Figure 3Size exclusion chromatography ( SEC ) profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) and presence of Tat - C5 ( purple , Absmax = 11 . 7 ml ) SEC profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) and presence of Tat - P4 - ( C5 ) 2 ( blue , Absmax = 10 . 7 ml ) SEC profiles of PICK1 in absence ( pink , Absmax = 11 . 7 ml ) and presence of TMR - Tat - P4 - ( C5 ) 2 ( blue ( abs280 nm ) , red ( abs544 nm ) , Absmax = 10 . 7 ml ) . Representative SEC profiles of PICK1 ( 40 µM ) incubated with increasing concentration of Tat - P4 - ( C5 ) 2 , colors indicate Gaussian fits of respective population , ( red ) aggregates / Void , ( blue ) dimeric PICK1 , ( green ) oligomeric PICK1 , ( orange ) unbound Tat - P4 - ( C5 ) 2 . Quantification of abs280 at Evol = 11 . 7 ml ( dimer - blue ) and Evol = 10 . 7 ml ( tetramer - green ) as a function of concentration ( 0 - 10 µM ) of Tat - P4 - ( C5 ) 2 . Light blue line indicates tetramer fraction of total PICK1 . SDS - PAGE sedimentation assay showing increased amounts of PICK1 in the pellet ( P ) fraction with increasing concentrations of Tat - P4 - ( C5 ) 2 . PICK1 ~ 50kDa , Tat - P4 - ( C5 ) 2 ~ 10kDa . S denotes supernatant fraction . Small angle X - ray scattering ( SAXS ) derived scattering curves of a concentration range of PICK1 on absolute scale show concentration dependent aggregation . Pair distance distribution function ( pddf ) of PICK1 in absence of Tat - P4 - ( C5 ) 2 , indicating very large protein complexes present in sample . SAXS derived scattering curves of a concentration range of PICK1 in complex with Tat - P4 - ( C5 ) 2 on absolute scale show minor concentration dependent aggregation . Pair distance distribution function ( pddf ) of a concentration range of PICK1 in presence of Tat - P4 - ( C5 ) 2 , indicating a smaller complex than for PICK1 in absence of Tat - P4 - ( C5 ) 2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Size exclusion chromatography (SEC) profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-C5 (purple, Absmax = 11.7 ml)SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of Tat-P4-(C5)2 (blue, Absmax = 10.7 ml)SEC profiles of PICK1 in absence (pink, Absmax = 11.7 ml) and presence of TMR-Tat-P4-(C5)2 (blue (abs280 nm), red (abs544 nm), Absmax=10.7 ml).Representative SEC profiles of PICK1 (40 µM) incubated with increasing concentration of Tat-P4-(C5)2, colors indicate Gaussian fits of respective population, (red) aggregates/Void, (blue) dimeric PICK1, (green) oligomeric PICK1, (orange) unbound Tat-P4-(C5)2.Quantification of abs280 at Evol = 11.7 ml (dimer - blue) and Evol = 10.7 ml (tetramer - green) as a function of concentration (0-10 µM) of Tat-P4-(C5)2. Light blue line indicates tetramer fraction of total PICK1.SDS-PAGE sedimentation assay showing increased amounts of PICK1 in the pellet (P) fraction with increasing concentrations of Tat-P4-(C5)2. PICK1~50kDa, Tat-P4-(C5)2~10kDa. S denotes supernatant fraction.Small angle X-ray scattering (SAXS) derived scattering curves of a concentration range of PICK1 on absolute scale show concentration dependent aggregation. Pair distance distribution function (pddf) of PICK1 in absence of Tat-P4-(C5)2, indicating very large protein complexes present in sample.SAXS derived scattering curves of a concentration range of PICK1 in complex with Tat-P4-(C5)2 on absolute scale show minor concentration dependent aggregation. Pair distance distribution function (pddf) of a concentration range of PICK1 in presence of Tat-P4-(C5)2, indicating a smaller complex than for PICK1 in absence of Tat-P4-(C5)2.


Figure 4Representative confocal images of hippocampal neurons showing membrane penetration of TMR - Tat - P4 - ( C5 ) 2 and TMR - Tat - C5 ( 5µM ) ( magenta ) , but not the control TMR - C5 ( all 20µM ) . The cell membrane was stained with DiO ( grey ) . Scale bars : 10µm and 5µm on the zooms / orthogonal views . Representative confocal images of hippocampal neurons transduced with the viral vector encoding GFP - PICK1 and incubated with 5nM TMR - Tat - P4 - ( C5 ) 2 . Partial co - localization between TMR - Tat - P4 - ( C5 ) 2 ( red ) , GFP - PICK1 ( green ) and GluA2 ( blue ) is seen in insert zooms . Experiment was done three times . Scale bars : 10µm and 1µm on the zooms . Representative immunoblots from hippocampal slices following treatment with Tat - P4 - ( C5 ) 2 or Tat - C5 ( bath application , 20µM ) . Densitometric analysis of immunoblots from C show reduced PICK1 : GluA2 Co - IP following incubation with Tat - P4 - ( C5 ) 2 but not Tat - C5 . Bar graphs show mean with error - bars as SEM . ( One - way ANOVA followed by Dunnett ´ s multiple comparisons test , * * * p ≤ 0 . 001 , n = 4 / group ) Representative immunoblots from hippocampal slices following Tat - P4 - ( C5 ) 2 bath application ( 20µM ) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD ( cLTD ) . Bar graphs show densitometric analysis of immunoblots from ( E ) . Representative immunoblots from hippocampal slices following Tat - C5 bath application ( 20µM ) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD ( cLTD ) . Bar graphs ( right panel ) show densitometric analysis of immunoblots from ( G ) . Representative confocal images of hippocampal neurons stained for surface GluA2 ( sGluA2 , light blue ) and internalized GluA2 ( iGluA2 , red ) . Treatment with Tat - P4 - ( C5 ) 2 ( 20 µM ) significantly increases the constitutive , but not PMA induced internalization . Scale bars 10µm . Quantification of iGluA2 / sGluA2 within the region of interest for individual neuron ( see I , top right corner ) . Tat - C5 did not increase GluA2 internalization ( see Appendix Fig S9D - F ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative confocal images of hippocampal neurons showing membrane penetration of TMR-Tat-P4-(C5)2 and TMR-Tat-C5 (5µM) (magenta), but not the control TMR-C5 (all 20µM). The cell membrane was stained with DiO (grey). Scale bars: 10µm and 5µm on the zooms/orthogonal views.Representative confocal images of hippocampal neurons transduced with the viral vector encoding GFP-PICK1 and incubated with 5nM TMR-Tat-P4-(C5)2. Partial co-localization between TMR-Tat-P4-(C5)2 (red), GFP-PICK1 (green) and GluA2 (blue) is seen in insert zooms. Experiment was done three times. Scale bars: 10µm and 1µm on the zooms.Representative immunoblots from hippocampal slices following treatment with Tat-P4-(C5)2 or Tat-C5 (bath application, 20µM).Densitometric analysis of immunoblots from C show reduced PICK1:GluA2 Co-IP following incubation with Tat-P4-(C5)2 but not Tat-C5. Bar graphs show mean with error-bars as SEM. (One-way ANOVA followed by Dunnett´s multiple comparisons test, *** p≤0.001, n = 4/group)Representative immunoblots from hippocampal slices following Tat-P4-(C5)2 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD). Bar graphs show densitometric analysis of immunoblots from (E).Representative immunoblots from hippocampal slices following Tat-C5 bath application (20µM) show reduction in the phosphorylation level of S880 GluA2 both under basal condition and after chemical LTD (cLTD). Bar graphs (right panel) show densitometric analysis of immunoblots from (G).Representative confocal images of hippocampal neurons stained for surface GluA2 (sGluA2, light blue) and internalized GluA2 (iGluA2, red). Treatment with Tat-P4-(C5)2 (20 µM) significantly increases the constitutive, but not PMA induced internalization. Scale bars 10µm. Quantification of iGluA2/sGluA2 within the region of interest for individual neuron (see I, top right corner). Tat-C5 did not increase GluA2 internalization (see Appendix Fig S9D-F).


Figure 5Representative coronal slices from lumbar spinal cord show the presence of both TMR - Tat - P4 - ( C5 ) 2 and TMR - Tat - C5 peptides ( magenta ) 1 hour after i . t . administration in naïve mice . Scale bar 200µm . DH dorsal horn , VH ventral horn . Insert zooms of merged channels ( right panels ) , scale bar 50µm . Representative immunoblots of co - immunoprecipited PICK1 : GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i . t . injection of Tat - P4 - ( C5 ) 2 . Densitometric analysis of immunoblots indicate a significant effect following Tat - P4 - ( C5 ) 2 treatment , graph show mean + / - SEM and individual points , unpaired t - test , * * p ≤ 0 . 01 , n = 4 / groupRepresentative immunoblots of co - immunoprecipited PICK1 : GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i . t . injection Tat - C5 . Densitometric analysis of immunoblots indicate no significant effect of Tat - C5 , graph show mean + / - SEM and individual points , unpaired t - test , p = 0 . 10 , n = 4 / groupRepresentative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880 - GluA2 total levels following 20 µM Tat - P4 - ( C5 ) 2 , and Tat - C5 i . t . injections compared to saline injected animals . Densitometric analysis of immunoblot indicates a significant effect only for Tat - P4 - ( C5 ) 2 ( graph show mean + / - SEM and individual points , One - way ANOVA followed by Dunnett ´ s multiple comparisons test , * * p ≤ 0 . 01 , n = 5 - 6 / group as indicated by individual values ) Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 and GluA2 surface level upon Tat - P4 - ( C5 ) 2 , Tat - C5 peptides incubation compared to the untreated condition ( CTR ) . Densitometric analysis of immunoblots show that the reduction in mean surface for GluA2 in not significant ( graph show mean + / - SEM and individual points , One - way ANOVA followed by Dunnett ´ s multiple comparisons test , n = 5 / group as indicated by individual values ) . Immunoblot of PICK1 following pull - down from lumbar spinal cord with Biotin - Tat - P4 - ( C5 ) 2 or Biotin following 20 µM i . t . administration demonstrating in vivo target engagement .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Representative coronal slices from lumbar spinal cord show the presence of both TMR-Tat-P4-(C5)2 and TMR-Tat-C5 peptides (magenta) 1 hour after i.t. administration in naïve mice. Scale bar 200µm. DH dorsal horn, VH ventral horn. Insert zooms of merged channels (right panels), scale bar 50µm.Representative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrate partial disruption of this interaction 1 hour after 20 µM i.t. injection of Tat-P4-(C5)2. Densitometric analysis of immunoblots indicate a significant effect following Tat-P4-(C5)2 treatment, graph show mean +/- SEM and individual points, unpaired t-test, **p≤0.01, n=4/groupRepresentative immunoblots of co-immunoprecipited PICK1:GluA2 from spinal cord lumbar tract total lysates demonstrates no disruption of this interaction 1 hour after 20 µM i.t. injection Tat-C5. Densitometric analysis of immunoblots indicate no significant effect of Tat-C5, graph show mean +/- SEM and individual points, unpaired t-test, p=0.10, n=4/groupRepresentative immunoblots of lumbar spinal cord total lysates exhibit decreased pS880-GluA2 total levels following 20 µM Tat-P4-(C5)2, and Tat-C5 i.t. injections compared to saline injected animals. Densitometric analysis of immunoblot indicates a significant effect only for Tat-P4-(C5)2 (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, ** p≤0.01, n=5-6/group as indicated by individual values)Surface biotinylation of spinal cord slices under basal condition demonstrates unchanged GluA1 and GluA2 surface level upon Tat-P4-(C5)2, Tat-C5 peptides incubation compared to the untreated condition (CTR). Densitometric analysis of immunoblots show that the reduction in mean surface for GluA2 in not significant (graph show mean +/- SEM and individual points, One-way ANOVA followed by Dunnett´s multiple comparisons test, n=5/group as indicated by individual values).Immunoblot of PICK1 following pull-down from lumbar spinal cord with Biotin-Tat-P4-(C5)2 or Biotin following 20 µM i.t. administration demonstrating in vivo target engagement.


Figure 6i . t . administration ( 20µM ) of Tat - P4 - ( C5 ) 2 significantly reduced ipsilateral paw hypersensitivity of SNI mice in the initiation stage ( 2 days after injury ) of the spared nerve injury model at time points 1 and 3 hours ( n = 7 ) i . t . administration ( 20µM ) of Tat - C5 did not affect ipsilateral paw hypersensitivity of SNI mice in the initiation stage ( 2 days after injury ) of the spared nerve injury model ( n = 8 ) Representative western blots ( top ) for GluA1 and GluA2 following surface biotinylation of spinal cord slices . Densitometric analysis ( bottom ) of immunoblots . We observed up - regulation of both GluA1 and GluA2 surface level following SNI surgery compared to non - operated animal CTL ( unpaired t test GluA1 : t ( 4 ) = 3 . 372 , GluA2 : t ( 4 ) = 3 . 991 , # p ≤ 0 . 05 , n = 3 / group ) . Tat - P4 - ( C5 ) 2 but not Tat - C5 significantly reduced the SNI - induced GluA2 surface expression and shows a strong tendency for GluA1 as well . Representative immunoblot shows unaltered PICK1 total level in the total lysate in the four conditions . Images of lumbar spinal cord sagittal sections showing the time course of TMR - Tat - P4 - ( C5 ) 2 ( magenta ) after i . t . administration ( 20 µM ) in SNI mice showing maximal accumulation in cells after 60 min . Scale bar 1000 µm and 100 µm in Zooms . Overlay with immunohistochemical staining for the neuronal marker NeuN ( yellow ) highlights strong neuronal tropism of TMR - Tat - P4 - ( C5 ) 2 ( magenta ) . Scale bar 100 µmDose - dependent Von Frey test in the acute phase ( 2 days after injury ) of the spared nerve injury model following i . t . administration of Tat - P4 - ( C5 ) 2 ( n = 8 / group ) did not affect ipsilateral paw hypersensitivity of SNI mice and lasts up to 4h post injection . i . t . administration of Tat - P4 - ( C5 ) 2 ( 20 µM ) at day 14 and 28 after injury in the maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in male and female mice ( compared to time 0 pre - injection , D14 n = 8 / group / gender , D28 n = 6 male , n = 7 female ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6i.t. administration (20µM) of Tat-P4-(C5)2 significantly reduced ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model at time points 1 and 3 hours (n = 7)i.t. administration (20µM) of Tat-C5 did not affect ipsilateral paw hypersensitivity of SNI mice in the initiation stage (2 days after injury) of the spared nerve injury model (n = 8)Representative western blots (top) for GluA1 and GluA2 following surface biotinylation of spinal cord slices. Densitometric analysis (bottom) of immunoblots. We observed up-regulation of both GluA1 and GluA2 surface level following SNI surgery compared to non-operated animal CTL (unpaired t test GluA1: t(4)=3.372, GluA2: t(4)=3.991,#p≤0.05, n=3/group). Tat-P4-(C5)2 but not Tat-C5 significantly reduced the SNI-induced GluA2 surface expression and shows a strong tendency for GluA1 as well. Representative immunoblot shows unaltered PICK1 total level in the total lysate in the four conditions.Images of lumbar spinal cord sagittal sections showing the time course of TMR-Tat-P4-(C5)2 (magenta) after i.t. administration (20 µM) in SNI mice showing maximal accumulation in cells after 60 min. Scale bar 1000 µm and 100 µm in Zooms.Overlay with immunohistochemical staining for the neuronal marker NeuN (yellow) highlights strong neuronal tropism of TMR-Tat-P4-(C5)2 (magenta). Scale bar 100 µmDose-dependent Von Frey test in the acute phase (2 days after injury) of the spared nerve injury model following i.t. administration of Tat-P4-(C5)2 (n=8/group) did not affect ipsilateral paw hypersensitivity of SNI mice and lasts up to 4h post injection.i.t. administration of Tat-P4-(C5)2 (20 µM) at day 14 and 28 after injury in the maintenance phase of SNI model in mice shows significant increase in pain withdrawal threshold up to 3 hours with equal efficacy in male and female mice (compared to time 0 pre-injection, D14 n=8/group/gender, D28 n=6 male, n=7 female).


Figure 7Representative images of kainate - induced cobalt uptake in spinal cord slices from p14 mouse pups treated with i ) saline , ii ) TNFα and iii ) TNFα + Tat - P4 - ( C5 ) 2 . Scale Bar : 100 µm . Quantification of cobalt positive ( black soma ) cells . Each data point represents the average 4 - 6 25 µm sections from a single 400 µm slice . Bars show means of 4 - 6 slices and error bars represent SEM . ( one - way ANOVA followed by Bonferroni ' s multiple comparisons test , * P < 0 . 05 , * * P < 0 . 01 ) . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Representative images of kainate-induced cobalt uptake in spinal cord slices from p14 mouse pups treated with i) saline, ii) TNFα and iii) TNFα + Tat-P4-(C5)2. Scale Bar: 100 µm. Quantification of cobalt positive (black soma) cells. Each data point represents the average 4-6 25 µm sections from a single 400 µm slice. Bars show means of 4-6 slices and error bars represent SEM. (one-way ANOVA followed by Bonferroni's multiple comparisons test, *P <0.05, **P<0.01).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 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 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.


Figure 1Kaplan - 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 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 . 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Kaplan-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 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.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 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.


Figure 2Mass - 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 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 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 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 levels of Collagen I in H1299control and H1299RASSF1A cells . Western blot indicating levels of Collagen I in H1299control cells in the presence of ( 4μM ) prolylhydroxylase inhibitor ( DPCA ) , siRNA against P4HA2 or combination . 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 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 cells showing increased expression of collagen I after siRNA against RASSF1A . 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mass-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 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 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 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 levels of Collagen I in H1299control and H1299RASSF1A cells.Western blot indicating levels of Collagen I in H1299control cells in the presence of (4μM) prolylhydroxylase inhibitor (DPCA), siRNA against P4HA2 or combination.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 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 cells showing increased expression of collagen I after siRNA against RASSF1A.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.


Figure 3Invasion 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 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Invasion 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 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 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 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 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 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 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.


Figure 4Bar 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 ) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices ( 2mg / ml ) . 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 images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM . AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30 . 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Bar 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) images showing organization of extracellular matrix generated by H1299control and H1299RASSF1A cells in 3D collagen matrices (2mg/ml).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 images of stiffness map of H1299control and H1299RASSF1A lung primary tumours provided by AFM.AFM images of ECM fibre organization within primary lung tumours generated by H1299control and H1299RASSF1A cells on day 30.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 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 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.


Figure 5Left : 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 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 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 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 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 .

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Figure 5Left: 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 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 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 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 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.


Figure 6Relative 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 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 and H1299RASSF1A lysates for NANOG , SOX2 and OCT4 in the presence of siNT or siYAP . 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 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 and OCT4 in the presence of siNT , siP4HA2 or 4μM DPCA .

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Figure 6Relative 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 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 and H1299RASSF1A lysates for NANOG, SOX2 and OCT4 in the presence of siNT or siYAP.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 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 and OCT4 in the presence of siNT, siP4HA2 or 4μM DPCA.


Figure 7Western 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 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 . MRepresentative 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 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 .

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Figure 7Western 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 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.MRepresentative 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 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.


Figure 1A 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 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 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 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 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 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 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 and HK1 / HK2 specific activities are measured . Effect of cl - HK2pep on glucose phosphorylation in 4T1 cell extracts ( J ) , where both total hexokinase activity and HK1 / HK2 specific activities are measured .

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Figure 1A 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 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 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 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 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 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 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 and HK1/HK2 specific activities are measured.Effect of cl-HK2pep on glucose phosphorylation in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.


Figure 2Effect 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 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 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 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 hereEffect 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 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 membrane potential assessed with the TMRM probe . Kinetic experiments ( G , single cell analysis , n = 216 cells analyzed in at least 10 independent experimentsEffect 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 - AMEffect 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

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Effect 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 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 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 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 hereEffect 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 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 membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n=216 cells analyzed in at least 10 independent experimentsEffect 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-AMEffect 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


Figure 3Effects 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 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 ) 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 testcell 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 ) 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 . 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 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 + 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Effects 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 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)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 testcell 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)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.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 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+ 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.


Figure 4A 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 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 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 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 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 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 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A 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 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 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 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 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 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 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.