Bar chart showing mean amplitude of field potential at lateral positions in lamina 1 recorded before approximately 30 minutes after peptide administration which was significantly reduced ( Wilcoxon matched pairs , * P = 0 . 0313 , W = - 26 , n = 7 mice , error bars : ) . chart to show data from ( E ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen that response was variable across mice with just over half showing a large reduction remaining mice only showing a relatively minor reduction ( with no reduction in one mouse ) . von Frey thresholds for mice before experiment are shown next to each . Bar chart showing mean amplitude of field potential at lateral positions in lamina 2 recorded before 30 minutes after peptide administration which was significantly reduced ( Wilcoxon matched pairs , * P = 0 . 0313 , W = - 26 , n = 7 mice , error bars : ) . chart to show data from ( G ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen there was reduction in but 1 mouse , although magnitude of reduction was again variable across mice . von Frey thresholds for mice before experiment are shown next to each .

O O O O O O O O O O O O B-TISSUE O O O O O O O O O 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 B-ORGANISM 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 B-ORGANISM O O O O O O O O O O O O B-ORGANISM 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 B-TISSUE O O O O O O O O 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 B-ORGANISM 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 B-ORGANISM O O O O O B-ORGANISM O O O O O O O O

Bar chart showing the mean amplitude of the field potential at lateral positions in lamina 1 recorded before and approximately 30 minutes after peptide administration which was significantly reduced (Wilcoxon matched pairs, *P = 0.0313, W = -26, n = 7 mice, error bars: SEM). Line chart to show data from (E), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen that the response was variable across mice with just over half showing a large reduction and the remaining mice only showing a relatively minor reduction (with no reduction in one mouse). The von Frey thresholds for the individual mice before the experiment are shown next to each line. Bar chart showing the mean amplitude of the field potential at lateral positions in lamina 2 recorded before and 30 minutes after peptide administration which was significantly reduced (Wilcoxon matched pairs, *P = 0.0313, W=-26, n=7 mice, error bars: SEM). Line chart to show data from (G), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen there was reduction in all but 1 mouse, although the magnitude of the reduction was again variable across mice. The von Frey thresholds for the individual mice before the experiment are shown next to each line.


Bar chart showing that mean amplitude of incoming volley is reduced post administration ( Wilcoxon matched pairs , * P = 0 . 0156 , W = - 28 , n = 7 mice , error bar : ) . chart to show data from ( J ) , as separate data points for mice expressed as a relative to baseline measurements . From this it can be seen that response was variable across mice with half showing a large reduction remaining mice only a minor reduction .

O O O O O O O O O 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 B-ORGANISM 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 B-ORGANISM O O O O O

Bar chart showing that the mean amplitude of the incoming volley is reduced post administration (Wilcoxon matched pairs, *P = 0.0156, W = -28, n = 7 mice, error bar: SEM). Line chart to show data from (J), as separate data points for individual mice expressed as a percentage relative to baseline measurements. From this it can be seen that the response was variable across mice with half showing a large reduction and the remaining mice only a minor reduction.


Amplitude of incoming volley expressed as a of baseline recording prior to peptide administration . In these 3 mice , a reduction in incoming volley at 30 minutes post - administration is seen . After this recording , remaining peptide was removed by washing spinal cord two further recordings made at 1 2 hours . Here it can be seen that incoming volley does not deteriorate further but stabilises or starts to return towards baseline confirming that initial reduction was not due to a deterioration in health of or damage to roots .

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 B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Amplitude of the incoming volley expressed as a percentage of the baseline recording prior to peptide administration. In these 3 mice, a reduction in the incoming volley at 30 minutes post-administration is seen. After this recording, the remaining peptide was removed by washing the spinal cord and two further recordings made at 1 and 2 hours. Here it can be seen that the incoming volley does not deteriorate further but stabilises or starts to return towards baseline levels confirming that the initial reduction was not due to a deterioration in the health of the animal or damage to the roots.


Kaplan - Meier curves for overall survival ( OS ) in lung adenocarcinoma TCGA _ LUAD ( RASSF1 mRNA high / low cutoff FKPM 5 . 85 ) squamous carcinoma TCGA _ LUSC ( RASSF1 mRNA high / low cutoff FKPM 6 . 52 ) . Significance derived from log - rank .

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

Kaplan-Meier curves for overall survival (OS) in lung adenocarcinoma TCGA_LUAD (RASSF1 mRNA high/low cutoff FKPM 5.85) and squamous cell carcinoma patients TCGA_LUSC (RASSF1 mRNA high/low cutoff FKPM 6.52). Significance derived from log-rank test.


Western blot with indicated antibodies of isogenic H1299 stably transfected with either empty vector pcDNA3 ( H1299control ) or RASSF1A ( H1299RASSF1A ) . Bottom : proliferation resazurin . ( n = 2 ) . Error bars represent mean ± S . E . M .

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

Western blot with indicated antibodies of isogenic H1299 cells stably transfected with either empty vector pcDNA3 (H1299control) or RASSF1A (H1299RASSF1A). Bottom: cell proliferation resazurin assay. (n=2). Error bars represent mean ± S.E.M.


of lung tumours measured at day 30 after lung orthotopic injection either with H1299control ( n = 14 mice per group ; experimental groups of 6 8 different shading within groups represents two independent experiments ) or with H1299RASSF1A stably re - expressing RASSF1A , ( n = 12 mice per group ; experimental groups of 6 6 ) . of tumours was measured by MRI software ITK - SNAP . Statistical significance via 2 - Student ' s t - . Error bars represent mean ± S . E . M .

O B-TISSUE B-DISEASE O O O O O B-TISSUE O O O O B-CELL_LINE 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 B-CELL_LINE O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-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

Size of lung primary tumours measured at day 30 after lung orthotopic injection either with H1299control (n=14 mice per group; experimental groups of 6 and 8 different shading within the groups represents two independent experiments) or with H1299RASSF1A cells stably re-expressing RASSF1A, (n=12 mice per group; experimental groups of 6 and 6). The size of primary tumours was measured by MRI software ITK-SNAP. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.


Visual assessment of metastatic events on ( left ) or ( right ) lungs , generated by H1299control or H1299RASSF1A at day 30 . Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A . Statistical significance via 2 - Student ' s t - . Error bars represent mean ± S . E . M .

B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-TISSUE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O

Visual assessment quantification of metastatic events on the ipsilateral (left) and or contralateral (right) lungs, generated by H1299control or H1299RASSF1A at day 30. Result was calculated from 2 independent in vivo experiments Graph shows significant decreasing of metastases when lungs were injected with H1299RASSF1A. Statistical significance via 2-tailed Student's t-test. Error bars represent mean ± S.E.M.


Mass - spectrometry of purified from extracellular matrix isolated from H1299control H1299RASSF1A lung adenocarcinoma lines with summary of results . Only identified with two or more peptides were taken into consideration . resulting list of was restricted to ECM ( n = 3 ) .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE B-DISEASE I-DISEASE 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-SUBCELLULAR O O O O O O

Mass-spectrometry analysis of proteins purified from extracellular matrix isolated from H1299control and H1299RASSF1A lung adenocarcinoma cell lines with summary of results. Only proteins identified with two or more peptides were taken into consideration. The resulting list of proteins was restricted to ECM proteins (n=3).


Kaplan Meier plots depicting prognosis in TCGA _ LUAD ( lung adenocarcinoma ) with high low mRNA expression of ECM revealed by proteomics . Significance derived from log - rank . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

O O O O O O O O B-DISEASE O B-DISEASE I-DISEASE O O O O B-EXP_ASSAY I-EXP_ASSAY 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

Kaplan Meier plots depicting prognosis in TCGA_LUAD (lung adenocarcinoma) with high and low mRNA expression of ECM proteins revealed by proteomics. Significance derived from log-rank test. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of H & E for P4HA2 , ( bars ) of two independent regions of n = 5 H1299control n = 3 H1299RASSF1A lung tumours . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Representative images of H&E and IHC staining for P4HA2, and quantification (bars) of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Hydroxyproline ( biolabs Inc . ) for total hydroxyproline activity of . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Hydroxyproline assay (Cell biolabs Inc.) for total hydroxyproline activity of cells. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Western blot indicating of Collagen I in H1299control H1299RASSF1A .

Western blot indicating levels of Collagen I in H1299control and H1299RASSF1A cells.


Western blot indicating of Collagen I in H1299control in presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination .

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

Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.


of immunofluorescence of Collagen I in H1299control in presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination . H1299RASSF1A was used as negative control for comparison . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Quantification of immunofluorescence staining of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination. H1299RASSF1A cell line was used as negative control for comparison. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Immunofluorescence for Collagen I P4HA2 ( right graph ) in HOP92 after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HOP92 cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Western blot analyses in HOP92 showing increased expression of collagen I after siRNA against RASSF1A .

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

Western blot analyses in HOP92 cells showing increased expression of collagen I after siRNA against RASSF1A.


Immunofluorescence for Collagen I P4HA2 ( right graph ) in HeLa after siRNA against RASSF1A . Scale bars 20µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 experiments error bars represent mean ± S . E . M .

Immunofluorescence staining for Collagen I and P4HA2 and quantification (right graph) in HeLa cells after siRNA against RASSF1A. Scale bars 20µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test (2-tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Invasion of H1299control H1299RASSF1A through three - dimensional collagen matrix coated inserts , over 24 hours ( n = 3 ) . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

B-EXP_ASSAY O B-CELL_LINE I-CELL_LINE 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

Invasion of H1299control and H1299RASSF1A cells through three-dimensional collagen matrix coated inserts, over 24 hours (n=3). Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of H1299 treated with siNT , siRNA against P4HA2 or in presence of prolylhydroxylase inhibitor DPCA ( 4μM ) , allowed to invade for 24 hours through a three - dimensional Matrigel matrix Boyden chamber . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Representative images and quantification of H1299 cells treated with siNT, siRNA against P4HA2 or in the presence of the prolylhydroxylase inhibitor DPCA (4μM), and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative of HOP92 stably transfected with shcontrol or shRASSF1A allowed to invade for 24 hours through a three - dimensional Matrigel matrix Boyden chamber . Scale bars 100µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Representative images and quantification of HOP92 cells stably transfected with shcontrol or shRASSF1A and allowed to invade for 24 hours through a three-dimensional Matrigel matrix Boyden chamber. Scale bars 100µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


3D collagen contraction : 5x105 / ml were embedded into collagen rat tail I matrix ( 2mg / ml ) analysed after 4 days . circles indicates diameter of gel plugs at time 0 . Representative bright - field ( bottom ) with , showing effect of H1299RASSF1A or H1299control on collagen gel remodelling after pre - treatment with siRNA against P4HA2 , 4μM DPCA or combination as indicated . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

3D collagen contraction assay: 5x105/ml cells were embedded into collagen rat tail I matrix (2mg/ml) and analysed after 4 days. White circles indicates diameter of gel plugs at time 0. Representative bright-field images (bottom) with quantification, showing the effect of H1299RASSF1A or H1299control cells on collagen gel remodelling and after cells pre-treatment with siRNA against P4HA2, 4μM DPCA or combination as indicated. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Upper image : Cartoon of hanging drops method for spheroids formation embedding collagen rat tail I matrix ( 2mg / ml ) . Bottom : Representative immunofluorescence of YAP distribution its ( right bars ) by Pearson ' s coefficient for correlation of YAP DAPI co - in H1299control H1299RASSF1A 3D spheroids grown in 3D collagen matrix ( 2mg / ml ) . Scale bars 10µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Upper image: Cartoon of hanging drops method for spheroids formation and embedding collagen rat tail I matrix (2mg/ml). Bottom: Representative immunofluorescence images of YAP distribution and its nuclear quantification (right bars) by Pearson's coefficient for correlation of YAP and DAPI co-staining in H1299control and H1299RASSF1A 3D spheroids grown in 3D collagen matrix (2mg/ml). Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Relative mRNA expression of P4HA2 YAP target genes CYR61 CTGF in H1299control in presence of siNT or siRNA targeting YAP1 . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Relative mRNA expression levels of P4HA2 and YAP target genes CYR61 and CTGF in H1299control cells in presence of siNT or siRNA targeting YAP1. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Representative immunofluorescence of YAP distribution ( right ) its by Pearson ' s coefficient for correlation ( left ) of YAP DAPI co - in H1299control H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A with or without treatment with siP4HA2 or 4μM DPCA . ( Correlation , Pearson ' s coefficient ) . ( n = 3 , 300 per experiment ) Scale bars 10µm . Data information : Unless otherwise indicated statistical analyses were performed using Student ' s t - 2 - ( ) of n = 3 experiments error bars represent mean ± S . E . M .

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

Representative immunofluorescence images of YAP distribution (right) and its nuclear quantification by Pearson's coefficient for correlation (left) of YAP and DAPI co-staining in H1299control and H1299RASSF1A plated on extracted ECM from H1299control or H1299RASSF1A cells with or without treatment with siP4HA2 or 4μM DPCA. (Correlation, Pearson's coefficient). (n=3, 300 cells per experiment) Scale bars 10µm. Data information: Unless otherwise indicated all statistical analyses were performed using Student's t-test 2-(tailed) of n=3 experiments and error bars represent the mean ± S.E.M.


Bar graph representing of stiffness measured by atomic force microscopy ( Young modulus ) of ECM generated by H1299control or H1299RASSAF1A in vitro . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O B-SUBCELLULAR O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph representing quantification of stiffness measured by atomic force microscopy (Young modulus) of ECM area generated by H1299control or H1299RASSAF1A cells in vitro. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative atomic force microscopy ( AFM ) showing organization of extracellular matrix generated by H1299control H1299RASSF1A in 3D collagen matrices ( 2mg / ml ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE O O B-SMALL_MOLECULE O O O O O O O

Representative atomic force microscopy (AFM) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).


Bar graph representing of stiffness by atomic force microscopy ( Young modulus ) in of n = 5 H1299control n = 3 H1299RASSF1A lung tumours on day 30 . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-CELL_LINE O O O B-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graph representing quantification of stiffness by atomic force microscopy (Young modulus) in of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours on day 30. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative topographic of stiffness map of H1299control H1299RASSF1A lung tumours provided by AFM .

Representative topographic images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.


AFM of ECM fibre organization within lung tumours generated by H1299control H1299RASSF1A on day 30 .

B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-DISEASE I-DISEASE O O B-CELL_LINE I-CELL_LINE O O O O

AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.


Second harmonic generation ( SHG ) representative ( bars ) showing deposition organization of collagen fibres produced by H1299control H1299RASSF1A spheroids embedded in collagen I matrix ( 2mg / ml ) , treated with siNT , siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity . Red arrowheads show highly organized , long collagen fibres . Scale bars 20µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

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

Second harmonic generation (SHG) representative Images and Quantification (bars) showing deposition and organization of collagen fibres produced by H1299control and H1299RASSF1A spheroids embedded in collagen I matrix (2mg/ml), treated with siNT, siRNA against P4HA2 or 4μM DPCA to restrict P4HA2 activity. Red arrowheads show highly organized, long collagen fibres. Scale bars 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


SHG of organization ( bars ) of collagen fractions in lung tumours metastases at day 30 . Scale bars 10µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

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SHG images of organization and quantification (bars) of collagen fractions in primary lung tumours and metastases at day 30. Scale bars 10µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Representative H & E ( bars ) of picrosirius red of two independent regions of n = 5 H1299control n = 3 H1299RASSF1A lung tumours . Scale bars 100µm , zoom 20µm . Data information : statistical analyses were performed using Student ' s t - ( 2 - ) of n = 3 for in vitro experiments error bars represent mean ± S . E . M .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-CELL_LINE O O O B-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative H&E and quantification (bars) of picrosirius red staining of two independent regions of n=5 H1299control and n=3 H1299RASSF1A primary lung tumours. Scale bars 100µm, zoom 20µm. Data information: All statistical analyses were performed using Student's t-test (2-tailed) of n=3 for in vitro experiments and error bars represent the mean ± S.E.M.


Left : Representative of H1299 on 3D collagen wells with defined stiffness with NANOG ( green ) or DAPI ( blue ) . Scale bars 10µm . Right : of NANOG : DAPI co - localization ( upper graph ) total NANOG ( bottom graph ) . of co - localization ( n = 300cells / experiment ) is represented by Pearson ' s coefficient . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

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

Left: Representative images of H1299 cells on 3D collagen wells with defined stiffness with NANOG (green) or DAPI (blue). Scale bars 10µm. Right: Quantification of NANOG:DAPI nuclear co-localization (upper graph) and total NANOG fluorescence intensity (bottom graph). Quantification of nuclear co-localization (n=300cells/experiment) is represented by Pearson's coefficient. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence show merge of NANOG ( green ) , DAPI ( blue ) distribution in H1299control H1299RASSF1A cultured on 2D glass . Scale bars 10µm . Right : of co - localization ( n = 200cells / experiment ) is represented by Pearson ' s coefficient .

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

Representative immunofluorescence images show merge of NANOG (green), DAPI (blue) distribution in H1299control and H1299RASSF1A cells cultured on 2D glass. Scale bars 10µm. Right: Quantification of nuclear co-localization (n=200cells/experiment) is represented by Pearson's coefficient.


Representative immunofluorescence of CD133 in H1299control H1299RASSF1A grown on 3D collagen wells with defined stiffness . Scale bars 10µm . Bottom : of n = 200 / experiment . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

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

Representative immunofluorescence images of CD133 in H1299control and H1299RASSF1A cells grown on 3D collagen wells with defined stiffness. Scale bars 10µm. Bottom: Quantification of n=200 cells/experiment. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative of immunofluorescence for pluripotency marker NANOG β - catenin in three - dimensional spheroids embedded in collagen matrix ( 2mg / ml ) . Scale bars 50µm .

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

Representative images of immunofluorescence staining for the pluripotency marker NANOG and β-catenin in three-dimensional spheroids embedded in collagen matrix (2mg/ml). Scale bars 50µm.


Representative of H & E immunohistochemical for NANOG YAP1 in lung tumours , ( day 17 ) . Scale bars 100µm . Right : Graph bars represent of NANOG YAP1 on strong ( 3 + , 2 + ) ( % ) for at least 2 independent regions of n = 4 H1299control n = 2 H1299RASSF1A tumours at day 17 . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

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

Representative images of H&E and immunohistochemical staining for NANOG and YAP1 in primary lung tumours, (day 17). Scale bars 100µm. Right: Graph bars represent quantification of NANOG and YAP1 staining based on strong (3+, 2+) nuclear intensity (%) for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Relative mRNA expression of NANOG , OCT4 SOX2 in H1299control H1299RASSF1A lung adenocarcinoma . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

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

Relative mRNA expression levels of NANOG, OCT4 and SOX2 in H1299control and H1299RASSF1A lung adenocarcinoma cells. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence of NANOG YAP in presence of siNT or siYAP in H1299control H1299RASSF1A embedded grown in three - dimensional collagen matrix ( 2mg / ml ) . Scale bars 10µm .

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Representative immunofluorescence images of NANOG and YAP in the presence of siNT or siYAP in H1299control and H1299RASSF1A cells embedded and grown in three-dimensional collagen matrix (2mg/ml). Scale bars 10µm.


Western blots of H1299control H1299RASSF1A lysates for NANOG , SOX2 OCT4 in presence of siNT or siYAP .

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Western blots of H1299control and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.


Relative H1299control mRNA expression NANOG , OCT4 SOX2 in H1299control in presence of siNT or siYAP . Data information : Statistical significance was determined by Student ' s t - ( 2 - ) of n = 3 experiments unless otherwise stated . Error bars represent mean ± S . E . M .

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

Relative H1299control mRNA expression levels NANOG, OCT4 and SOX2 in H1299control cells in the presence of siNT or siYAP. Data information: Statistical significance was determined by Student's t-test (2-tailed) of n=3 experiments unless otherwise stated. Error bars represent the mean ± S.E.M.


Representative immunofluorescence of H1299control H1299RASSF1A spheroids embedded grown in collagen matrix ( 2mg / ml ) stained for NANOG ( Green ) YAP ( Red ) with or without 4μM DPCA . Scale bars 10µm .

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Representative immunofluorescence images of H1299control and H1299RASSF1A spheroids embedded and grown in collagen matrix (2mg/ml) and stained for NANOG (Green) and YAP (Red) with or without 4μM DPCA. Scale bars 10µm.


Western blots of H1299control lysates for NANOG , SOX2 OCT4 in presence of siNT , siP4HA2 or 4μM DPCA .

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Western blots of H1299control lysates for NANOG, SOX2 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.


Western blot analyses of expression of TTF - 1 Mucin 5B in H1299control H1299RASSF1A with band determine by ImageJ analyser .

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Western blot analyses of expression levels of TTF-1 and Mucin 5B in H1299control and H1299RASSF1A cells with band intensity quantification ratio determine by ImageJ analyser.


Representative of H & E immunohistochemical for lung differentiation markers TTF - 1 Mucin 5B for at least 2 independent regions of n = 4 H1299control n = 2 H1299RASSF1A tumours at day 17 with ( right ) of TTF - 1 on . Scale bars 100µm . Statistical significance was determined by Student ' s t - ( 2 - ) . Error bars represent mean ± S . E .

O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-CELL_LINE O O O B-CELL_LINE B-DISEASE O O O O O O O O B-GENEPROD I-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

Representative images of H&E and immunohistochemical staining for lung differentiation markers TTF-1 and Mucin 5B for at least 2 independent regions of n=4 H1299control and n=2 H1299RASSF1A primary tumours at day 17 with quantification (right) of TTF-1 based on nuclear intensity. Scale bars 100µm. Statistical significance was determined by Student's t-test (2-tailed). Error bars represent the mean ± S.E.M


Representative of immunofluorescence of spheroids grown in collagen matrix ( 2mg / ml ) for differentiation markers TTF - 1 ( Green ) Mucin 5B ( Red ) . Scale bars 50µm , Zoom 10µm .

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

Representative images of immunofluorescence staining of spheroids grown in collagen matrix (2mg/ml) for differentiation markers TTF-1 (Green) and Mucin 5B (Red). Scale bars 50µm, Zoom 10µm.


Bright field of H1299control H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three - dimensional substrate . Scale bars 200µm .

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Bright field images of H1299control and H1299RASSF1A spheroid differentiation on Matrigel matrix after 24 hours when 3D spheroids were seeded on three-dimensional substrate. Scale bars 200µm.


A Immunofluorescence of HK2 with an AlexaFluor488 - conjugated antibody in HeLa expressing mitochondria - targeted RFP . Yellow signals in merge indicate mitochondrial localization of HK2 . Scale bar : 15 µm .

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A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.


B Immunofluorescence of HK2 with a secondary AlexaFluor555 - conjugated antibody in HeLa expressing both mitochondria - targeted YFP ER - targeted CFP . merged signal indicates MAM localization of HK2 is quantified in bar graph on right ( n = 24 ) . Image magnifications are shown in lower part of panel ; arrows indicate HK2 dots in mito - ER contact sites . Scale bar : 15 µm .

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

B Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n=24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 µm.


C co - of HK2 split - GFP - probe for ER - mitochondria contacts ( SPLICSL ) on HeLa ; HK2 is revealed with a secondary AlexaFluor555 - conjugated antibody , merged signal is . Scale bar : 15 µm .

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C Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, the merged signal is white. Scale bar: 15 µm.


D experiment showing of HK2 dots that merge with MAMs in HeLa ( n = 24 analyzed from three independent experiments ; mean ± SD ) .

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D Quantification experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells (n=24 cells analyzed from three independent experiments; mean±SD).


E experiment showing of SPLICSL dots positive for HK2 in HeLa ( n = 10 analyzed from three independent experiments ; mean ± SD ) .

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E Quantification experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells (n=10 cells analyzed from three independent experiments; mean±SD).


F of HK2 dots positive for SPLICSL in HeLa , COLO 741 , MDA - MB - 231 , PN 04 . 4 S462 ( n ≥ 6 for each ; mean ± SD ) .

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

F Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4 and S462 cells (n≥6 cells for each cell line; mean±SD).


H HK2 displacement from HeLa MAMs after a 2 min treatment with cl - HK2pep is shown by loss of merging signal analyzed as in ( C ) . cl - SCRpep is used as a negative control Pearson ' s co - localization coefficient is indicated in figure ( cl - SCRpep n = 26 ; cl - HK2pep n = 24 ; p < 0 . 001 with a Student ' s t ) . Scale bar : 10 µm .

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H HK2 displacement from HeLa MAMs after a 2 min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in (C). cl-SCRpep is used as a negative control and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n=26 cells; cl-HK2pep n=24 cells; p<0.001 with a Student's t test). Scale bar: 10 µm.


Effect of cl - HK2pep on glucose phosphorylation by human recombinant HK2 ( I ) where both total hexokinase activity HK1 / HK2 specific activities are measured .

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

Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) where both total hexokinase activity and HK1/HK2 specific activities are measured.


Effect of cl - HK2pep on glucose phosphorylation in 4T1 extracts ( J ) , where both total hexokinase activity HK1 / HK2 specific activities are measured .

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Effect of cl-HK2pep on glucose phosphorylation in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.


Effect of cl - HK2pep on Ca2 + dynamics ER Ca2 + are measured by FRET - , D4ER probe expressed in lumen of ER ( A ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O

Effect of cl-HK2pep on cellular Ca2+ dynamics ER Ca2+ levels are measured by the FRET-based, D4ER fluorescent probe expressed in the lumen of ER (A) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep on IP3 . IP3 are assessed with GFP - PHD probe ; histamine ( 100μM ) is used as a positive control for IP3 generation ; data are reported as mean of signals ± ( n = 3 independent experiments ; B ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at 5 μM used here [

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O 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 B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O

Effect of cl-HK2pep on IP3 levels. IP3 levels are assessed with the GFP-PHD probe; histamine (100μM) is used as a positive control for IP3 generation; data are reported as mean of fluorescent signals±SEM (n=3 independent experiments; B) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [


Effect of cl - HK2pep on Ca2 + dynamics changes in mitochondrial Ca2 + are recorded ( C ; scale bar : 20µm ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

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Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded (C; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep on Ca2 + dynamics changes in mitochondrial Ca2 + are recorded quantified using GCAMP6f sensor ( in D , as mean of 475 / 410 nm ; signal ± of at least 5 independent experiments more than 20 analized ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at 5 μM used

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE 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 B-CELL_LINE O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O

Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal±SEM of at least 5 independent experiments and more than 20 cells analized) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here


Effect of cl - HK2pep on Ca2 + dynamics ( E ) as of with increased Ca2 + in mitochondria , with a threshold 475 / 410 for positivity > 3 ; baseline mean = 1 . 84 ± 0 . 54 ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at 5 μM used here [

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

Effect of cl-HK2pep on cellular Ca2+ dynamics (E) as percentage of cells with increased Ca2+ in mitochondria, with a threshold 475/410 ratio for positivity >3; baseline mean ratio=1.84±0.54) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [


Effect of cl - HK2pep on Ca2 + dynamics with mitochondria - targeted aequorin ( F , where data are reported as mean of [ Ca2 + ] ± of 3 independent experimental days ) . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O

Effect of cl-HK2pep on cellular Ca2+ dynamics with mitochondria-targeted aequorin (F, where data are reported as mean of [Ca2+]±SEM of 3 independent experimental days). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep treatment on mitochondrial potential assessed with TMRM probe . Kinetic experiments ( G , single , n = 216 analyzed in at least 10 independent

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

Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n=216 cells analyzed in at least 10 independent experiments


Effect of cl - HK2pep treatment on mitochondrial potential assessed with TMRM probe . Kinetic experiments H , representative field ; scale bar : 20µm ) Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Where indicated , are kept in Ca2 + free medium plus 500 µM EGTA with or without 10 µM BAPTA -

O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O B-SMALL_MOLECULE 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 B-CELL_LINE 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 I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE

Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments H, representative field; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM


Effect of cl - HK2pep treatment on mitochondrial potential assessed with TMRM probe . quantified ( I J ; TMRM is normalized to initial value expressed in for each time point , with a depolarization threshold placed at 40 % of initial value ) . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Where indicated , are kept in Ca2 + free medium plus 500 µM EGTA with or without 10 µM BAPTA - AM ; Xe - C is Xestospongin C , which selectively inhibits IP3R at 5 μM used

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

Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM; Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here


Effects of PTP desensitizers CsA or C63 ( 5 µM each , 1 h preincubation ) on mitochondrial Ca2 + recorded with GCAMP6f ( A ) in treated with cl - HK2pep . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

O O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O

Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial Ca2+ levels recorded with GCAMP6f (A) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


Effects of PTP desensitizers CsA or C63 ( 5 µM each , 1 h preincubation ) on mitochondrial potential assessed with TMRM ( B ) in treated with cl - HK2pep . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

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

Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial membrane potential assessed with TMRM (B) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


induction by cl - HK2pep ; in cytofluorimetric analyses viable are double negative for Annexin V - FITC 7 - AAD measured 15 min after peptide treatment data are presented as mean ± SD they were obtained from 3 different experiments or more ; 3 technical replicates in analyzed experiment . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) data are presented as mean ± SD of at least 3 independent experiments . * * * p < 0 . 001 with a Student ' s t

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) data are presented as mean±SD of at least 3 independent experiments. *** p<0.001 with a Student's t test


induction by cl - HK2pep ; in cytofluorimetric analyses in kinetic experiment shown in ( D ; scale bar : 50µm ) viable are double negative for Annexin V - FITC 7 - AAD TMRM positive . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses in the kinetic experiment shown in (D; scale bar: 50µm) viable cells are double negative for Annexin V-FITC and 7-AAD and TMRM positive. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


induction by cl - HK2pep ; in cytofluorimetric analyses viable are double negative for Annexin V - FITC 7 - AAD measured 15 min after peptide treatment Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated , caspase inhibitor Z - VAD - fmk or calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment .

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated, the caspase inhibitor Z-VAD-fmk or the calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.


induction by cl - HK2pep ; in cytofluorimetric analyses viable are double negative for Annexin V - FITC 7 - AAD measured 15 min after peptide treatment data are presented as mean ± SD they were obtained from 3 different experiments or more ; 3 technical replicates in analyzed experiment . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment . data are presented as mean ± SD of at least 3 independent experiments . p < 0 . 0001 with a two - way ANOVA ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; Bonferroni post - in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep cl - HK2pep vs PD150606 + cl - HK2pep ) .

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).


Effect of calpain inhibition on mitochondrial potential assessed with TMRM ( G , normalized signal mean ± SD ; 3 different experiments including 3 technical replicates each ) in treated with cl - HK2pep Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment . data are presented as mean ± SD of at least 3 independent experiments . p < 0 . 0001 with a two - way ANOVA ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; Bonferroni post - in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep cl - HK2pep vs PD150606 + cl - HK2pep ) .

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

Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM (G, normalized fluorescence signal mean±SD; 3 different experiments including 3 technical replicates each) in cells treated with cl-HK2pep Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).


Effect of calpain inhibition on mitochondrial Ca2 + recorded with GCAMP6f ( H ) in treated with cl - HK2pep . Data information : Experiments throughout Figure are carried out on HeLa ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment .

O O B-GENEPROD O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O 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

Effect of calpain inhibition on mitochondrial Ca2+ levels recorded with GCAMP6f (H) in cells treated with cl-HK2pep . Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.


A Representative IHCs in human reactive lymphoid tissue , where HK2 expression is mainly restricted to proliferating blasts within germinal centers ( center ) , in B - CLL . Scale bar 100 µm .

O O B-EXP_ASSAY O B-ORGANISM B-TISSUE I-TISSUE I-TISSUE O O B-GENEPROD O O O O O B-EXP_ASSAY O O B-TISSUE I-TISSUE O O O O O B-DISEASE I-DISEASE I-DISEASE O O O O O O

A Representative IHCs in human reactive lymphoid tissue, where HK2 expression is mainly restricted to proliferating blasts within germinal centers (center), and in patient B-CLL cells. Scale bar 100 µm.


Effect of cl - HK2pep on human B - CLL freshly isolated from . HK2 - expressing B - CLL ( B ; GAPDH respiratory III subunit UQCRC2 are cytosol mitochondrial loading controls , respectively ) are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 on CD19 + , B lymphocytes from 5 healthy controls .

O O O O O O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE O O O O B-GENEPROD O O B-DISEASE I-DISEASE I-DISEASE O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR 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 O O O O O O O O O O O O O O O O O O O O O O

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells (B; GAPDH and respiratory complex III subunit UQCRC2 are cytosol and mitochondrial loading controls, respectively) Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.


Effect of cl - HK2pep on human B - CLL freshly isolated from . HK2 - expressing B - CLL undergo mitochondrial depolarization assessed by TMRM ( C ) Experiments are carried out on samples from at least 10 on CD19 + , B lymphocytes from 5 healthy controls . data are presented as mean ± ; data were obtained from 3 technical replicates in analyzed samples . two - way ANOVA : p < 0 . 0001 ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) Student ' s t ; * * * p < 0 . 01 , * p < 0 . 05 .

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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells undergo mitochondrial depolarization assessed by TMRM staining (C) Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. two-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments) Student's t test; ***p<0.01, *p<0.05.


ffect of cl - HK2pep on human B - CLL freshly isolated from . , measured by cytofluorimetric of Annexin V - FITC 7 - AAD are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 on CD19 + , B lymphocytes from 5 healthy controls . In C , D , F G data are presented as mean ± ; data were obtained from 3 technical replicates in analyzed samples . wo - way ANOVA : p < 0 . 0001 ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; tudent ' s t ; * * * p < 0 . 01 , * p < 0 . 05 .

O O O O O O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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 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 O O O O O O O O O O O O O O O O O

ffect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. In C, D, F and G data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. wo-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); tudent's t test; ***p<0.01, *p<0.05.


Effect of cl - HK2pep on human B - CLL freshly isolated from . , measured by cytofluorimetric of Annexin V - FITC 7 - AAD are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 on CD19 + , B lymphocytes from 5 healthy controls .

O O O O O O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.


Effect of cl - HK2pep on human B - CLL freshly isolated from . , measured by cytofluorimetric of Annexin V - FITC 7 - AAD are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 on CD19 + , B lymphocytes from 5 healthy controls . data are presented as mean ± ; data were obtained from 3 technical replicates in analyzed samples . Bonferroni post - in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep cl - HK2pep vs PD150606 + cl - HK2pep ) Student ' s t ; * * * p < 0 . 01 , * p < 0 . 05 .

O O O O O O B-ORGANISM B-DISEASE I-DISEASE I-DISEASE O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O 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 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 O O O O O O O O O O O O O O O

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. Bonferroni post-test in graph ***p<0.001(cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep) Student's t test; ***p<0.01, *p<0.05.


Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 shHK2 - 2 ) in CT26 colon cancer ( A ) Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control .

O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O O O O B-CELL_LINE B-DISEASE I-DISEASE O O O O O O O O O O O O O O O O O O O O O

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells (A) Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 shHK2 - 2 ) in CT26 colon cancer inhibits growth in soft agar ( B ; colony ± ; n = 6 replicates obtained from 3 independent experiments ; Student ' s t * * * p < 0 . 001 ; A . U . , arbitrary units ) . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells inhibits growth in soft agar (B; colony area±SEM; n=6 replicates obtained from 3 independent experiments; Student's t test ***p<0.001; A.U., arbitrary units). Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with cl - HK2pep ( 50 µM , 6 h ; C , scale bar : 100 µm ) reverts formation of foci ; dead are highlighted by Trypan Blue . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with uncleaved HK2pep ( 2 µM , 48 h ; D , scale bar : 200 µm ) reverts formation of foci ; dead are highlighted by Trypan Blue . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . HK2 - MMP - 2 - expressing CT26 ( E , scale bar : 100 µm ) are allografted in Balb / c mice . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2-targeting on in vivo neoplastic growth. HK2- and MMP-2-expressing CT26 cells (E, scale bar: 100 µm) are allografted in Balb/c male mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . Intratumor administration ( 5 injections of 2 nmol peptide every 12 h ) of either cl - HK2pep or HK2pep ( F ; CNT n = 4 ; cl - HK2pep n = 5 ; HK2pep n = 5 mice ) in Balb / c mice . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control . In F , mean volume ± SD is reported ; Student ' s t * * * p < 0 .

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

Effect of HK2-targeting on in vivo neoplastic growth. Intratumor administration (5 injections of 2 nmol peptide every 12 h) of either cl-HK2pep or HK2pep (F; CNT n=4; cl-HK2pep n=5; HK2pep n=5 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In F, mean tumor volume±SD is reported; Student's t test ***p<0.001


Effect of HK2 - targeting on in vivo neoplastic growth . intraperitoneal administration of HK2pep ( G ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 6 ; HK2pep n = 7 mice ) reduce neoplastic growth in Balb / c mice . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control . In G , representative ultrasound inspections 3D reconstructions of masses are shown ( grey : SCRpep treated ; blue : HK2pep treated ) . normalized volume ± ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - in graphs * p < 0 . 05 ; * * * p < 0 . 001 .

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

Effect of HK2-targeting on in vivo neoplastic growth. intraperitoneal administration of HK2pep (G; 5 injections of 60 nmol peptide every 12 h; SCRpep n=6; HK2pep n=7 mice) reduce neoplastic growth in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In G, representative ultrasound inspections and 3D reconstructions of tumor masses are shown (light grey: SCRpep treated animal; light blue: HK2pep treated animal). normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


Effect of HK2 - targeting on in vivo neoplastic growth . same effect is observed on allografts of breast carcinoma 4T1 ( H ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 5 ; HK2pep n = 7 mice ) in Balb / c mice . Data information : Throughout Figure , cl - SCRpep or SCRpep are used as peptide negative control . normalized volume ± ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - in graphs * p < 0 . 05 ; * * * p < 0 . 001 .

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

Effect of HK2-targeting on in vivo neoplastic growth. the same effect is observed on allografts of breast carcinoma 4T1 cells (H; 5 injections of 60 nmol peptide every 12 h; SCRpep n=5; HK2pep n=7 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


( D ) RNF145 ADIPOR2 respond to a range of FAs . HEK - 293T were treated for 6h with saturated FAs ( SFAs ; 400 µM ( C12 : 0 - C18 : 0 ) ; 200 µM ( C20 : 0 ) ) or unsaturated FAs ( UFAs ; 400 µM ( C18 : 1 - n9 ) ; 200 µM ( C20 : 4 - n6 , C20 : 5 - n3 ) ) . * non - specific bands .

(D) RNF145 and ADIPOR2 respond to a range of FAs. HEK-293T cells were treated for 6h with saturated FAs (SFAs; 400 µM (C12:0 - C18:0); 200 µM (C20:0)) or unsaturated FAs (UFAs; 400 µM (C18:1-n9); 200 µM (C20:4-n6, C20:5-n3)). * non-specific bands.


( E ) Inhibition of lipid desaturase SCD1 affects RNF145 ADIPOR2 . HEK - 293T were treated with SCD1 inhibitor MF - 438 ( SCDi , 100 nM , 18h ) RNF145 / ADIPOR2 expression analysed by immunoblotting . * non - specific bands .

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

(E) Inhibition of the primary lipid desaturase SCD1 affects RNF145 and ADIPOR2 levels. HEK-293T cells were treated with the SCD1 inhibitor MF-438 (SCDi, 100 nM, 18h) and RNF145/ADIPOR2 expression analysed by immunoblotting. * non-specific bands.


( F ) SCD1 overexpression affects RNF145 / ADIPOR2 . HEK - 293T containing an empty control vector or expressing SCD1 - HA were cultured in FCS - free DMEM for 24h whole - lysates analysed . * non - specific bands .

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

(F) SCD1 overexpression affects RNF145/ADIPOR2. HEK-293T cells containing an empty control vector or expressing SCD1-HA were cultured in FCS-free DMEM for 24h and whole-cell lysates analysed. * non-specific bands.


( A ) Endogenous ADIPOR2 is stabilised in RNF145 knockout . Representative 35S - chase in HeLa WT , RNF145 knockout ( ∆ RNF145 ) or ADIPOR2 knockout ( ∆ AR2 ) . of 35S - labelled ADIPOR2 normalised to t = 0 h is shown below .

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

(A) Endogenous ADIPOR2 is stabilised in RNF145 knockout cells. Representative 35S pulse-chase analysis in HeLa WT, RNF145 knockout (∆RNF145) or ADIPOR2 knockout (∆AR2) cells. Quantification of 35S-labelled ADIPOR2 normalised to t = 0 h is shown below.


( B ) ADIPOR2 regulation requires E3 ligase activity of RNF145 . HEK - 293T RNF145 knockout ( ∆ RNF145 ) were stably complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . * non - specific bands .

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

(B) ADIPOR2 regulation requires the E3 ligase activity of RNF145. HEK-293T RNF145 knockout cells (∆RNF145) were stably complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). * non-specific bands.


( C ) RNF145 binds ADIPOR2 . RNF145 was immunoprecipitated from HeLa RNF145 knockout complemented with WT RNF145 ( RNF145 - V5 ) or an inactive RNF145C552A , H554A RING domain mutant ( mRNF145 - V5 ) . were treated with VCP inhibitor ( NMS - 873 , 10 µM , 90 min ) prior to immunoprecipitation with an anti - V5 antibody .

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

(C) RNF145 binds ADIPOR2. RNF145 was immunoprecipitated from HeLa RNF145 knockout cells complemented with WT RNF145 (RNF145-V5) or an inactive RNF145C552A,H554A RING domain mutant (mRNF145-V5). Cells were treated with VCP inhibitor (NMS-873, 10 µM, 90 min) prior to immunoprecipitation with an anti-V5 antibody.


( D ) E2 ubiquitin - conjugating enzyme UBE2G2 is involved in RNF145 - mediated ADIPOR2 degradation . Immunoblot of HEK - 293T depleted of RNF145 ( gRNF145 ) , UBE2G2 ( gUBE2G2 ) or B2M ( control ; gCTR ) .

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

(D) The E2 ubiquitin-conjugating enzyme UBE2G2 is involved in RNF145-mediated ADIPOR2 degradation. Immunoblot analysis of HEK-293T cells depleted of RNF145 (gRNF145), UBE2G2 (gUBE2G2) or B2M (control; gCTR).


( E ) ADIPOR2 degradation depends on UBXD8 . HEK - 293T were depleted of B2M ( gCTR ) or UBXD8 ( gUBXD8 ) analysed by immunoblotting .

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

(E) ADIPOR2 degradation depends on UBXD8. HEK-293T cells were depleted of B2M (gCTR) or UBXD8 (gUBXD8) and analysed by immunoblotting.


( A ) FA - mediated regulation of RNF145 is proteasome - dependent . HEK - 293T were treated with BSA , PA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml , 6h ) analysed by immunoblotting . * non - specific bands .

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

(A) FA-mediated regulation of RNF145 is proteasome-dependent. HEK-293T cells were treated with BSA, PA (400 µM, 6h) ± MG132 (10 µg/ml, 6h) and analysed by immunoblotting. *non-specific bands.


( B ) UBXD8 participates in PA - modulated RNF145 degradation . CRISPR / Cas9 - mediated depletion of UBXD8 ( gUBXD8 ) or B2M ( gB2M , control ) in HEK - 293T , followed by exposure to PA ( 400 µM , 6h ) or BSA ( 6h , vehicle control ) .

O O O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O O O O O

(B) UBXD8 participates in PA-modulated RNF145 degradation. CRISPR/Cas9-mediated depletion of UBXD8 (gUBXD8) or B2M (gB2M, control) in HEK-293T cells, followed by exposure to PA (400 µM, 6h) or BSA (6h, vehicle control).


( C ) PA OA treatment alter RNF145 ubiquitination . Immunoprecipitation of RNF145 from treated with BSA ( control ) , PA or OA ( each 200 µM , 3h ) . During FA treatments , VCP inhibitor ( NMS - 873 , 10 µM ) was present to stabilise ubiquitinated . RNF145 ubiquitination ( ubi ) was detected using an anti - ubiquitin antibody ( VU - 1 ) .

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

(C) PA and OA treatment alter RNF145 ubiquitination. Immunoprecipitation of RNF145 from cells treated with BSA (control), PA or OA (each 200 µM, 3h). During FA treatments, VCP inhibitor (NMS-873, 10 µM) was present to stabilise ubiquitinated proteins. RNF145 ubiquitination (ubi) was detected using an anti-ubiquitin antibody (VU-1).


( D ) HEK - 293T were treated with cycloheximide ( CHX , 1 µg / ml ) OA ( 400 µM , 6h ) ± MG132 ( 10 µg / ml ) for indicated times . LE , long exposure ; * non - specific bands .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE 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

(D) HEK-293T cells were treated with cycloheximide (CHX, 1 µg/ml) and OA (400 µM, 6h) ± MG132 (10 µg/ml) for the indicated times. LE, long exposure; * non-specific bands.


( E ) Lysine - less RNF145 is stabilised under PA treatment . RNF145 knockout HEK - 293T ( ∆ RNF145 ) were complemented with RNF145 - V5 or a lysine - less variant of RNF145 ( RNF145 - V5 K > R ) treated BSA or PA ( 400 µM , 6h ) . Dotted lines demarcate different lines .

O O O O O O B-GENEPROD O O O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O

(E) Lysine-less RNF145 is stabilised under PA treatment. RNF145 knockout HEK-293T cells (∆RNF145) were complemented with RNF145-V5 or a lysine-less variant of RNF145 (RNF145-V5 K > R) and treated BSA or PA (400 µM, 6h). Dotted lines demarcate the different cell lines.


( F ) Adjustment of RNF145 to PA requires its ubiquitin ligase activity . Immunoblot of whole - lysates from RNF145 knockout HEK - 293T ( ∆ RNF145 ) stably complemented with RNF145 - V5 or RNF145C552A , H554A - V5 ( mRNF145 - V5 ) treated with BSA or PA ( 400 µM , 6h ) . LE , long exposure .

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

(F) Adjustment of RNF145 levels to PA requires its ubiquitin ligase activity. Immunoblot of whole-cell lysates from RNF145 knockout HEK-293T (∆RNF145) cells stably complemented with RNF145-V5 or RNF145C552A, H554A-V5 (mRNF145-V5) and treated with BSA or PA (400 µM, 6h). LE, long exposure.


( A ) ADIPOR2 regulation by FAs is completely dependent on RNF145 . WT or RNF145 knockout HEK - 293T ( ∆ RNF145 ) were treated with BSA , PA or OA ( 400 µM , 6h ) analysed by immunoblotting . Non - specific bands are indicated ( * ) .

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

(A) ADIPOR2 regulation by FAs is completely dependent on RNF145. WT or RNF145 knockout HEK-293T cells (∆RNF145) were treated with BSA, PA or OA (400 µM, 6h) and analysed by immunoblotting. Non-specific bands are indicated (*).


( B ) RNF145 - ADIPOR2 interaction is FA - modulated . Immunoprecipitation of endogenous RNF145 HEK - 293T depleted of UBE2G2 ( gUBE2G2 ) / or RNF145 ( gRNF145 ) treated with 400 µM of indicated PA , OA or BSA ( control ) for 3h . LE , long exposure . * non - specific bands .

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

(B) RNF145 - ADIPOR2 interaction is FA-modulated. Immunoprecipitation of endogenous RNF145 HEK-293T cells depleted of UBE2G2 (gUBE2G2) and/or RNF145 (gRNF145) and treated with 400 µM of the indicated PA, OA or BSA (control) for 3h. LE, long exposure. * non-specific bands.


( C ) Immunoprecipitation ( IP ) of endogenous ADIPOR2 from WT or ∆ RNF145 ( see ( C ) ) treated with MG132 ( 5 µg / ml , 6h ) ± OA ( 200 µM , 6h ) . Ubiquitinated ADIPOR2 was detected using a specific anti - ubiquitin antibody ( clone VU - 1 ) . Note that ADIPOR2 in RNF145 knockout HEK - 293T are increased .

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

(C) Immunoprecipitation (IP) of endogenous ADIPOR2 from WT or ∆RNF145 cells (see (C)) treated with MG132 (5 µg/ml, 6h) ± OA (200 µM, 6h). Ubiquitinated ADIPOR2 was detected using a specific anti-ubiquitin antibody (clone VU-1). Note that ADIPOR2 levels in RNF145 knockout HEK-293T cells are increased.


( D ) ADIPOR2 knockout HEK - 293T were stably complemented with ADIPOR2 - HA , an ADIPOR2 mutant in which lysines were mutated to arginines ( K - less ADIPOR2 - HA ) , or an empty vector control exposed to OA ( 200 µM , 6h ) or BSA ( control ) . ADIPOR2 variants were visualised by immunoblotting using a specific anti - HA antibody . LE , long exposure .

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

(D) ADIPOR2 knockout HEK-293T cells were stably complemented with ADIPOR2-HA, an ADIPOR2 mutant in which all lysines were mutated to arginines (K-less ADIPOR2-HA), or an empty vector control and exposed to OA (200 µM, 6h) or BSA (control). ADIPOR2 variants were visualised by immunoblotting using a specific anti-HA antibody. LE, long exposure.


( A ) Loss of ADIPOR2 sensitises to PA - induced cytotoxicity . HEK - 293T were depleted of RNF145 ( purple ) , ADIPOR2 ( blue ) , B2M ( CTR ; grey ) , or both ADIPOR2 RNF145 ( blue + purple ) , exposed to PA ( 200 µM , 96h ) stained with crystal violet . of crystal - violet stained is shown alongside a representative micrograph ( below ) . response of each to PA was normalised to its BSA - treated condition ( dotted ) . Statistically significant differences to containing gCTR are indicated ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) . Mean ± SD are shown . Boxplots represent median , first third quantiles . Upper lower whiskers extend to values up to 1 . 5 - fold interquartile range . N = 3 biological replicates , * * q ≤ 0 . 01 . Double knockdowns were generated by sequential constitutive CRISPR / Cas9 - mediated depletion of RNF145 or ADIPOR2 . Knockdown efficiencies are shown in Appendix Fig S1A .

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

(A) Loss of ADIPOR2 sensitises cells to PA-induced cytotoxicity. HEK-293T cells were depleted of RNF145 (purple), ADIPOR2 (blue), B2M (CTR; grey), or both ADIPOR2 and RNF145 (blue + purple), exposed to PA (200 µM, 96h) and stained with crystal violet. Quantification of crystal-violet stained cells is shown alongside a representative micrograph (below). The response of each cell line to PA was normalised to its BSA-treated condition (dotted line). Statistically significant differences to cells containing gCTR cells are indicated (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction). Mean ± SD are shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. N = 3 biological replicates, ** q ≤ 0.01. Double knockdowns were generated by sequential constitutive CRISPR/Cas9-mediated depletion of RNF145 or ADIPOR2. Knockdown efficiencies are shown in Appendix Fig S1A.


( B ) were treated as in ( A ) lysates analysed by immunoblotting . LE , long exposure ; * non - specific bands .

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

(B) Cells were treated as in (A) and lysates analysed by immunoblotting. LE, long exposure; *non-specific bands.


( C ) HEK - 293T depleted of B2M ( gCTR ) or ADIPOR2 ( gADIPOR2 ) were complemented with empty vector ( blue ) , full - ADIPOR2 - HA ( green ) , or a catalytically inactive variant of ADIPOR2 - HA ( dead ADIPOR2 ; red ) treated with PA ( 200 µM , 96h ) or BSA ( vehicle control , 96h ) . Measurements from PA - treated were normalised to their BSA - treated condition ( dotted ) . growth was quantified , representative crystal violet is shown . Boxplots represent median , first third quantiles . Upper lower whiskers extend to values up to 1 . 5 - fold interquartile range . * * q ≤ 0 . 01 ( robust one - way ANOVA with Benjamini - Hochberg multiple testing correction ) .

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

(C) HEK-293T cells depleted of B2M (gCTR) or ADIPOR2 (gADIPOR2) were complemented with empty vector (blue), full-length ADIPOR2-HA (green), or a catalytically inactive variant of ADIPOR2-HA (dead ADIPOR2; red) and treated with PA (200 µM, 96h) or BSA (vehicle control, 96h). Measurements from PA-treated cells were normalised to their BSA-treated condition (dotted line). Cell growth was quantified, and representative crystal violet staining is shown. Boxplots represent the median, first and third quantiles. Upper and lower whiskers extend to values up to 1.5-fold interquartile range. ** q ≤ 0.01 (robust one-way ANOVA with Benjamini-Hochberg multiple testing correction).