μg/ml).


HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42 ˚ C for 2 h , alone or combined with CHX , in GFP - NCL HeLa Kyoto cells .

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HSPA8 subcellular distribution and recruitment inside nucleoli following exposure to HS at 42˚C for 2 h, alone or combined with CHX, in GFP-NCL HeLa Kyoto cells.


DRiP labelling in HeLa cells treated with MG132 ( 10 µM ) alone or with CHX ( 50 μg / ml ) for 8 h ; where indicated , OP - puro ( 25 µM ) was added during the last 45 min , prior to cell fixation .

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DRiP labelling in HeLa cells treated with MG132 (10 µM) alone or with CHX (50 μg/ml) for 8 h; where indicated, OP-puro (25 µM) was added during the last 45 min, prior to cell fixation.


Amylo - glo staining of HeLa cells left untreated or exposed to MG132 ( 10 µM ) alone or combined with CHX ( 50 μg / ml ) for 8 or 13 h .

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Amylo-glo staining of HeLa cells left untreated or exposed to MG132 (10 µM) alone or combined with CHX (50 μg/ml) for 8 or 13 h.


Amylo - glo staining of HeLa cells left untreated or subjected to transcriptional stress ( MG132 10 µM and Act . D 4 µM ) for 6 hrs . Where indicated , translation was concomitantly inhibited with CHX ( 50 μg / ml ) . Amylo - Glo staining is also shown in HeLa cells treated with Act . D alone or with CHX for 6 hrs , as control .

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Amylo-glo staining of HeLa cells left untreated or subjected to transcriptional stress (MG132 10 µM and Act. D 4 µM) for 6 hrs. Where indicated, translation was concomitantly inhibited with CHX (50 μg/ml). Amylo-Glo staining is also shown in HeLa cells treated with Act.D alone or with CHX for 6 hrs, as control.


Amylo - glo staining of HeLa cells exposed to HS at 42 ˚ C for 3 h and let to recover for 3 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated cells were treated with VER alone ( VER 3 h ) .

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Amylo-glo staining of HeLa cells exposed to HS at 42˚C for 3 h and let to recover for 3 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 3 h).


Amylo - glo staining of HeLa cells treated with MG132 ( 10 µM ) for 13 h and let to recover for 5 h in drug - free medium ( + rec . control ) or with VER ( 40 µM ; + rec . VER ) . Where indicated cells were treated with VER alone ( VER 5 h ) .

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Amylo-glo staining of HeLa cells treated with MG132 (10 µM) for 13 h and let to recover for 5 h in drug-free medium (+ rec. control) or with VER (40 µM; + rec. VER). Where indicated cells were treated with VER alone (VER 5 h).


HeLa cells were transfected with vectors encoding for GFP , GFP - GR50 or RPL23a - GFP . 24 hrs post - transfection cells were fixed and stained with Amylo - glo . The percentage of transfected cells with Amylo - Glo positive nucleoli is shown . Number of cells counted / condition : GFP , 412 ; RPL23a - GFP , 482 ; GFP - GR50 , 359 , in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 7 , ± s . e . m .

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HeLa cells were transfected with vectors encoding for GFP, GFP-GR50 or RPL23a-GFP. 24 hrs post-transfection cells were fixed and stained with Amylo-glo. The percentage of transfected cells with Amylo-Glo positive nucleoli is shown. Number of cells counted/condition: GFP, 412; RPL23a-GFP, 482; GFP-GR50, 359, in three independent experiments; statistical significance via One-way ANOVA; p < 10-7, ± s.e.m.


DRiPs , polyUb proteins ( FK1 ) and PML labelling in HeLa cells treated with OP - puro ( 25 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h ; where indicated cycloheximide was also added ( CHX ; 50 μg / ml ) . Scale bars : 5 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa cells treated with a low concentration of OP - puro ( 5 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h . PML - NBs were visualized using a PML specific antibody , followed by incubation with alexa fluor 647 . For convenience the alexa fluor 647 signal is shown in green . Scale bars : 5 µm .

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DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h; where indicated cycloheximide was also added (CHX; 50 μg/ml). Scale bars: 5 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and heat shock (HS) at 42˚C for 2 h. PML-NBs were visualized using a PML specific antibody, followed by incubation with alexa fluor 647. For convenience the alexa fluor 647 signal is shown in green. Scale bars: 5 µm.


PolyUb proteins ( FK1 ) labelling in HeLa cells left untreated or treated with HS at 42 ˚ C for 2 h , alone or combined with CHX ( 50 μg / ml ) . Scale bars : 5 µm .

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PolyUb proteins (FK1) labelling in HeLa cells left untreated or treated with HS at 42˚C for 2 h, alone or combined with CHX (50 μg/ml). Scale bars: 5 µm.


STED super - resolution microscopy showing colocalization of endogenous polyUb proteins ( FK1 ) and PML - NBs in PML - GFP HeLa cells treated with HS at 42 ˚ C for 2 h . Scale bars : 5 µm .

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STED super-resolution microscopy showing colocalization of endogenous polyUb proteins (FK1) and PML-NBs in PML-GFP HeLa cells treated with HS at 42˚C for 2 h. Scale bars: 5 µm.


Quantitation of cells shown in A - C . Number of cells counted / condition : 712 - 1343 in three independent experiments ; statistical significance via One - way ANOVA ; p < 10 - 5 , ± s . e . m .

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Quantitation of cells shown in A-C. Number of cells counted/condition: 712 - 1343 in three independent experiments; statistical significance via One-way ANOVA; p < 10-5, ± s.e.m.


DRiPs , polyUb proteins ( FK1 ) and PML labelling in HeLa cells treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h ; where indicated , cycloheximide was also added ( CHX ; 50 μg / ml ) . Asterisks ( * ) indicate nucleoli . Scale bars : 10 µm . Lower panel : STED super - resolution microscopy showing colocalization of DRiPs with PML - NBs in PML - GFP HeLa cells treated with a low concentration of OP - puro ( 5 µM ) and MG132 ( 10 µM ) for 4 hrs . Scale bars : 5 µm . Quantitation of cells shown in E . Number of cells counted / condition : 298 - 1713 in three independent experiments ; statistical significance via One - way ANOVA ; p ≤ 0 . 001 , ± s . e . m .

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DRiPs, polyUb proteins (FK1) and PML labelling in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h; where indicated, cycloheximide was also added (CHX; 50 μg/ml). Asterisks (*) indicate nucleoli. Scale bars: 10 µm. Lower panel: STED super-resolution microscopy showing colocalization of DRiPs with PML-NBs in PML-GFP HeLa cells treated with a low concentration of OP-puro (5 µM) and MG132 (10 µM) for 4 hrs. Scale bars: 5 µm. Quantitation of cells shown in E. Number of cells counted/condition: 298 - 1713 in three independent experiments; statistical significance via One-way ANOVA; p ≤ 0.001, ± s.e.m.


Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Asterisks ( * ) indicate nucleoli . Scale bars : 10 µm .

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Intranuclear distribution of 20S proteasomes in HeLa cells left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Asterisks (*) indicate nucleoli. Scale bars: 10 µm.


HeLa cells were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , total proteins were extracted and expression levels of PML were analyzed by immunoblotting . TUBA4A was used as loading control .

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HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, total proteins were extracted and expression levels of PML were analyzed by immunoblotting. TUBA4A was used as loading control.


HeLa cells were lipofected with non - targeting siRNA control or a specific siRNA against PML . 72 h post - transfection , cells were treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Left panel : automated quantitation of the number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 334 - 364 ; p < 10 - 10 . Right panel : automated quantitation of the number of DRiP foci / nucleus . Cell number analyzed : 334 - 364 ; p < 10 - 10 . Automated DRiP segmentation is based on azide signal .

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HeLa cells were lipofected with non-targeting siRNA control or a specific siRNA against PML. 72 h post-transfection, cells were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Left panel: automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 334 - 364; p < 10-10. Right panel: automated quantitation of the number of DRiP foci/nucleus. Cell number analyzed: 334 - 364; p < 10-10. Automated DRiP segmentation is based on azide signal.


DRiPs and polyUb ( FK1 ) protein distribution in cells treated as described in ( A ) . Automated DRiP segmentation is based on azide signal . Quantitation of the number of DRiP foci enriched for polyUb ( > 2 ) is shown ; n = 590 - 2114 ; statistical significance via 2 ‐ tailed Student ' s t ‐ test ; p < 10 - 10 . Scale bars : 10 µm .

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DRiPs and polyUb (FK1) protein distribution in cells treated as described in (A). Automated DRiP segmentation is based on azide signal. Quantitation of the number of DRiP foci enriched for polyUb (>2) is shown; n = 590 - 2114; statistical significance via 2‐tailed Student's t‐test; p < 10-10. Scale bars: 10 µm.


Immunoblotting on protein extracts from HeLa cells lipofected with non - targeting siRNA control or a specific siRNA against Ubc9 . A dilution curve of the control sample is shown . TUBA4A was used as loading control . Quantitation of the number of PML - NBs / nucleus from cells treated as described in ( D ) . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal .

Immunoblotting on protein extracts from HeLa cells lipofected with non-targeting siRNA control or a specific siRNA against Ubc9. A dilution curve of the control sample is shown. TUBA4A was used as loading control. Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal.


Cells lipofected for 72 h with non - targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Automated quantitation of the number of PML - NBs / nucleus is reported . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 151 - 225 ; p < 10 - 10 ( see panel G ) .

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Cells lipofected for 72 h with non-targeting siRNA control or a specific siRNA against Ubc9 were treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Automated quantitation of the number of PML-NBs/nucleus is reported. Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 151 - 225; p < 10-10 (see panel G).


Quantitation of the number of PML - NBs / nucleus from cells treated as described in ( D ) . Statistical significance via 2 ‐ tailed Student ' s t ‐ test . Cell number analyzed : 192 - 430 ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal . Representative pictures showing that Ubc9 - depleted cells cannot efficiently induce the self - assembly of PML - NBs upon treatment with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Scale bars : 10 µm .

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Quantitation of the number of PML-NBs/nucleus from cells treated as described in (D). Statistical significance via 2‐tailed Student's t‐test. Cell number analyzed: 192 - 430; p < 10-10. Automated PML-NB segmentation is based on PML signal. Representative pictures showing that Ubc9-depleted cells cannot efficiently induce the self-assembly of PML-NBs upon treatment with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Scale bars: 10 µm.


PolyUb protein ( FK1 ) labelling in PML - GFP HeLa Kyoto cells that were either left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Cells were either immediately fixed or let to recover for 5 h in drug - free medium ( Control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Scale bars : 10 µm . Quantitation of the % of cells with polyUb ( FK1 ) nuclear foci shown in ( A ) . Number of cells counted / condition : 310 - 1258 in three independent experiments ; statistical significance via One - way ANOVA ; p = 0 . 0003 or 10 - 6 , ± s . e . m .

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PolyUb protein (FK1) labelling in PML-GFP HeLa Kyoto cells that were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Cells were either immediately fixed or let to recover for 5 h in drug-free medium (Control), with VER (40 µM) or Eeyarestatin I (5 µM). Scale bars: 10 µm. Quantitation of the % of cells with polyUb (FK1) nuclear foci shown in (A). Number of cells counted/condition: 310 - 1258 in three independent experiments; statistical significance via One-way ANOVA; p = 0.0003 or 10-6, ± s.e.m.


Quantitation of the number of PML - NBs enriched for polyUb ( > 2 ) ; n = 611 - 2612 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal .

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Quantitation of the number of PML-NBs enriched for polyUb (>2); n = 611 - 2612; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.


Quantitation of the % of cells with polyUb proteins ( FK1 ) sequestered in nuclear foci . HeLa cells were treated with OP - puro ( 25 µM ) and heat shock ( HS ) at 42 ˚ C for 2 h . Then , cells were either immediately fixed or let to recover for 3 h in drug - free medium ( control ) , with VER ( 40 µM ) or Eeyarestatin I ( 5 µM ) . Number of cells counted / condition : 593 - 1016 in three independent experiments ; statistical significance via One - way ANOVA ; p = 10 - 3 , ± s . e . m .

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Quantitation of the % of cells with polyUb proteins (FK1) sequestered in nuclear foci. HeLa cells were treated with OP-puro (25 µM) and heat shock (HS) at 42˚C for 2 h. Then, cells were either immediately fixed or let to recover for 3 h in drug-free medium (control), with VER (40 µM) or Eeyarestatin I (5 µM). Number of cells counted/condition: 593 - 1016 in three independent experiments; statistical significance via One-way ANOVA; p = 10-3, ± s.e.m.


Quantitation of the number of PML - NBs enriched for polyUb ( > 2 ) in HeLa cells treated as described in ( D ) but with a recovery time of 2 h ; n = 1155 - 3283 ; statistical significance via One - way ANOVA ; p < 10 - 10 . Automated PML - NB segmentation is based on PML signal .

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Quantitation of the number of PML-NBs enriched for polyUb (>2) in HeLa cells treated as described in (D) but with a recovery time of 2 h; n = 1155 - 3283; statistical significance via One-way ANOVA; p < 10-10. Automated PML-NB segmentation is based on PML signal.


Amylo - Glo staining of PML - GFP HeLa Kyoto cells that were subjected to the following treatments : untreated ( Control ) ; MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h , alone or combined with CHX ( 50 μg / ml ) . Where indicated cells were allowed to recover for 5 hrs in drug - free medium ( Control ) or with VER ( 40 µM ) . Confocal images showing Amylo - Glo and PML - GFP . Scale bars : 5 µm .

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Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to the following treatments: untreated (Control); MG132 (10 µM) and OP-puro (25 µM) for 4 h, alone or combined with CHX (50 μg/ml). Where indicated cells were allowed to recover for 5 hrs in drug-free medium (Control) or with VER (40 µM). Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.


Amylo - Glo staining of PML - GFP HeLa Kyoto cells that were subjected to heat shock ( HS ) at 42 ˚ C for 2 hrs . Confocal images showing Amylo - Glo and PML - GFP . Scale bars : 5 µm .

Amylo-Glo staining of PML-GFP HeLa Kyoto cells that were subjected to heat shock (HS) at 42˚C for 2 hrs. Confocal images showing Amylo-Glo and PML-GFP. Scale bars: 5 µm.


PML - GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry - VHL . 24 h post - transfection , cells were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h . Where indicated ( OP - puro + MG132 + CHX ) , translation was inhibited during stress with cycloheximide ( CHX ; 50 μg / ml ) . In the lower panel , cells were allowed to recover for 5 h in drug - free medium ( Control ) , or with VER ( 40 µM ) . The subcellular distribution of PML - GFP and mCherry - VHL was studied by live - cell confocal imaging . Representative pictures are shown . Scale bars : 5 µm . Quantitation of the fluorescence intensity recovery after bleach of PML - GFP . PML - GFP HeLa Kyoto overexpressing mCherry - VHL for 24 h and treated as described in ( A ) were analyzed . The mean of 19 - 23 FRAP curves and the fitting curves are shown in black and red respectively . In gray , the SD is shown .

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PML-GFP HeLa Kyoto cells were lipofected with a cDNA encoding for mCherry-VHL. 24 h post-transfection, cells were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h. Where indicated (OP-puro + MG132 + CHX), translation was inhibited during stress with cycloheximide (CHX; 50 μg/ml). In the lower panel, cells were allowed to recover for 5 h in drug-free medium (Control), or with VER (40 µM). The subcellular distribution of PML-GFP and mCherry-VHL was studied by live-cell confocal imaging. Representative pictures are shown. Scale bars: 5 µm. Quantitation of the fluorescence intensity recovery after bleach of PML-GFP. PML-GFP HeLa Kyoto overexpressing mCherry-VHL for 24 h and treated as described in (A) were analyzed. The mean of 19-23 FRAP curves and the fitting curves are shown in black and red respectively. In gray, the SD is shown.


Intranuclear distribution of 20S proteasomes , HSP70 , VCP and DRiPs in HeLa cells treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h and allowed to recover for 5 h in drug - free medium ( control ) , with VER ( 40 µM ) , Eeyarestatin I ( 5 µM ) or MG132 ( 10 µM ) . Scale bars : 5 µm .

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Intranuclear distribution of 20S proteasomes, HSP70, VCP and DRiPs in HeLa cells treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h and allowed to recover for 5 h in drug-free medium (control), with VER (40 µM), Eeyarestatin I (5 µM) or MG132 (10 µM). Scale bars: 5 µm.


GFP - PSMA7 HeLa Kyoto cells were left untreated or treated as described in A . Cells were subjected to FRAP to analyze GFP - PSMA7 mobility . Quantitation of three populations of GFP - PSMA7 molecules is shown : fast moving , slow moving and immobile ; statistical significance via One - way ANOVA ; n = 12 - 14 ± SEM .

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GFP-PSMA7 HeLa Kyoto cells were left untreated or treated as described in A. Cells were subjected to FRAP to analyze GFP-PSMA7 mobility. Quantitation of three populations of GFP-PSMA7 molecules is shown: fast moving, slow moving and immobile; statistical significance via One-way ANOVA; n = 12 - 14 ± SEM.


Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) for 4 h , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) . Where indicated , cells were treated with MG132 , VER or EeyI alone for 5 h . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( A ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 1112 - 1537 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

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Subcellular distribution of 53BP1 and DRiPs in HeLa cells that were left untreated or treated with MG132 (10 µM) and OP-puro (25 µM) for 4 h, followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI). Where indicated, cells were treated with MG132, VER or EeyI alone for 5 h. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (A) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 1112 - 1537 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


HeLa cells were lipofected with cDNAs encoding for HA - Ubiquitin , Flag - Ubiquitin or Flag - VCP 24 h post - transfection , cells were either left untreated or treated with MG132 ( 10 µM ) and OP - puro ( 25 µM ) . Cells were processed for staining of HA , Flag and endogenous 53BP1 . * indicates cells that do not express HA - or Flag - Ubiquitin and show no rescue of 53BP1 foci formation after stress . Scale bars : 20 µm . Quantitation of the % of cells with 53BP1 foci . Cells were treated as described in ( C ) and divided in three categories , based on the number of 53BP1 foci / nucleus : 0 , 1 - 2 and > 3 . Number of cells counted / condition : 710 - 938 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 01 .

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HeLa cells were lipofected with cDNAs encoding for HA-Ubiquitin, Flag-Ubiquitin or Flag-VCP 24 h post-transfection, cells were either left untreated or treated with MG132 (10 µM) and OP-puro (25 µM). Cells were processed for staining of HA, Flag and endogenous 53BP1. * indicates cells that do not express HA- or Flag-Ubiquitin and show no rescue of 53BP1 foci formation after stress. Scale bars: 20 µm. Quantitation of the % of cells with 53BP1 foci. Cells were treated as described in (C) and divided in three categories, based on the number of 53BP1 foci/nucleus: 0, 1-2 and > 3. Number of cells counted/condition: 710 - 938 in three independent experiments; statistical significance via One-way ANOVA; p < 0.01.


Colony formation assay . The number of colonies surviving 10 days after plating is shown . Control cells or cells treated with MG132 ( 10 µM ) and puromycin ( 5 µg / ml ) , followed by 5h recovery in drug - free medium ( + rec . control ) , with VER ( 40 µM ; + rec . VER ) or Eeyarestatin I ( 5 µM ; + rec . EeyI ) are shown . Where indicated , cells were treated with VER or EeyI alone for 5 h . Number of colonies counted / condition : up to 526 in three independent experiments ; statistical significance via One - way ANOVA ; p < 0 . 03 , ± s . e . m .

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Colony formation assay. The number of colonies surviving 10 days after plating is shown. Control cells or cells treated with MG132 (10 µM) and puromycin (5 µg/ml), followed by 5h recovery in drug-free medium (+ rec. control), with VER (40 µM; + rec. VER) or Eeyarestatin I (5 µM; + rec. EeyI) are shown. Where indicated, cells were treated with VER or EeyI alone for 5 h. Number of colonies counted/condition: up to 526 in three independent experiments; statistical significance via One-way ANOVA; p < 0.03, ± s.e.m.


A , B Functional freeMitos association with PMN . Overlay dot plot population , representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN ( anti - CD11b - PE ) and freeMitos . Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed . CD11b - labeled PMN were incubated in absence ( A ) or presence ( B ) of freeMitos at a 1 : 5 ratio ( PMN : mitochondria ) .

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A, B Functional freeMitos association with PMN. Overlay dot plot population, representative of 4 biological replicates of the flow cytometry gating strategy used to quantify the interaction between PMN (anti-CD11b-PE) and freeMitos. Freshly isolated platelets were labeled with MitoTrackerTM Deep Red and freeMitos isolation was subsequently performed. CD11b-labeled PMN were incubated in absence (A) or presence (B) of freeMitos at a 1:5 ratio (PMN:mitochondria).


C Confocal microscopy imaging of PMN cell membrane ( red ) labeled with CellMask Orange in absence ( upper panel ) or presence ( lower panel ) of MitoTrackerTM Deep Red - labeled freeMitos ( magenta ) . PMN nuclei were stained with DAPI ( cyan ) . The white scale bars shown in the lower right of the images represent 10 μm in size .

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C Confocal microscopy imaging of PMN cell membrane (red) labeled with CellMask Orange in absence (upper panel) or presence (lower panel) of MitoTrackerTM Deep Red-labeled freeMitos (magenta). PMN nuclei were stained with DAPI (cyan). The white scale bars shown in the lower right of the images represent 10 μm in size.


D , E FreeMitos associate with PMN and modulate gene expression . Volcano plot , of 4 biological replicates , representing the gene expression modulation induced by mitochondrial DAMPs preparation ( D ) and freshly isolated functional freeMitos ( E ) in PMN .

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D, E FreeMitos associate with PMN and modulate gene expression. Volcano plot, of 4 biological replicates, representing the gene expression modulation induced by mitochondrial DAMPs preparation (D) and freshly isolated functional freeMitos (E) in PMN.


FreeMitos effect on the respiratory state of PMN . O2 consumption rates of resting or activated ( incubated with TNF - a and GM - CSF ) PMN were measured using high - resolution O2 respirometry ( Oroboros Oxygraph ) and revealed no significant differences between the basal state ( left panel ) , leak state ( middle panel ) and the maximum electron transport system capacity ( ETS , right panel ) of cellular respiration ( F ) . Data information : For panels F data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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FreeMitos effect on the respiratory state of PMN. O2 consumption rates of resting or activated (incubated with TNF-a and GM-CSF) PMN were measured using high-resolution O2 respirometry (Oroboros Oxygraph) and revealed no significant differences between the basal state (left panel), leak state (middle panel) and the maximum electron transport system capacity (ETS, right panel) of cellular respiration (F). Data information: For panels F data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels F Values without at least one common superscript letter are different (P < 0.05).


Immunoblot of mitochondrial cytochrome c oxidase ( COX IV ) and histone H3 as a loading control of PMN after co - incubations with freeMitos ( G ) with subsequent densitometry analysis ( H ) . Data information : For panels H , data are shown as means ± SEM , of 3 biological replicates . Two - way ANOVA tests followed by Tukey ' s multiple comparisons tests for each sample was performed for panels H . Values without at least one common superscript letter are different ( P < 0 . 05 ) .

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Immunoblot of mitochondrial cytochrome c oxidase (COX IV) and histone H3 as a loading control of PMN after co-incubations with freeMitos (G) with subsequent densitometry analysis (H). Data information: For panels H, data are shown as means ± SEM, of 3 biological replicates. Two-way ANOVA tests followed by Tukey's multiple comparisons tests for each sample was performed for panels H. Values without at least one common superscript letter are different (P < 0.05).


A , B FreeMitos induce PMN intracellular calcium release in PMN . Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co - incubated with 5 x 106 mitochondria ( A ) . Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo - 3 AM . The gray line represents PMN with the vehicle ( HBSS ) while the dark line represents PMN incubated with mitochondria . PMN intracellular calcium mobilization induced by either the vehicle ( HBSS ) , freeMitos or mitochondrial DAMPs and quantified by flow cytometry ( B ) . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel B , One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels B , Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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A, B FreeMitos induce PMN intracellular calcium release in PMN. Representative visualization of intracellular calcium monitoring by flow cytometry of 1 x 106 PMN co-incubated with 5 x 106 mitochondria (A). Calcium mobilization was monitored by flow cytometry for 120 seconds using the fluorescent probe Fluo-3 AM. The gray line represents PMN with the vehicle (HBSS) while the dark line represents PMN incubated with mitochondria. PMN intracellular calcium mobilization induced by either the vehicle (HBSS), freeMitos or mitochondrial DAMPs and quantified by flow cytometry (B). Data information: Data are shown as means ± SEM of 3 biological replicates (panel B, One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels B, Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


Functional freeMitos induce the release of PMN - derived microvesicles . A dose - response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles . PMN microvesicles labeling was performed with either anti - CD66b - FITC labelling ( C , left panel ) or anti - CD11b - PE ( C , right panel ) . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel C two - way ( panel C ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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Functional freeMitos induce the release of PMN-derived microvesicles. A dose-response analysis of various mitochondria to PMN ratios was performed to measure the release of PMN microvesicles. PMN microvesicles labeling was performed with either anti-CD66b-FITC labelling (C, left panel) or anti-CD11b-PE (C, right panel). Data information: Data are shown as means ± SEM of 4 biological replicates (panel C two-way (panel C) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


PMN - derived microparticle gating strategy is shown in panel D . Flow cytometry panel established to distinguish the PMNL - MVs from the background using the leucocyte specific integrin alpha - M beta - 2 ( CD11b ) , the granulocyte specific carcinoembryonic antigen - related cell adhesion molecule 8 ( CD66b ) , and size reference beads . PMN were either incubated with the vehicle ( Tyrode ' s Buffer , upper panels ) or freeMitos ( lower panels ) . Following co - incubation , PMN were washed twice and resuspended in HBSS before processing on flow cytometer . Only single events , identified by the singlets gate ( left upper and lower panels ) using side scatter area ( SSC - A ) vs side scatter heights ( SSC - H ) were analyzed . Light scatter ( side scatter ( SSC ) and forward scatter ( FSC ) ) approach for determining the background noise in HBSS . Histogram displaying the fluorescence intensity of samples labelled with either CD11b - FITC or CD66b - PE . Overlay dot plot of PMNL - MVs population and size reference beads , representative of 4 biological replicates , each performed in 2 technical replicates .

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PMN-derived microparticle gating strategy is shown in panel D. Flow cytometry panel established to distinguish the PMNL-MVs from the background using the leucocyte specific integrin alpha-M beta-2 (CD11b), the granulocyte specific carcinoembryonic antigen-related cell adhesion molecule 8 (CD66b), and size reference beads. PMN were either incubated with the vehicle (Tyrode's Buffer, upper panels) or freeMitos (lower panels). Following co-incubation, PMN were washed twice and resuspended in HBSS before processing on flow cytometer. Only single events, identified by the singlets gate (left upper and lower panels) using side scatter area (SSC-A) vs side scatter heights (SSC-H) were analyzed. Light scatter (side scatter (SSC) and forward scatter (FSC)) approach for determining the background noise in HBSS. Histogram displaying the fluorescence intensity of samples labelled with either CD11b-FITC or CD66b-PE. Overlay dot plot of PMNL-MVs population and size reference beads, representative of 4 biological replicates, each performed in 2 technical replicates.


E Visual confirmation of PMN and PMN - derived microvesicles . Transmission electron microscopy ( TEM ) imaging of PMN - derived microvesicles ( black arrowhead ) , representative of 3 biological replicates , from resting or activated PMN ( 106 cells ) incubated with either mitochondria , mitochondrial DAMPs or fMLP as positive control . The black scale bars are shown in the lower right of the images for the vehicles ( 400 nm ) , mitochondria ( 600 nm ) , DAMPs ( 600 nm ) and fMLP ( 500 nm ) conditions .

E Visual confirmation of PMN and PMN-derived microvesicles. Transmission electron microscopy (TEM) imaging of PMN-derived microvesicles (black arrowhead), representative of 3 biological replicates, from resting or activated PMN (106 cells) incubated with either mitochondria, mitochondrial DAMPs or fMLP as positive control. The black scale bars are shown in the lower right of the images for the vehicles (400 nm), mitochondria (600 nm), DAMPs (600 nm) and fMLP (500 nm) conditions.


F Quantification of resting PMN microvesicles produced by PMN ( 106 cells ) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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F Quantification of resting PMN microvesicles produced by PMN (106 cells) incubated with 107 freeMitos or 25 µg of mitochondrial DAMPs for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


G Quantification of calpain activity , by fluorescence assay , in cell lysate of either resting ( left panel ) or activated ( right panel ) PMN ( 2 x 105 cells ) incubated in presence of freeMitos for 2 hr . Data information : Data are shown as means ± SEM of 4 biological replicates ( panel G ) . One - way ANOVA tests , followed by Tukey ' s multiple comparisons tests were performed for panels G . Values without one common superscript letter were different ( P < 0 . 05 ) . ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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G Quantification of calpain activity, by fluorescence assay, in cell lysate of either resting (left panel) or activated (right panel) PMN (2 x 105 cells) incubated in presence of freeMitos for 2 hr. Data information: Data are shown as means ± SEM of 4 biological replicates (panel G). One-way ANOVA tests, followed by Tukey's multiple comparisons tests were performed for panels G. Values without one common superscript letter were different (P < 0.05). ; *P<0.05, **P<0.01 and ***P<0.001.


H ELISA quantification of IL - 8 concentrations present in the supernatant following the incubation of PMN ( 106 cells ) with freeMitos ( 107 ) , DAMPs ( 25 µg ) or fMLP ( 1 µM ) for 2 hr . Data information : Data are shown as means ± SEM of 3 biological replicates ( panel H ) One - way ( panel H ) ANOVA tests , followed by Dunnett ' s multiple comparisons test were performed and compared to the vehicle ( HBSS ) ; * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 .

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H ELISA quantification of IL-8 concentrations present in the supernatant following the incubation of PMN (106 cells) with freeMitos (107), DAMPs (25 µg) or fMLP (1 µM) for 2 hr. Data information: Data are shown as means ± SEM of 3 biological replicates (panel H) One-way (panel H) ANOVA tests, followed by Dunnett's multiple comparisons test were performed and compared to the vehicle (HBSS); *P<0.05, **P<0.01 and ***P<0.001.


( C ) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA . The dCas9 - ω was verified in the ∆ rpoZ strain background , and the rest of candidates were verified in the wild - type strain ( BW25113 ) .

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(C) Fold induction of the fluorescence of CasTA candidates using different gRNAs targeting to different locations of the GFP reporter gene compared to a strain without CasTA. The dCas9-ω was verified in the ∆rpoZ strain background, and the rest of candidates were verified in the wild-type strain (BW25113).


( D ) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9 - AsiA with gRNA - H3 under kanamycin selection ( 2 . 5 μg / ml ) .

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(D) Survival of cells containing upregulated antibiotic resistance reporter induced by dCas9 or dCas9-AsiA with gRNA-H3 under kanamycin selection (2.5 μg/ml).


( E ) Different gRNAs were paired with dCas9 - AsiA and dCas9 - ω to profile the optimal gRNA binding distance . The dCas9 - AsiA was verified in the wild - type strain ( BW25113 ) , and dCas9 - ω was examined in the ∆ rpoZ strain background .

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(E) Different gRNAs were paired with dCas9-AsiA and dCas9-ω to profile the optimal gRNA binding distance. The dCas9-AsiA was verified in the wild-type strain (BW25113), and dCas9-ω was examined in the ∆rpoZ strain background.


( F ) Comparing single and multiple gRNA with dCas9 - AsiA .

(F) Comparing single and multiple gRNA with dCas9-AsiA.


( C ) Distribution of fluorescence signal of the GFP reporter induced by different dCas9 - AsiA variants ( top ) . Fold induction by different dCas9 - AsiA variants is shown ( bottom ) .

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(C) Distribution of fluorescence signal of the GFP reporter induced by different dCas9-AsiA variants (top). Fold induction by different dCas9-AsiA variants is shown (bottom).


( D ) CRISPRa induction of promoters with varying basal expression levels . CRISPRa - , basal expression of the promoter ; CRISPRa + , expression activated by dCas9 - AsiA - m2 . 1 and associated gRNA - H4 .

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(D) CRISPRa induction of promoters with varying basal expression levels. CRISPRa-, basal expression of the promoter; CRISPRa+, expression activated by dCas9-AsiA-m2.1 and associated gRNA-H4.


( A ) CasTA2 . 1 upregulates a genomically inserted GFP reporter .

(A) CasTA2.1 upregulates a genomically inserted GFP reporter.


( B ) Chromosomal gene targets activated by CasTA2 . 1 with bars showing the activation fold change and dots showing basal expression of each gene . Expression was quantified using RT - qPCR .

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(B) Chromosomal gene targets activated by CasTA2.1 with bars showing the activation fold change and dots showing basal expression of each gene. Expression was quantified using RT-qPCR.


( C ) CasTA2 . 1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene . A non - specific gRNA is used as the negative control .

(C) CasTA2.1 can mediate CRISPRi with appropriate gRNA designs by positioning different gRNAs relative to the target gene. A non-specific gRNA is used as the negative control.


( D ) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2 . 1 on a reporter containing GFP and mScarlet . Parental cells had low basal GFP and high basal mScarlet expression .

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(D) Demonstration of multiplexed CRISPRa and CRISPRi using CasTA2.1 on a reporter containing GFP and mScarlet. Parental cells had low basal GFP and high basal mScarlet expression.


( B ) Promoters significantly activated by CasTA2 . 1 using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted with basal expression level on x - axis against fold activation by CRISPRa on y - axis . N is the total number of promoters shown . Red box corresponds to highly activated promoters ( fold change > 10 ) .

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(B) Promoters significantly activated by CasTA2.1 using gRNA-H23 (left) or gRNA-H22 (right) are plotted with basal expression level on x-axis against fold activation by CRISPRa on y-axis. N is the total number of promoters shown. Red box corresponds to highly activated promoters (fold change >10).


( C ) Highly activated promoters ( > 10 - fold ) using gRNA - H23 ( left ) or gRNA - H22 ( right ) are plotted . Basal expression levels ( purple lines ) , induced expression level ( orange lines on left or red lines on right , activated with gRNA - H23 or gRNA - H22 , respectively ) and induced fold changes ( gray bars ) are shown . Promoters were first ranked and grouped by basal expression levels . Within each group , promoters were ranked by activation fold changes .

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(C) Highly activated promoters (>10-fold) using gRNA-H23 (left) or gRNA-H22 (right) are plotted. Basal expression levels (purple lines), induced expression level (orange lines on left or red lines on right, activated with gRNA-H23 or gRNA-H22, respectively) and induced fold changes (gray bars) are shown. Promoters were first ranked and grouped by basal expression levels. Within each group, promoters were ranked by activation fold changes.


( B ) CRISPRi in S . enterica and K . oxytoca using CasTA2 . 1 .

(B) CRISPRi in S. enterica and K. oxytoca using CasTA2.1.


( C ) CRISPRa in S . enterica and K . oxytoca using CasTA1 . 0 ( dCas9 - AsiA _ wt ) , CasTA2 . 1 ( dCas9 - AsiA _ m2 . 1 ) or ancestral strain with basal promoter expression ( none ) .

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(C) CRISPRa in S. enterica and K. oxytoca using CasTA1.0 (dCas9-AsiA_wt), CasTA2.1 (dCas9-AsiA_m2.1) or ancestral strain with basal promoter expression (none).


B Expression levels of RBM15 and AS - RBM15 in different blood lineages by meta - analysis of RNA - seq data [ 38 ] : HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitor cells ) , CMP ( common myeloid progenitor cells ) , CLP ( common lymphoid progenitor cells ) , GMP ( Granulocyte - macrophage progenitor cells ) , MEP ( megakaryocyte - erythrocyte progenitor cells ) , MK ( megakaryocyte ) , and EB ( erythroid body ) .

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B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).


C Relative expression levels of AS - RBM15 ( left ) and RBM15 ( right ) in MEG - 01 cells treated with PMA were measured by real - time PCR assays ( n = 4 , mean ± SD ) . SD : standard deviation .

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C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.


D Relative expression levels of AS - RBM15 and RBM15 in CD34 + cells grown in pro - MK medium were measured by real - time PCR . GAPDH was used as an internal control ( n = 3 , mean ± SD ) . Also see Fig EV 1 .

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D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.


A CFU - MK assays with CD34 + cells expressing AS - RBM15 . MK colonies staining for acetylcholinesterase activity were counted ( right ) . P value was calculated using Student ' s t test ( n = 3 , mean ± SD ) .

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A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student's t test (n=3, mean ± SD).


B The number of CD41 + CD42 + mature MK cells was determined by FACS analysis . CD34 + cells expressing sh - AS - RBM15 were grown in pro - MK medium containing thrombopoietin and stem cell factor ( TPO / SCF ) for five days ( n = 4 , mean ± SD ) .

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B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).


D FACS analysis of CD34 + cells grown in pro - MK medium and expressing AS - RBM15 full length or Δ5 or Δ3 . The CD41 + CD42 + cell percentages were analyzed using one way ANOVA , * * P < 0 . 01 , ( n = 4 , mean ± SD ) .

D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or Δ5 or Δ3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).


E The percentage of CD41 + CD42 + cells in CD34 + cells expressing AS - RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro - MK medium for five days . P value was calculated by t test , p < 0 . 01 , ( n = 3 , mean ± SD ) .

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E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).


A RBM15 protein levels in leukemia cell lines ( MEG - 01 , K562 , HEL ) were assessed by Western blotting ( WB ) ( left ) ( n = 3 ) . The mRNA levels of RBM15 in cells overexpressing AS - RBM15 were measured by real - time PCR ( right , n = 3 , mean ± SD ) . GAPDH was used as an internal control for WB and PCR .

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A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.


B Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing two different shRNAs against AS - RBM15 respectively were measured using WBs and real - time PCR assays , respectively ( n = 3 , mean ± SD ) .

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B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).


C Protein levels ( left ) and mRNA levels ( right ) of RBM15 in MEG - 01 cells expressing truncated AS - RBM15 RNAs ( the schematic as shown on Fig 2C ) were measured using WB and real - time PCR respectively ( n = 3 , mean ± SD ) .

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C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).


D The role of AS - RBM15 in protein translation was assessed by reporter assays . The reporter under control of the CMV promoter contains the 5 ' UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control . The reporter plasmid was cotransfected with plasmids expressing full - length , exon 1 and Δ5 AS - RBM15 respectively . The vector reporter without 5 ' UTR is a negative control . ( n = 3 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 ) .

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D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5'UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5'UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).


A Cytoplasmic extracts from MEG - 01 cell lines stably expressing AS - RBM15 , Exon 1 , or shRNA - 02 against AS - RBM15 were fractionated through sucrose gradients ( 15 % - 60 % ) ( n = 4 ) . Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm ( A254 ) . The arrow indicates increasing polyribosome weights . Low molecular weight , LMW ; high molecular weight , HMW .

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A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.


B The distributions of RBM15 mRNA ( top ) , GAPDH mRNA ( middle ) , and AS - RBM15 ( bottom ) were measured by real - time PCR and represented as percentages of total RNA in the fractions ( n = 3 ) .

B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).


C MS2 pull - down assays : AS - RBM15 full - length ( FL ) and exon 1 were fused to MS2RNA as baits . Plasmids expressing MS2 binding protein ( HA - tagged ) and fusion baits were co - transfected into 293T cells . HA antibody was used to immunoprecipitateHA - MS2 binding protein as shown by a WB on the bottom panel . RBM15 mRNA and MS2 fusions were assessed by real - time PCR ( n = 3 , mean ± SD ) . Fold enrichment was calculated as 2 to the power of [ ( CI . R - Cb . R ) - ( CI . MS2 - Cb . MS2 ) ] . I : input i . e . the RNA in solution before immunoprecipitation , R : RBM15 mRNA , b : bead - bound mRNA , MS2 : RNA levels of MS2 fusions , C : number of cycles in real - time PCR . One - way ANONA for p value : p < 0 . 05 .

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C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2RNA as baits. Plasmids expressing MS2 binding protein ( HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitateHA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.


D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS - RBM15 - MS2 and MS2 ( n = 3 ) .

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D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).


E RBM15 mRNA interacts with AS - RBM15 via RNA - RNA interaction . AS - RBM15 - MS2 - associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15 , AS - RBM15 and MS2 under denaturing ( heat ) and non - denaturing conditions ( n = 3 ) .

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E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).


F RNase A protection assays for detecting endogenous association of AS - RBM15 and RBM15 mRNA ( n = 4 ) . RNA was extracted from MEG - 01 cells with phenol and digested with RNase A . cDNA was synthesized after RNase A treatment and detected using AS - RBM15 exon - specific primers as shown in the schematic diagram on top . RT : reverse transcriptase .

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F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.


G MEG - 01 cells expressing AS - RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes . Cells were harvested for WB using antibodies against GAPDH and RBM15 ( n = 2 ) .

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G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).


H The RBM15 5 ' UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS - RBM15 ( n = 4 , mean ± SD ) . P values were calculated by one - way ANOVA test ( * * p < 0 . 01 and * * * p < 0 . 001 ) .

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H The RBM15 5'UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).


The subcellular distribution of RBM15 and AS - RBM15 in CD34 + cells grown in TPO - containing medium for 5 days . Cytoplasmic and nuclear RNA levels of AS - RBM15 and RBM15 were normalized to GAPDH ( control for cytoplasmic fraction ) and MALAT1 ( control for nuclear fraction ) , respectively , using the ΔΔCt method ( n = 4 , mean ± SD ) .

The subcellular distribution of RBM15 and AS-RBM15 in CD34+ cells grown in TPO-containing medium for 5 days. Cytoplasmic and nuclear RNA levels of AS-RBM15 and RBM15 were normalized to GAPDH (control for cytoplasmic fraction) and MALAT1 (control for nuclear fraction), respectively, using the ΔΔCt method (n=4, mean ± SD).