A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM , the neuronal specific anti - NCAM antibody and the nuclear staining Hoechst . Left panel , basal conditions . Right panel , after addition of the mitochondrial uncoupler FCCP . Scale bar 20 μm . Plot showing the quantification of TMRM fluorescence signal from NCAM + neurons . Data presented as means + SEM of three independent experiments ( unpaired , two - tailed t - test ) .

O O O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O

A Representative images of humanneurons cells stained with the mitochondrial membrane potential sensible fluorescent probe TMRM, the neuronal specific anti-NCAM antibody and the nuclear staining Hoechst. Left panel, basal conditions. Right panel, after addition of the mitochondrial uncoupler FCCP. Scale bar 20 μm. Plot showing the quantification of TMRM fluorescence signal from NCAM+neurons. Data presented as means + SEM of three independent experiments (unpaired, two-tailed t-test).


B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope . PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein . Scale bar 500nm . Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in > 30 field ( 200 mitochondria in total ) for each sample ( one - way ANOVA ) .

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B Representative images of ultrastructural analysis of fixed neurons examined with electron microscope. PANK2 panel represents PKAN neuronal cells overexpressing PANK2 protein. Scale bar 500nm. Mitochondrial size was measured at level of the larger diameter along the perpendicular axis for all the mitochondria in >30 field (200 mitochondria in total) for each sample (one-way ANOVA).


C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls , PKAN patients and PKAN patients overexpressing PANK2 . The plots show OCR normalization to cell number . OCR was measured in basal conditions , and after oligomycin and FCCP addition . Bars indicate means + SEM of three independent experiments ( Two - way ANOVA ) .

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C OCR measurements of controls and each PKAN patients analyzed individually and D data obtained by individual analysis and plotted as grouped controls, PKAN patients and PKAN patients overexpressing PANK2. The plots show OCR normalization to cell number. OCR was measured in basal conditions, and after oligomycin and FCCP addition. Bars indicate means + SEM of three independent experiments (Two-way ANOVA).


A Representative images of neurons stained with the neuron specific anti - NCAM antibody , ROS sensible fluorescent probe DCF , and the nuclei dye Hoechst . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM positive controls - and PKAN humanneurons , infected or not with Ngn2 - PANK2 - LV . Data presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O B-CELL_TYPE O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O B-SUBCELLULAR O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-ORGANISM O O O O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

A Representative images of neurons stained with the neuron specific anti-NCAM antibody, ROS sensible fluorescent probe DCF, and the nuclei dye Hoechst. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM positive controls- and PKAN humanneurons, infected or not with Ngn2-PANK2-LV. Data presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1 . Scale bar 20 μm . ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots . Data presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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B Representative images of neurons stained with ThiolTracker Violet and the anti Tuj1. Scale bar 20 μm. ThiolTracker Violet fluorescence signal from Tuj1 positive neurons were quantified and shown in the plots. Data presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Upper panel : representative images of in - gel enzymatic activity of mitochondrial and cytosolic aconitase ( mAco and cAco , respectively ) . The lower part of the gel was cut , stained with Coomassie blue , and a protein band was used as loading control ( Loading ) . Lower panel : quantification of mAco and cAco enzymatic activity by densitometry .

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A Upper panel: representative images of in-gel enzymatic activity of mitochondrial and cytosolic aconitase (mAco and cAco, respectively). The lower part of the gel was cut, stained with Coomassie blue, and a protein band was used as loading control (Loading). Lower panel: quantification of mAco and cAco enzymatic activity by densitometry.


B Upper panel : Western blot analysis of mitochondrial and cytosolic aconitases . Lower panel : quantification of mAco and cAco by densitometry .

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B Upper panel: Western blot analysis of mitochondrial and cytosolic aconitases. Lower panel: quantification of mAco and cAco by densitometry.


C Hemequantification by absorbance at 400 nm of the soluble cell lysates .

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C Hemequantification by absorbance at 400 nm of the soluble cell lysates.


D Upper panel : western blot analysis of transferrin receptor ( TfR1 ) and ferritin ( FtH ) . Lower panel : quantification of TfR1 or FtH normalized on actin by densitometry . All the data are presented as means + SEM of at least three independent experiments ( unpaired , two - tailed t - test ) .

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D Upper panel: western blot analysis of transferrin receptor (TfR1) and ferritin (FtH). Lower panel: quantification of TfR1 or FtH normalized on actin by densitometry. All the data are presented as means + SEM of at least three independent experiments (unpaired, two-tailed t-test).


A Representative images of co - cultures of control and PKAN NPCs infected with GFP - LV and tdT - LV expressing vectors , respectively , at the beginning ( Day 1 ) , after 120 ( Day 120 ) and 150 days of differentiation ( Day 150 ) . Scale bar 20 μm . Plots show the number of green and red humanneurons counted at different time points . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Representative images of co-cultures of control and PKAN NPCs infected with GFP-LV and tdT-LV expressing vectors, respectively, at the beginning (Day 1), after 120 (Day 120) and 150 days of differentiation (Day 150). Scale bar 20 μm. Plots show the number of green and red humanneurons counted at different time points. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days . Repetitive firing activity ( left ) and relatively large Na + and K + currents ( right ) , were restored by CoA . The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs . CoA - treated neurons from control and PKAN patients ( z - test ) .

O O O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O B-CELL_TYPE O O O O B-ORGANISM O O O O O O

B Example of electrophysiological properties of cultured PKAN humanneurons with or without CoA incubation for 30 days. Repetitive firing activity (left) and relatively large Na+ and K+ currents (right), were restored by CoA. The histogram on the right shows fractions of repetitively firing cells recorded in untreated vs. CoA-treated neurons from control and PKAN patients (z-test).


C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst . Anti - NCAM antibody was used to detect neurons . Scale bar 20 μm . Plots of the DCF fluorescence signal from NCAM + controls - and PKAN humanneurons differentiated or not in the presence of CoA ( 25 μM ) in the medium for 3 weeks . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-SUBCELLULAR O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C An example of neurons stained with the ROS sensible fluorescent probe DCF and the nuclei dye Hoechst. Anti-NCAM antibody was used to detect neurons. Scale bar 20 μm. Plots of the DCF fluorescence signal from NCAM+ controls- and PKAN humanneurons differentiated or not in the presence of CoA (25 μM) in the medium for 3 weeks. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


D Oxygen consumption rate ( OCR ) with and without CoA on controls and PKAN patients . Basal and uncoupled ( FCCP ) respiration increased after CoA supplementation . Data presented as means + SEM of 24 independent replicates for each condition ( Two - way ANOVA ) .

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D Oxygen consumption rate (OCR) with and without CoA on controls and PKAN patients. Basal and uncoupled (FCCP) respiration increased after CoA supplementation. Data presented as means + SEM of 24 independent replicates for each condition (Two-way ANOVA).


E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates . All data are presented as means + SEM of at least three independent experiments ( One - way ANOVA ) .

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E Heme quantification by absorbance at 400 nm of the soluble NPC cell lysates. All data are presented as means + SEM of at least three independent experiments (One-way ANOVA).


Representative images of changes in intracellular calcium , as measured in fluorescence ratio images ( F340 / 380 ) ( A ) Examples show a TRPM8 - expressing DRG neuron ( left in A responding to both the TRPM8 agonist menthol ( 300 μM ) and to a cold stimulus from 32 ° C to 4 ° C a neuron that responds to cold ( right in A but does not express any of TRPM8 , TRPA1 or TRPC5 , as shown by lack of response to the TRPM8 agonist menthol ( 300 μM ) Calcium increase is due to influx from extracellular

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE B-CELL_TYPE O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O

Representative images of changes in intracellular calcium, as measured in fluorescence ratio images (F340/380) (A) Examples show a TRPM8-expressing DRG neuron (left in A responding to both the TRPM8 agonist menthol (300 μM) and to a cold stimulus from 32°C to 4°C a neuron that responds to cold (right in A but does not express any of TRPM8, TRPA1 or TRPC5, as shown by lack of response to the TRPM8 agonist menthol (300 μM) Calcium increase is due to influx from extracellular solution


experiments showing an SCG neuron responding to the TRPA1 agonist AITC ( left in E and a novel cold - sensitive SCG neuron that responds to cold but does not express any of TRPM8 , TRPA1 or TRPC5 ( right in E Many glial cells are also visibly activated by AITC but not by cold .

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experiments showing an SCG neuron responding to the TRPA1 agonist AITC (left in E and a novel cold-sensitive SCG neuron that responds to cold but does not express any of TRPM8, TRPA1 or TRPC5 (right in E Many glial cells are also visibly activated by AITC but not by cold.


( G ) Mean current voltage relation of 10 SCG neurons at 32 ° C ( dashed line ) , 4 ° C ( blue ) and 32 ° C ( red dots ) . ( H ) Difference trace from ( G ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Mean current voltage relation of 10 SCG neurons at 32°C (dashed line), 4°C (blue) and 32°C (red dots). (H) Difference trace from (G).


( C ) Mean cold - sensitive increase in calcium - dependent fluorescence ratio F340 / 380 in SCG neurons is significantly smaller in neurons recorded in voltage - clamp at - 60mV ( 0 . 56 ± 0 . 08 , mean ± SEM , n = 21 neurons ) than in neurons recorded in current - clamp mode ( 1 . 16 ± 0 . 29 , n = 11 neurons , p = 0 . 0165 , unpaired t - test ) .

O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-TISSUE B-CELL_TYPE O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O

(C) Mean cold-sensitive increase in calcium-dependent fluorescence ratio F340/380 in SCG neurons is significantly smaller in neurons recorded in voltage-clamp at -60mV (0.56 ±0.08, mean ± SEM, n=21 neurons) than in neurons recorded in current-clamp mode (1.16 ±0.29, n=11 neurons, p=0.0165, unpaired t-test).


( F ) Summary of results of experiments on SCG neurons ( mean ± SEM , n = 115 neurons , 4 separate cultures ) . YM58483 caused a significant decrease in cold - response amplitude ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) , with only partial recovery . Temperature trace below . ( G ) In neurons with a small response to cold ( F340 / 380 increase < 0 . 2 ; n = 24 neurons ) , many responses are potentiated by verapamil ( 31 % of cold - insensitive neurons ) and are fully blocked by ORAI channel antagonist YM58483 ( p < 0 . 0001 , RM one - way ANOVA + Dunnett ' s test ) .

O O O O O O O O O B-TISSUE B-CELL_TYPE O O O O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O B-CELL_TYPE O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(F) Summary of results of experiments on SCG neurons (mean ±SEM, n=115 neurons, 4 separate cultures). YM58483 caused a significant decrease in cold-response amplitude (p<0.0001, RM one-way ANOVA + Dunnett's test), with only partial recovery. Temperature trace below. (G) In neurons with a small response to cold (F340/380 increase <0.2; n=24 neurons), many responses are potentiated by verapamil (31% of cold-insensitive neurons) and are fully blocked by ORAI channel antagonist YM58483 (p<0.0001, RM one-way ANOVA + Dunnett's test).


( D ) Representative fluorescence images of SCG neurons from neonatal mice ( age P5 - 7 ) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1 . Neurons were cooled from 35 ° C to 7 ° C in the presence of 100μM verapamil . Electroporation was indicated by expression of mCherry ( # in left - hand panel ) ; in these neurons a cold response was absent ( # in right - hand panel ) while in neurons not expressing mCherry ( * ) cold responses were still observed . Scale bar 20μm .

O O O O O O O B-TISSUE B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O

(D) Representative fluorescence images of SCG neurons from neonatal mice (age P5-7) electroporated with plasmids coding for mCherry and for siRNA targeting STIM1. Neurons were cooled from 35°C to 7°C in the presence of 100μM verapamil. Electroporation was indicated by expression of mCherry (# in left-hand panel); in these neurons a cold response was absent (# in right-hand panel) while in neurons not expressing mCherry (*) cold responses were still observed. Scale bar 20μm.


( F ) Cold response amplitudes ( F340 / 380 , mean ± SEM ) . Successfully electroporated neurons ( mCherry positive , red bar , n = 111 neurons from four separate cultures ) had a significantly lower cold response amplitude than neurons not electroporated ( mCherry negative , black bar , n = 104 neurons from same cultures , p = 0 . 0082 , unpaired t - test ) . ( G ) Percentage of neurons that responded to cold ramp with F340 / 380 increase > 0 . 2 . Only 5 % of mCherry - positive neurons ( successfully electroporated , red bar ) responded to cold , compared to 19 % of mCherry - negative neurons in same cultures ( non - electroporated , black bar , p < 0 . 001 , chi - square test ) .

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(F) Cold response amplitudes (F340/380, mean ±SEM). Successfully electroporated neurons (mCherry positive, red bar, n=111 neurons from four separate cultures) had a significantly lower cold response amplitude than neurons not electroporated (mCherry negative, black bar, n=104 neurons from same cultures, p=0.0082, unpaired t-test). (G) Percentage of neurons that responded to cold ramp with F340/380 increase >0.2. Only 5% of mCherry-positive neurons (successfully electroporated, red bar) responded to cold, compared to 19% of mCherry-negative neurons in same cultures (non-electroporated, black bar, p<0.001, chi-square test).


( A ) Representative TIRF microscopy images ( imaging depth 100nm ) showing that cooling from 35 ° C to 13 ° C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1 - CFP and STIM1 - YFP . Cooling to 13 ° C causes the appearance of new ORAI1 puncta ( examples shown by * ) that are not present in the 35 ° C image and are colocalised with STIM1 . Cooling also causes STIM1 to move towards the cell surface in edge - on views ( arrows ) . To reduce cell motility , the experiment was conducted in nominal 0 [ Ca ] e , which potentially caused partial formation of STIM1 puncta before the images shown . Scale bar = 5μm . Temperature trace shown below . Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling ( see Fig . S12 ) .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(A) Representative TIRF microscopy images (imaging depth 100nm) showing that cooling from 35°C to 13°C increases ORAI1 puncta formation and colocalization of ORAI1 with STIM1 in HEK293 cells transfected with ORAI1-CFP and STIM1-YFP. Cooling to 13°C causes the appearance of new ORAI1 puncta (examples shown by *) that are not present in the 35°C image and are colocalised with STIM1. Cooling also causes STIM1 to move towards the cell surface in edge-on views (arrows). To reduce cell motility, the experiment was conducted in nominal 0[Ca]e, which potentially caused partial formation of STIM1 puncta before the images shown. Scale bar = 5μm. Temperature trace shown below. Mean fluorescence intensity adjusted to compensate for increase in intrinsic fluorescence of CFP and YFP caused by cooling (see Fig. S12).


( A ) Representative immunofluorescence images showing adult mouse stellate ganglion ( SG ) sections stained for nNOS ( red ) , to label nitrergic neurons , βIII Tubulin ( green ) to label all neurons , and DAPI to label nuclei . nNOS is widely expressed in neurons of the stellate ganglion , but is absent from glial cells ( glial nuclei identifiable in merged image , similar results in n = 5 animals ) . Antibody against nNOS from Abcam ; similar results obtained with antibodies from ECM and Millipore . All three antibodies show nNOS widely expressed in SG cell bodies and neurites .

O O O O B-EXP_ASSAY O O O B-ORGANISM B-TISSUE I-TISSUE O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE O O B-SMALL_MOLECULE O O B-SUBCELLULAR O B-GENEPROD O O O O B-CELL_TYPE O O B-TISSUE I-TISSUE O O O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-TISSUE B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O

(A) Representative immunofluorescence images showing adult mouse stellate ganglion (SG) sections stained for nNOS (red), to label nitrergic neurons, βIII Tubulin (green) to label all neurons, and DAPI to label nuclei. nNOS is widely expressed in neurons of the stellate ganglion, but is absent from glial cells (glial nuclei identifiable in merged image, similar results in n=5 animals). Antibody against nNOS from Abcam; similar results obtained with antibodies from ECM and Millipore. All three antibodies show nNOS widely expressed in SG cell bodies and neurites.


( B ) Adult mouse SCG ( n = 2 animals , upper ) and SG ( n = 5 animals , lower ) sections stained for nNOS ( red ) , tyrosine hydroxylase ( TH , green ) and DAPI ( blue ) to label nitrergic neurons , noradrenergic neurons and nuclei respectively . Many noradrenergic cells also express nNOS .

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(B) Adult mouse SCG (n=2 animals, upper) and SG (n=5 animals, lower) sections stained for nNOS (red), tyrosine hydroxylase (TH, green) and DAPI (blue) to label nitrergic neurons, noradrenergic neurons and nuclei respectively. Many noradrenergic cells also express nNOS.


( B ) The ratio of unique histone iCLIP - seq read population to unique histone RNA - seq read population was calculated and shown , with the unique histone RNA - seq read population set as " 1 " . Error bars represent standard deviations from three replicates of ALYREF iCLIP data . Statistical analysis was performed using Student ' s t - test . * * * P < 0 . 001 .

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(B) The ratio of unique histone iCLIP-seq read population to unique histone RNA-seq read population was calculated and shown, with the unique histone RNA-seq read population set as "1". Error bars represent standard deviations from three replicates of ALYREF iCLIP data. Statistical analysis was performed using Student's t-test. ***P< 0.001.


( C ) ALYREF RIP - qPCRs to examine ALYREF binding on histone mRNAs . Relative RIP efficiencies are shown . A tRNA was used as a negative control for ALYREF binding . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O B-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) ALYREF RIP-qPCRs to examine ALYREF binding on histone mRNAs. Relative RIP efficiencies are shown. A tRNA was used as a negative control for ALYREF binding. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01, ***P< 0.001, n.s., not significant.


( D ) The distribution of histone reads in 5 ' UTR , CDS and 3 ' UTR in rRNA - depleted nuclear RNA - seq and ALYREF - iCLIP - seq .

O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O

(D) The distribution of histone reads in 5' UTR, CDS and 3' UTR in rRNA-depleted nuclear RNA-seq and ALYREF-iCLIP-seq.


( E ) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Metagene analysis of normalized ALYREF signals on histone mRNA based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( F ) Screenshots of two histone mRNAs showing clustered ALYREF binding sites . The 3 ' UTR regions are highlighted .

O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O O O O O

(F) Screenshots of two histone mRNAs showing clustered ALYREF binding sites. The 3' UTR regions are highlighted.


( G ) Distribution profile of clustered ALYREF binding sites on histone mRNA 3 ' UTRs relative to the cleavage site ( CS ) . The CS is set as " 0 " in the x axis , and the relative position to the CS is marked . The y axis shows the total occurrence of binding sites in three repeats at each nucleotide .

O O O B-EXP_ASSAY O O O B-GENEPROD B-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O

(G) Distribution profile of clustered ALYREF binding sites on histone mRNA 3' UTRs relative to the cleavage site (CS). The CS is set as "0" in the x axis, and the relative position to the CS is marked. The y axis shows the total occurrence of binding sites in three repeats at each nucleotide.


IPs from RNase A - treated S - phase HeLa cell lysate using the SLBP antibody or IgG ( A ) Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IPs from RNase A-treated S-phase HeLa cell lysate using the SLBP antibody or IgG (A) Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


IPs from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody or IgG ( B ) . Western blotting was performed with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IPs from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody or IgG (B). Western blotting was performed with indicated antibodies. 2% of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blots


( C ) Pull - downs of in vitro translated 35S labelled SLBP and luciferase ( Cntl ) using MBP or MBP - ALYREF in the presence of RNase A . The proteins pulled down were visualized by coomassie staining ( left ) or phosphoimager ( right ) . 3 % of input was loaded .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O O O O O

(C) Pull-downs of in vitro translated 35S labelled SLBP and luciferase (Cntl) using MBP or MBP-ALYREF in the presence of RNase A. The proteins pulled down were visualized by coomassie staining (left) or phosphoimager (right). 3% of input was loaded.


( D ) GST - SLBP , GST - UAP56 and GST were used for pull - down of purified MBP - ALYREF or MBP in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE , followed by coomassie staining . 37 . 5 % of input proteins were loaded .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O

(D) GST-SLBP, GST-UAP56 and GST were used for pull-down of purified MBP-ALYREF or MBP in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE, followed by coomassie staining. 37.5% of input proteins were loaded.


( E ) ( Top ) Domain schematic representation of SLBP . ( Bottom ) Flag IPs from RNase A - treated HeLa cell lysate individually expressing indicated Flag tagged proteins , followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same

O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD B-EXP_ASSAY O B-GENEPROD I-GENEPROD O O B-CELL_LINE O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O

(E) (Top) Domain schematic representation of SLBP. (Bottom) Flag IPs from RNase A-treated HeLa cell lysate individually expressing indicated Flag tagged proteins, followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( E ) ( Top ) Domain schematic representation of SLBP . followed by western blotting using Flag and ALYREF antibodies . 3 % of input was loaded . The * indicates a band probably resulted from degradation of Flag - SLBP . The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA - SLBP stable expression cells transfected with plasmids expressing ALYREF fragments .

O O O O O O O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY B-GENEPROD O O

(E) (Top) Domain schematic representation of SLBP. followed by western blotting using Flag and ALYREF antibodies. 3% of input was loaded. The * indicates a band probably resulted from degradation of Flag-SLBP. The white line delineates the boundary where irrelevant lanes have been removed from the same blot except that Flag IPs were carried out from HA-SLBP stable expression cells transfected with plasmids expressing ALYREF fragments.


( G ) Western blotting to examine the KD efficiency of SLBP . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(G) Western blotting to examine the KD efficiency of SLBP. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


Cntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to western analysis ( H ) Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O B-GENEPROD O O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to western analysis (H) Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


Cntl - or SLBP - siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody . The immunoprecipitates were subjected to RT - qPCRs ( I ) . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , n . s . , not significant .

O O O B-GENEPROD O O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cntl- or SLBP-siRNA treated HeLa cells were used for IPs with IgG or the ALYREF antibody. The immunoprecipitates were subjected to RT-qPCRs (I). Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001, n.s., not significant.


( J ) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP - seq data . iCLIP - seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA ' s contribution to the plot .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Metagene analysis of normalized ALYREF signals on histone mRNAs in Cntl and SLBP KD cells based on iCLIP-seq data. iCLIP-seq signal at each position of an mRNA is divided by the sum of signal of the mRNA to normalize each mRNA's contribution to the plot.


( B ) List of histone genes with defective 3 ' - end processing detected by polyA + and polyA - RNA - seq . PolyA + ALYREF / Cntl shows polyA + RNA - seq RPM ratio of each histone gene in ALYREF KD versus Cntl .

O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-GENEPROD O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-GENEPROD O O O O

(B) List of histone genes with defective 3'-end processing detected by polyA+ and polyA- RNA-seq. PolyA+ ALYREF/Cntl shows polyA+ RNA-seq RPM ratio of each histone gene in ALYREF KD versus Cntl.


( C ) Screenshots of two exemplified histone mRNAs with 3 ' - end processing defects are shown . Transcription direction is indicated . Black arrowheads and the corresponding lines mark the position of CS .

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

(C) Screenshots of two exemplified histone mRNAs with 3'-end processing defects are shown. Transcription direction is indicated. Black arrowheads and the corresponding lines mark the position of CS.


( D ) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3 ' READS + .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O

(D) RPM of each histone mRNA in Cntl cells and RPM ratio of each histone mRNA in KDs to Cntl detected in 3'READS+.


( E , F ) RT - qPCRs to detect histone mRNA levels in the polyA + ( E ) or the polyA - fraction ( F ) in cells treated with Cntl , ALYREF or ARS2 siRNA . The arrowhead and arrows indicate the cleavage site and primer location , respectively . The bars show the relative abundance of polyA + forms to the actin mRNA ( E ) and that of polyA - forms relative to the 18S rRNA ( F ) . Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) RT-qPCRs to detect histone mRNA levels in the polyA+ (E) or the polyA- fraction (F) in cells treated with Cntl, ALYREF or ARS2 siRNA. The arrowhead and arrows indicate the cleavage site and primer location, respectively. The bars show the relative abundance of polyA+ forms to the actin mRNA (E) and that of polyA- forms relative to the 18S rRNA (F). Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western blotting to examine ALYREF protein level ( G ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The white line delineates the boundary where irrelevant lanes have been removed from the same

B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

Western blotting to examine ALYREF protein level (G) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


RT - qPCRs to detect polyA + histone mRNA levels ( H ) in Flag - Cntl ( eIF4A3 ) or siRNA - resistant Flag - ALYREF stable expression cells treated with Cntl or ALYREF siRNA . The bars show the relative abundance of histone mRNAs to the GAPDH mRNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCRs to detect polyA+ histone mRNA levels (H) in Flag-Cntl (eIF4A3) or siRNA-resistant Flag-ALYREF stable expression cells treated with Cntl or ALYREF siRNA. The bars show the relative abundance of histone mRNAs to the GAPDH mRNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


IPs were carried out from RNase A - treated S - phase HeLa cell lysate using the Lsm11 antibody and IgG ( A ) followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the Lsm11 antibody and IgG (A) followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


IPs were carried out from RNase A - treated S - phase HeLa cell lysate using the ALYREF antibody and IgG ( B ) , followed by western blotting with indicated antibodies . 2 % of input was loaded . * indicates a nonspecific band . The white line delineates the boundary where irrelevant lanes have been removed from the same

B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD O O O O O B-CELL_LINE O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

IPs were carried out from RNase A-treated S-phase HeLa cell lysate using the ALYREF antibody and IgG (B), followed by western blotting with indicated antibodies. 2 % of input was loaded. * indicates a nonspecific band. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( C ) Pull - down of purified His - Cntl ( PGK1 ) or His - Lsm11 using equal amount of GST and GST - ALYREF proteins in the presence of RNase A . Proteins pulled down were separated by SDS - PAGE followed by coomassie staining . * indicates a nonspecific band .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(C) Pull-down of purified His-Cntl (PGK1) or His-Lsm11 using equal amount of GST and GST-ALYREF proteins in the presence of RNase A. Proteins pulled down were separated by SDS-PAGE followed by coomassie staining. * indicates a nonspecific band.


Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected western analysis ( D ) . * indicates the antibody heavy chain .

O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O

Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected western analysis (D). * indicates the antibody heavy chain.


Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody . The immunoprecipitates were subjected to RT - qPCRs ( E ) * indicates the antibody heavy chain . The bars show the relative IP efficiencies of histone pre - mRNAs to U7 snRNA . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected to RT-qPCRs (E) * indicates the antibody heavy chain. The bars show the relative IP efficiencies of histone pre-mRNAs to U7 snRNA. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( A ) ( Top ) Illustration of the HIST2H2AA3 reporter construct . ( Bottom ) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . DAPI staining served as a nuclear marker . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O B-GENEPROD O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-SMALL_MOLECULE B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( B ) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript - specific probe in HeLa cells depleted of Cntl , ALYREF , THOC2 ( THO ) and UAP56 . N and C indicate nuclear and cytoplasmic FISH signals , respectively . N / C ratios were calculated Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O B-SUBCELLULAR O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western analysis to examine the purities of nuclear and cytoplasmic fractions ( C ) in Cntl and UAP56 KD cells . MTR4 and Tubulin were used as the nuclear and cytoplasmic marker , respectively .

B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O

Western analysis to examine the purities of nuclear and cytoplasmic fractions (C) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively.


RT - qPCRs to examine N / C distribution of each histone mRNA ( D ) in Cntl and UAP56 KD cells . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( F ) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl , ALYREF and SLBP KD cells . Data information : Error bars represent standard deviations from biological repeats n = 2 Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells. Data information: Error bars represent standard deviations from biological repeats n=2 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( G ) NXF1 iCLIP signals along the histone mRNA . The histone mRNA is divided into 10 fractions based on their mRNA size . Each fraction represents 10 % of each histone mRNA . The tenth fractions that contains the SL is boxed by green dashed lines .

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(G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.


( A ) ( Top ) Illustration of the reporter constructs . The black and red lines indicate H2AA3 - SL and H2AA3 - SLD PCR products , respectively . The dashed line indicates the FISH probe . ( Bottom ) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3 - SL and H2AA3 - SLD PCR products . C and N indicate cytoplasmic and nuclear FISH signals , respectively . C / N ratios were determined for 20 cells in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(A) (Top) Illustration of the reporter constructs. The black and red lines indicate H2AA3-SL and H2AA3-SLD PCR products, respectively. The dashed line indicates the FISH probe. (Bottom) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3-SL and H2AA3-SLD PCR products. C and N indicate cytoplasmic and nuclear FISH signals, respectively. C/N ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( B ) ( Top ) Illustration of the reporter constructs . The dashed line indicates the FISH probe . FISH to detect mRNA distribution at 24 hr after transfection of the H1C - SL - Rb ( cH1C ) and H1C - SLD - Rb ( pH1C ) constructs . C / N ratios were quantified Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( Top ) Illustration of the reporter constructs . PCR products of cG - SL and cG - SLD were used instead . cG , β - globin cDNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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(Top) Illustration of the reporter constructs. PCR products of cG-SL and cG-SLD were used instead. cG, β-globin cDNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis to detect ALYREF ( D )

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D)


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . RT - qPCRs to detect RNAs ( E ) . The bars in the graph shows the relative ALYREF IP efficiency , with that for the H1C - SL - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. RT-qPCRs to detect RNAs (E). The bars in the graph shows the relative ALYREF IP efficiency, with that for the H1C-SL-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( F ) H1C - SLD - Rb construct with wild - type ( H1C - SLDWT - Rb ) or mutant ( H1C - SLDmut - Rb ) U7 - binding sequence , together with VSV M , was co - transfected into HeLa cells , followed by IPs with IgG or the ALYREF antibody at 24 hr post - transfection . The immunoprecipitates were subjected to western analysis ( left panel ) and RT - qPCRs ( right panel ) . The bars in the right graph show the relative ALYREF IP efficiency , with that for the H1C - SLDWT - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 .

O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O B-ORGANISM O O O O O O O B-CELL_LINE O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) H1C-SLD-Rb construct with wild-type (H1C-SLDWT-Rb) or mutant (H1C-SLDmut-Rb) U7-binding sequence, together with VSV M, was co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis (left panel) and RT-qPCRs (right panel). The bars in the right graph show the relative ALYREF IP efficiency, with that for the H1C-SLDWT-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( A ) Western blotting to examine the expression levels of H2B and H4 in Cntl , ALYREF and ARS2 KD cells . GAPDH was used as a loading control . The white line delineates the boundary where irrelevant lanes have been removed from the same

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(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( B ) Growth curve of Cntl and ALYREF KD cells . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical analysis was performed using Student ' s t - test . * * P < 0 . 01 .

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(B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01.


( C ) Cell cycle analysis of Cntl , ALYREF and ARS2 KD cells . Quantifications of cells in G0 / G1 ( 1N ) , G2 ( 2N ) and S - phase ( intermediate ) of the cell cycle are shown in the upper right corner of each histogram .

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(C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram.


Results from multiplex ACE2 competition assay are shown for the three spike - derived antigens : RBD , S1 - domain ( S1 ) and homotrimeric spike ( Spike ) . Color - coded beads coated with the respective antigens were co - incubated with biotinylated ACE2 and dilution series of NM1267 ( 8 pM to 126 nM ) followed by measuring residual binding of ACE2 . MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2 - only control . IC50 values were calculated from a four - parametric sigmoidal model . Data are presented as mean ± s . d . of three technical replicates .

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Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.


E Neutralization potency of NM1267 was analyzed in Caco ‑ 2 cells using the SARS - CoV - 2 - mNG infectious clones . Infection rate normalized to virus - only infection control is illustrated as percent of infection ( % Infection ) . IC50 value was calculated from a four - parametric sigmoidal model and data are presented as mean ± s . e . m . of three biological replicates ( n = 3 ) .

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E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).


B For the NeutrobodyPlex , antigen - coated beads comprising RBD , S1 or spike were co - incubated with serum samples from 5 patients and a dilution series of NM1267 ( 1 µM to 6 pM ) ( n = 1 ) . Mean fluorescent intensities ( MFI ) derived from antigen - bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum - only samples , illustrated as MFI ( % control ) , are shown .

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B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.


A , B Total IgGs derived from spike - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . C , D Total IgGs derived from N - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in the presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . F

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A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F


( A ) Graph derived from two published data available in the PubMed GEO database ( GSE6919 and GSE35988 ) . The box charts depict the relative expression of ESM1 in benign , primary , and metastatic prostate cancer patients . The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset . The whiskers mark the 5th and 95th percentiles , and values beyond these upper and lower bounds are considered outliers .

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(A) Graph derived from two published data available in the PubMed GEO database (GSE6919 and GSE35988). The box charts depict the relative expression of ESM1 in benign, primary, and metastatic prostate cancer patients. The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers.


( B ) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines . P : parental cells , M : metastatic cells .

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(B) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines. P: parental cells, M: metastatic cells.


( C ) Cell invasion assays of 22Rv1 - M and PC3 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . The data are shown as the relative fold change of invasive cells compared with the shScramble group . Scale bar : 100 μm .

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(C) Cell invasion assays of 22Rv1-M and PC3-M cells transfected with either Scramble-shRNA or ESM1-shRNA. The data are shown as the relative fold change of invasive cells compared with the shScramble group. Scale bar: 100 μm.


( D ) Representative images of lungs , livers , intestines , and kidneys from mice 8 weeks after orthotopical injection of 22Rv1 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . Scale bar : 0 . 5 cm . Tumors were removed and weighed . Quantitative summary of tumor luminescence in different metastases measured in photons / second are shown .

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(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.


( E ) Formalin - fixed , paraffin - embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis ( n = 16 and n = 49 , respectively ) were stained with an anti - ESM1 antibody . Tissue - bound ESM1 is shown in brown . Scale bar : 50 μm . Arrowheads indicate positively stained nucleus of cancer cells . Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis . The scores are calculated by intensity ( score 1 - 3 ) x percentage ( score 1 - 4 ) of stained cells . The line in the middle of the box is plotted at the median . The box extends from the 25th to 75th percentiles . Whiskers above and below the box indicate the 5th and 95th percentiles .

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(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles.


( A ) Immunoblotting analysis of ESM1 in supernatant ( S ) , nucleus ( N ) , and cytoplasm ( C ) was performed . Ponceau S , Lamin A / C , and α - tubulin were used as loading control in supernatant , nucleus , and cytoplasm , respectively . P : parental cells , M : metastatic cells .

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(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells.


( B ) Left , representative images of cells in the transwell invasion assay . Right , quantification of cells that invaded through the matrigel - coated membrane following treatment with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody . Scale bar : 100 μm .

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(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.


( D ) Elicitation of ESM1 overexpression using HA - tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1 - P cells . Lamin A / C and α - tubulin were used as loading control in the nucleus and cytoplasm , respectively .

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(D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively.


( E ) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant ( S ) of 22Rv1 - P cells .

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(E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells.


( F ) Left , representative images of 22Rv1 - P cells in the transwell invasion assay . Right , statistic data of invasive ability of ESM1 expressing cells . Scale bar : 100 μm .

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(F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm.


( G ) Left , representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1 - Luc cells with different ESM1 expression patterns . Scale bar : 0 . 5 cm . Right , quantification of the lung bioluminescence by photons / seconds .

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(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.


( A ) Tumor spheroid formation . Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody .

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(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody.


( B ) Representative images of tumor spheres . Spheres were cultured for 14 days before counting . Histogram shows the mean numbers of spheres cultured . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

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(B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( C ) 22Rv1 - P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h . * *

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(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. **


( D ) Cells with or without ESM1 - NLS overexpression were subcutaneously injected ( 10 , 100 , 1000 cells per mouse ) into NOD / SCID mice . Tumor formation ability and tumor weight were analyzed . Scale bar : 1 cm . Bars are the mean ± SD of indicated ( n ) independent experiments .

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(D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments.


( E ) Flow cytometry analysis of the ratio of CD44 + and CD133 + cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed .

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(E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed.


( F ) qRT - PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments . *

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(F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. *


( A ) IPA analysis of RNA - seq data showing the 14 top - predicted upstream regulators of differentially expressed genes . Positive Z - scores ( orange ) represent activated upstream regulators , negative Z - scores ( blue ) represent inhibited upstream regulators .

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(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.


( E ) The transcriptional activity of β - catenin in 22Rv1 - P cells with different patterns of ESM1 overexpression . Bars are the mean ± SD of three independent experiments .

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(E) The transcriptional activity of β-catenin in 22Rv1-P cells with different patterns of ESM1 overexpression. Bars are the mean ± SD of three independent experiments.


( F ) Manipulating ESM1 expression impacts transcriptional activity of NFκB . Bars are the mean ± SD of three independent experiments .

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(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the mean ± SD of three independent experiments.


( A ) qRT - PCR analysis of the mRNA levels of the genes related to β - catenin downstream signaling pathway . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments .

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(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( B ) qRT - PCR analysis of the mRNA levels of NFκB targeting genes . Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments .

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(B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( C ) Immunoblotting analysis of 22Rv1 - P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated .

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(C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated.


( D ) Suppression of β - catenin or NFκB in 22Rv1 - P cells stably expressing ESM1 - NLS were treated with docetaxel at the indicated concentrations for 48 h . * * P < 0 . 01 when compared to vector - shScramble cells by two - tailed Student ' s t test and error bars represent the standard deviation of three independent experiments .

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(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.


( E ) Tumor spheres formed by 22Rv1 - P cells stably expressing ESM1 - NLS with either β - catenin or NFκB shRNAs . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( F ) Representative images and quantitative data comparing cell invasion of the indicated cells . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( A ) Nuclear and cytosolic extracts of 22Rv1 - M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting .

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(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.


( B ) Nuclear or cytosolic extracts of 22Rv1 - M cells were immunoprecipitated with an ESM1 antibody .

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(B) Nuclear or cytosolic extracts of 22Rv1-M cells were immunoprecipitated with an ESM1 antibody.


( C ) Human recombinant His - ESM1 and GST - β - catenin protein were pull down with either Ni sepharose or Glutathione sepharose .

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(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.


( E ) HEK293T cells were co - transfected with the indicated β - catenin - Flag and ESM1 - HA constructs . Cell extracts were immunoprecipitated with Flag - M2 agarose beads . Light chain was labeled as l . c .

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(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.


( F ) HEK293T cells were transfected with ESM1 - HA constructs and pull - down was carried out with different purified GST - ARM fragments .

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(F) HEK293T cells were transfected with ESM1-HA constructs and pull-down was carried out with different purified GST-ARM fragments.


( G ) HEK293T cells were transfected with the indicated ESM1 - GFP constructs . Cell extracts were pulled down with purified GST - ARM .

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(G) HEK293T cells were transfected with the indicated ESM1-GFP constructs. Cell extracts were pulled down with purified GST-ARM.


( B ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM .

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(B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM.


( C ) Cell extracts of 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA were immunoprecipitated with an β - catenin antibody and blotted with anti - TCF4 and anti - BCL9 antibody .

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(C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody.


( E ) Upper , Schematic diagram of ESM1 - targeting short peptides . Lower , HEK293T cells were transfected with ESM1 - HA constructs . Cell extracts were treated with the short peptides , pulled down with purified GST - ARM and blotted with anti - HA and anti - TCF4 antibody .

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(E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody.


( F ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM and blotted with an anti - TCF4 antibody .

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(F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody.


( G ) ChIP - qPCR validation of β - catenin binding to the CCND1 and c - Myc promoter in 22Rv1 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . Bars are the mean ± SD of three independent experiments .

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(G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments.


( H ) Upper , Schematic diagram of ChIP - qPCR primers targeting regions upstream of the CCND1 and c - Myc transcription start sites ( TSSs ) . Bar graphs show β - catenin and ESM1 binding at the CCND1 and c - Myc promoter regions . Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments .

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(H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments.


( I ) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

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(I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( J ) The effects of ESM1 point mutants on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

(J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( B - C ) Fluorescent dye release from the mitochondria - mimic liposomes by the indicated wild - type ( wt ) or mutant Bax in the presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching the fluorescence of the released dyes by the dye - specific antibodies outside the liposomes during a time course . The fraction of dye release was normalized to that by detergent . The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes .

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(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.


( D ) Digitonin permeabilized BMK Bax - / - / Bak - / - cells expressing SMAC - mCherry in the mitochondria intermembrane space ( 50 μL , 500 , 000 total cells ) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37 ° C in a 96 - well plate . The samples were centrifuged for 10 min and separated into supernatant and pellet fractions . SMAC - mCherry release was calculated as the fraction of total ( supernatant + pellet ) mCherry fluorescence coming from the supernatant fraction . The data was normalized to the percent SMAC - mCherry release of wt Bax and cBid at 60 min . Each symbol represents the normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable the data was fit with the [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . The dotted lines represent the 95 % confidence interval of the fit .

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(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.


A , Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA , or 5 nM Actinomycin D ( ActD ) . GAPDH served as a loading control .

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A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.


B , HCT116 cells were treated for 24 hrs with vehicle alone ( - ) , 1 µM MPA , 10 µM MPA , or the combination of 10 µM MPA with 400 µM guanosine . Guanylate nucleotides levels were measured by LC - MS , and normalized to protein content . Mean ± SEM is representative of 3 independent experiments carried out in triplicate . Data information data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . mRNAs of IMPDH1 and IMPDH2 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to β - actin mRNA ( C ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.