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 1C , Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A . Total protein extracts ( ZT13 ) from 10 - day - old seedlings grown under 12h / 12h light / dark cycles were subject to heat ( 30ºC 20min ) , pyrophosphatase ( PP ) and NaF / Na3VO4 ( phosphatase inhibitors ) treatments . TOC1 - GFP at different phosphorylation states were separated by 8 % SDS - PAGE ( acrylamide : bisacrylamide 150 : 1 ) and subsequently detected by α - GFP .

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Figure 1C, Phosphorylation of TOC1 is eliminated or largely reduced in 5X and S175A. Total protein extracts (ZT13) from 10-day-old seedlings grown under 12h/12h light/dark cycles were subject to heat (30ºC 20min), pyrophosphatase (PP) and NaF/Na3VO4 (phosphatase inhibitors) treatments. TOC1-GFP at different phosphorylation states were separated by 8% SDS-PAGE (acrylamide:bisacrylamide 150:1) and subsequently detected by α-GFP.


Figure 2A - F Shown are hypocotyl lengths of cRL - grown 4 - day - old seedlings of toc1 complemented with wild - type TOC1 ( A ) , 5X ( B ) , T135A ( C ) , S175A ( D ) driven by TOC1 native promoter and wild - type TOC1 ( E ) , 5X ( F ) driven by 35S promoter at sequential fluent rates . Different letters indicate statistically significant differences between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) , error bars indicate SEM . Data are representative of three biological trials with similar results .

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Figure 2A-F Shown are hypocotyl lengths of cRL-grown 4-day-old seedlings of toc1 complemented with wild-type TOC1 (A), 5X (B), T135A (C), S175A (D) driven by TOC1 native promoter and wild-type TOC1 (E), 5X (F) driven by 35S promoter at sequential fluent rates. Different letters indicate statistically significant differences between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test), error bars indicate SEM. Data are representative of three biological trials with similar results.


Figure 3Hypocotyl lengths in SD condition of Col - 0 , toc1 - 101 and two independent native or 35S promoter lines of wild - type TOC1 and each phosphosite mutants . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week . C - E , Average CCA1 : LUC bioluminescence traces of indicated plant lines . Data information : 18 to 27 seedlings per line were entrained in 12h / 12h light / dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2 - h intervals for 1 week . White and gray regions indicate subjective light and dark period . Data are representative of at least two independent trials with similar results . Different letters indicate statistically significant differences in period between genotypes ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) .

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

Figure 3Hypocotyl lengths in SD condition of Col-0, toc1-101 and two independent native or 35S promoter lines of wild-type TOC1 and each phosphosite mutants. The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week.C-E, Average CCA1:LUC bioluminescence traces of indicated plant lines. Data information: 18 to 27 seedlings per line were entrained in 12h/12h light/dark cycles for 7 d and next transferred to constant red light at ZT2 for image acquisition with 2-h intervals for 1 week. White and gray regions indicate subjective light and dark period. Data are representative of at least two independent trials with similar results. Different letters indicate statistically significant differences in period between genotypes (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test).


Figure 4A - C , PIL1 , AT5G02580 and CDF5 expression pattern in 3 - d - old SD - grown seedlings from indicated plant lines at night . Data is representative of 3 biological trials .

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Figure 4A-C, PIL1, AT5G02580 and CDF5 expression pattern in 3-d-old SD-grown seedlings from indicated plant lines at night. Data is representative of 3 biological trials.


Figure 5A , Co - IP assay of PIF3 - TAP with TOC1 - GFP vs . 5X - GFP in N . benthamiana . Protein extracts were immunoprecipitated by IgG resins followed by HRV - 3C protease digestion . PIF3 and TOC1 were detected by anti - MYC and anti - GFP , respectively . B , Quantification of protein interaction in A . Results from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant difference ( * p < 0 . 05 , student ' s t test ) . C , ChIP - qPCR assay of TOC1 binding to promoter of pre - dawn - phased PIF3 target genes at ZT14 in Col - 0 , native promoter TOC1 and 5X lines . D , ChIP - qPCR assay of the chromatin residence of native promoter TOC1 - GFP at target promoters in toc1 vs . pif3 / 4 / 5 toc1 ( pif345t1 ) mutant backgrounds . Data information : 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 - GFP was immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicates significant differences ( * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

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Figure 5A, Co-IP assay of PIF3-TAP with TOC1-GFP vs. 5X-GFP in N. benthamiana. Protein extracts were immunoprecipitated by IgG resins followed by HRV-3C protease digestion. PIF3 and TOC1 were detected by anti-MYC and anti-GFP, respectively. B, Quantification of protein interaction in A. Results from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant difference (*p<0.05, student's t test). C, ChIP-qPCR assay of TOC1 binding to promoter of pre-dawn-phased PIF3 target genes at ZT14 in Col-0, native promoter TOC1 and 5X lines. D, ChIP-qPCR assay of the chromatin residence of native promoter TOC1-GFP at target promoters in toc1 vs. pif3/4/5 toc1 (pif345t1) mutant backgrounds. Data information: 3-d-old SD-grown seedlings were harvested at ZT14, TOC1-GFP was immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicates significant differences (** p<0.001, *** p<0.0001, Student's t test), n.s=not significant.


Figure 6B , Measurement of TOC1 - GFP protein turn - over in TOC1 , 5X , T135A , S175A native promoter lines by cycloheximide ( CHX ) treatment . 10 - d - old seedlings grown in 12h / 12h light / dark cycles were subject to cycloheximide ( CHX ) or ethanol ( Mock ) treatment for indicated time length . Data from 3 independent trials were fitted to non - linear Weibull regression curves . Data information : TOC1 protein was detected by α - GFP , ADK was used as loading control . TOC1 / ADK ratio was calculated by the intensities of TOC1 - GFP bands normalized by the intensities of ADK bands using ImageJ . C , Estimates of TOC1 - GFP protein half - life in B . Error bars indicate SEM , statistical analyses were performed with student ' s t test , different letters indicate significant differences ( p < 0 . 05 ) . Data from 3 independent trials were averaged .

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Figure 6B, Measurement of TOC1-GFP protein turn-over in TOC1, 5X, T135A, S175A native promoter lines by cycloheximide (CHX) treatment. 10-d-old seedlings grown in 12h/12h light/dark cycles were subject to cycloheximide (CHX) or ethanol (Mock) treatment for indicated time length. Data from 3 independent trials were fitted to non-linear Weibull regression curves. Data information: TOC1 protein was detected by α-GFP, ADK was used as loading control. TOC1/ADK ratio was calculated by the intensities of TOC1-GFP bands normalized by the intensities of ADK bands using ImageJ.C, Estimates of TOC1-GFP protein half-life in B. Error bars indicate SEM, statistical analyses were performed with student's t test, different letters indicate significant differences (p<0.05). Data from 3 independent trials were averaged.


Figure 7B , Quantification of interactions between TOC1 / 5X - TAP and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TOC1 / 5X were immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . TOC1 / 5X and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated TOC1 / 5X to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , Student ' s t test ) . C , ChIP - qPCR assay of native promoter driven TOC1 - GFP and 5X - GFP ' s residence at target promoters in toc1 vs . nf - yc3 / 4 / 9 toc1 ( c349t1 ) mutants . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP and magnetic protein G beads . Data from 3 independent trials were averaged . Error bars indicate SEM , asterisks indicate significant differences ( * p < 0 . 05 , * * * p < 0 . 0001 , Student ' s t test ) , n . s = not significant .

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Figure 7B, Quantification of interactions between TOC1/5X-TAP and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TOC1/5X were immunoprecipitated with IgG resins followed by HRV-3C protease digestion. TOC1/5X and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated TOC1/5X to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, Student's t test).C, ChIP-qPCR assay of native promoter driven TOC1-GFP and 5X-GFP's residence at target promoters in toc1 vs. nf-yc3/4/9 toc1 (c349t1) mutants. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP and magnetic protein G beads. Data from 3 independent trials were averaged. Error bars indicate SEM, asterisks indicate significant differences (* p<0.05, *** p<0.0001, Student's t test), n.s= not significant.


Figure 8A , Co - IP assay of HDA15 - TAP with TOC1 - GFP and 5X - GFP in N . benthamiana . HDA15 - TAP was immunoprecipitated with IgG resins and digested by HRV - 3C protease . HDA15 and TOC1 were detected by α - MYC and α - GFP , respectively . B , Quantification of the interaction strength in A . Results from 5 independent trials were averaged . Error bar indicates SEM , asterisk indicates significant difference ( * p < 0 . 05 , student ' s t test ) . C and D , Hypocotyl lengths and free - running periods of indicated plants lines . Data of ~ 75 plants from 3 independent experiments were averaged , different letters indicate statistically significant differences ( p < 0 . 01 , one - way ANOVA followed by Tukey - Kramer HSD test ) . The middle line of the box represents the median , the x in the box represents the mean . The bottom line and the top line of the box represent 1st and 3rd quartile , respectively . The whiskers extend from the ends of the box to the minimum value and maximum value . E , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 independent experiments were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 8A, Co-IP assay of HDA15-TAP with TOC1-GFP and 5X-GFP in N. benthamiana. HDA15-TAP was immunoprecipitated with IgG resins and digested by HRV-3C protease. HDA15 and TOC1 were detected by α-MYC and α-GFP, respectively. B, Quantification of the interaction strength in A. Results from 5 independent trials were averaged. Error bar indicates SEM, asterisk indicates significant difference (*p<0.05, student's t test). C and D, Hypocotyl lengths and free-running periods of indicated plants lines. Data of ~75 plants from 3 independent experiments were averaged, different letters indicate statistically significant differences (p<0.01, one-way ANOVA followed by Tukey-Kramer HSD test). The middle line of the box represents the median, the x in the box represents the mean. The bottom line and the top line of the box represent 1st and 3rd quartile, respectively. The whiskers extend from the ends of the box to the minimum value and maximum value.E, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 independent experiments were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


Figure 9A and B , ChIP - qPCR assay of HDA15 ' s ( A ) and NF - YC9 ' s ( B ) residence at target promoters in presence or absence of TOC1 and PRR5 . GFP - HDA15 and GFP - NF - YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col - 0 , toc1 single and prr5 toc1 double mutant grown in SD condition . Results were normalized by % input of PIL1 coding region ( PIL1 CDS ) . Data from at least 3 independent experiments were averaged , error bars indicate SEM . Asterisks indicate significant differences ( * p < 0 . 05 , * * p < 0 . 001 , * * * p < 0 . 0001 , Student ' s t test ) . C and D , Dual luciferase assay of the repression activity of GFP - TOC1 and GFP - 5X on promoter of AT5G02580 ( C ) and CDF5 ( D ) in protoplasts of toc1 and hda15toc1 ( h15t1 ) mutants . AT5G02580 : LUC , CDF5 : LUC reporters and the effectors ( empty GFP , GFP - TOC1 , and GFP - 5X ) were co - transfected to toc1 and hda15 toc1 protoplasts with 35S : Renilla - LUC as internal control . Data are means ± SEM ( n = 5 biological trials ) relative to empty GFP which is set as 1 . Different letters indicate statistically significant differences ( student ' s t test , p < 0 . 05 ) .

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Figure 9A and B, ChIP-qPCR assay of HDA15's (A) and NF-YC9's (B) residence at target promoters in presence or absence of TOC1 and PRR5. GFP-HDA15 and GFP-NF-YC9 driven by 35S promoter were transiently expressed in protoplasts isolated from Col-0, toc1 single and prr5 toc1 double mutant grown in SD condition. Results were normalized by % input of PIL1 coding region (PIL1 CDS). Data from at least 3 independent experiments were averaged, error bars indicate SEM. Asterisks indicate significant differences (* p<0.05, ** p<0.001, *** p<0.0001, Student's t test).C and D, Dual luciferase assay of the repression activity of GFP-TOC1 and GFP-5X on promoter of AT5G02580 (C) and CDF5 (D) in protoplasts of toc1 and hda15toc1 (h15t1) mutants. AT5G02580:LUC, CDF5:LUC reporters and the effectors (empty GFP, GFP-TOC1, and GFP-5X) were co-transfected to toc1 and hda15 toc1 protoplasts with 35S:Renilla-LUC as internal control. Data are means ± SEM (n = 5 biological trials) relative to empty GFP which is set as 1. Different letters indicate statistically significant differences (student's t test, p<0.05).


Figure 10A , Quantification of interactions between TAP - PRR5 and NF - YC proteins in presence or absence of NF - YB subunits . Proteins were transiently expressed in N . benthamiana . TAP - PRR5 was immunoprecipitated with IgG resins followed by HRV - 3C protease digestion . PRR5 and NF - YB / C were detected by α - MYC and α - HA , respectively . Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co - immunoprecipitated NF - YC to immunoprecipitated PRR5 to NF - YC input . Results from 3 independent trials were averaged . Error bars indicate SEM , different letters denote significant differences ( p < 0 . 05 , student ' s t test ) . C , ChIP - qPCR assay of native promoter TOC1 - GFP and 5X - GFP binding to the target promoters at ZT14 in toc1 vs . prr5 toc1 ( p5t1 ) mutant . 3 - d - old SD - grown seedlings were harvested at ZT14 , TOC1 and 5X were immunoprecipitated by α - GFP . Data from 3 independent trials were averaged , error bars indicate SEM . Asterisks indicate significant differences ( Student ' s t test ; * p < 0 . 05 ) , n . s = not significant . D , Expression of PIL1 , AT5G02580 and CDF5 in 3 - d - old SD - grown seedlings at ZT18 . Data from 3 biological trials were averaged . Error bars indicate SEM . Different letters denote statistically significant differences based on Wilcoxon test ( p < 0 . 05 ) .

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Figure 10A, Quantification of interactions between TAP-PRR5 and NF-YC proteins in presence or absence of NF-YB subunits. Proteins were transiently expressed in N. benthamiana. TAP-PRR5 was immunoprecipitated with IgG resins followed by HRV-3C protease digestion. PRR5 and NF-YB/C were detected by α-MYC and α-HA, respectively. Protein band intensity was measured by ImageJ and interactions were exhibited as ratio of co-immunoprecipitated NF-YC to immunoprecipitated PRR5 to NF-YC input. Results from 3 independent trials were averaged. Error bars indicate SEM, different letters denote significant differences (p<0.05, student's t test).C, ChIP-qPCR assay of native promoter TOC1-GFP and 5X-GFP binding to the target promoters at ZT14 in toc1 vs. prr5 toc1 (p5t1) mutant. 3-d-old SD-grown seedlings were harvested at ZT14, TOC1 and 5X were immunoprecipitated by α-GFP. Data from 3 independent trials were averaged, error bars indicate SEM. Asterisks indicate significant differences (Student's t test; * p<0.05), n.s= not significant.D, Expression of PIL1, AT5G02580 and CDF5 in 3-d-old SD-grown seedlings at ZT18. Data from 3 biological trials were averaged. Error bars indicate SEM. Different letters denote statistically significant differences based on Wilcoxon test (p<0.05).


Figure 2A HT - 29 cells were transfected with NC , siRIPK1 or miR - 324 - 5p . After 48 h , cells were treated with TNF - α ( T ) or T + S + Z for an additional 6 h . Western blotting analysis of p - RIPK1 , RIPK1 , p - RIPK3 , RIPK3 , p - MLKL , MLKL and β - actin . B Western blotting analysis of RIPK1 , RIPK3 , MLKL and β - actin levels in HT - 29 cells that were transfected with NC , siRIPK1 , siRIPK3 , MLKL siRNA oliogs ( siMLKL ) , and miR - 324 - 5p . C HT - 29 cells were harvested 48 h after transfection with NC , siMLKL , and miR - 324 - 5p . MLKL expression was analyzed by qPCR ( upper ) or RT - PCR ( lower ) . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . D , E MKN45 ( D ) and 174T ( E ) cells were harvested 48 h after transfection with NC , siMLKL , and miR - 324 - 5p . qPCR analysis for the expression of MLKL ( upper ) and western blotting analysis of MLKL and β - actin ( lower ) . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . F HT - 29 cells were transfected with NC , siMLKL , miR - 324 - 5p , or anti - miR - 324 - 5p . After 48 h , cells were treated with DMSO or T + S + Z for an additional 24 h . Cell survival was determined by measuring ATP levels . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . G Western blotting analysis of MLKL and β - actin in HT - 29 cells that were harvested 48 h after transfection with NC , siMLKL , miR - 324 - 5p , or an anti - miR - 324 - 5p .

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 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 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 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 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

Figure 2A HT-29 cells were transfected with NC, siRIPK1 or miR-324-5p. After 48 h, cells were treated with TNF-α (T) or T+S+Z for an additional 6 h. Western blotting analysis of p-RIPK1, RIPK1, p-RIPK3, RIPK3, p-MLKL, MLKL and β-actin.B Western blotting analysis of RIPK1, RIPK3, MLKL and β-actin levels in HT-29 cells that were transfected with NC, siRIPK1, siRIPK3, MLKL siRNA oliogs ( siMLKL), and miR-324-5p.C HT-29 cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. MLKL expression was analyzed by qPCR (upper) or RT-PCR (lower). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D, E MKN45 (D) and 174T (E) cells were harvested 48 h after transfection with NC, siMLKL, and miR-324-5p. qPCR analysis for the expression of MLKL (upper) and western blotting analysis of MLKL and β-actin (lower). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F HT-29 cells were transfected with NC, siMLKL, miR-324-5p, or anti-miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for an additional 24 h. Cell survival was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.G Western blotting analysis of MLKL and β-actin in HT-29 cells that were harvested 48 h after transfection with NC, siMLKL, miR-324-5p, or an anti-miR-324-5p.


Figure 3B HEK293T cells were transfected with NC , MLKL siRNA oligos targeting the 3 ' UTR of MLKL ( siMLKL - 3 ' UTR ) , or miR - 324 - 5p , together with pmirGLO - MLKL - 3 ' UTR or the mutated form of MLKL - 3 ' UTR shown in A . Relative luciferase activity analysis of MLKL - 3 ' UTR . Data are presented as firefly luciferase activity / renilla luciferase activity ± SD . In B , data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . C HEK293T cells were transfected with NC , miR - 324 - 5p , or the mutated form of miR - 324 - 5p together with pmirGLO - MLKL - 3 ' UTR . Relative luciferase activity analysis of MLKL - 3 ' UTR . Data are presented as firefly luciferase activity / renilla luciferase activity ± SD ( n = 5 , biological replicates ) . D Western blotting analysis of MLKL and β - actin in HeLa - endogenous MLKL cells ( HeLa cells stably expressing human RIPK3 ) and HeLa - exogenous MLKL cells ( Mlkl - / - HeLa cells stably expressing human RIPK3 and the coding sequence ( CDS ) of MLKL ) that were harvested 48 h after transfection with NC , miR - 324 - 5p , or MLKL siRNA oligos targeting the CDS region of MLKL ( siMLKL - CDS ) . E qPCR analysis for the expression of MLKL in HeLa - exogenous MLKL cells that were harvested 48 h after transfection with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . F , G HeLa - endogenous MLKL cells ( F ) and HeLa - exogenous MLKL ( G ) were transfected with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . After 48 h , cells were treated with T + S + Z for 24 h . Cell survival was determined by measuring ATP levels . Data information : The number of surviving cells was normalized to the number of surviving control cells , which were treated with DMSO . data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . H HeLa cells expressing MLKL ( 1 - 190 aa ) fused to DmrB ( HeLa - MLKL ( 1 - 190 ) - Dmir cells ) were transfected with NC , miR - 324 - 5p , siMLKL - 3 ' UTR , or siMLKL - CDS . After 48 h , cells were treated with DMSO or the dimerization agent AP20187 ( 60 nM ) for 24 h . Cell survival was determined by measuring ATP levels . Data information : The number of surviving cells was normalized to the number of surviving control cells , which were treated with DMSO . data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown .

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 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 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 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 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

Figure 3B HEK293T cells were transfected with NC, MLKL siRNA oligos targeting the 3'UTR of MLKL (siMLKL-3'UTR), or miR-324-5p, together with pmirGLO-MLKL-3'UTR or the mutated form of MLKL-3'UTR shown in A. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD. In B, data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.C HEK293T cells were transfected with NC, miR-324-5p, or the mutated form of miR-324-5p together with pmirGLO-MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n=5, biological replicates).D Western blotting analysis of MLKL and β-actin in HeLa-endogenous MLKL cells (HeLa cells stably expressing human RIPK3) and HeLa-exogenous MLKL cells (Mlkl-/- HeLa cells stably expressing human RIPK3 and the coding sequence (CDS) of MLKL) that were harvested 48 h after transfection with NC, miR-324-5p, or MLKL siRNA oligos targeting the CDS region of MLKL (siMLKL-CDS).E qPCR analysis for the expression of MLKL in HeLa-exogenous MLKL cells that were harvested 48 h after transfection with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F, G HeLa-endogenous MLKL cells (F) and HeLa-exogenous MLKL (G) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with T+S+Z for 24 h. Cell survival was determined by measuring ATP levels. Data information: The number of surviving cells was normalized to the number of surviving control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.H HeLa cells expressing MLKL(1-190 aa) fused to DmrB (HeLa-MLKL(1-190)-Dmir cells) were transfected with NC, miR-324-5p, siMLKL-3'UTR, or siMLKL-CDS. After 48 h, cells were treated with DMSO or the dimerization agent AP20187 (60 nM) for 24 h. Cell survival was determined by measuring ATP levels. Data information: The number of surviving cells was normalized to the number of surviving control cells, which were treated with DMSO. data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


Figure 4C African green monkey kidney epithelial ( Vero ) cells were transfected with NC , siMLKL , or miR - 324 - 5p for 48 h . qPCR analysis for the expression of MLKL . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . D MEFs were transfected with NC , siMLKL , or miR - 324 - 5p for 48 h . qPCR analysis for the expression of MLKL and western blotting analysis of MLKL and β - actin . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . β - actin serves as an internal control ( D ) . E MEFs were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were treated with DMSO or T + S + Z for 24 h . Cell viability was determined by measuring ATP levels . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . F HEK293T cells were transfected with NC or miR - 324 - 5p , together with pmirGLO - mouse MLKL - 3 ' UTR or the mutated form of mouse MLKL - 3 ' UTR . Relative luciferase activity analysis of MLKL - 3 ' UTR . Data are presented as firefly luciferase activity / renilla luciferase activity ± SD ( n = 5 , biological replicates ) .

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

Figure 4C African green monkey kidney epithelial (Vero) cells were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D MEFs were transfected with NC, siMLKL, or miR-324-5p for 48 h. qPCR analysis for the expression of MLKL and western blotting analysis of MLKL and β-actin. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown. β-actin serves as an internal control (D).E MEFs were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with DMSO or T+S+Z for 24 h. Cell viability was determined by measuring ATP levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F HEK293T cells were transfected with NC or miR-324-5p, together with pmirGLO-mouse MLKL-3'UTR or the mutated form of mouse MLKL-3'UTR. Relative luciferase activity analysis of MLKL-3'UTR. Data are presented as firefly luciferase activity/renilla luciferase activity ± SD (n = 5, biological replicates).


Figure 5A U937 cells were treated with 100 ng / ml IFN - α , 100 ng / ml IFN - β , 100 ng / ml IFN - γ , 100 ng / ml LPS , or 25 μg / ml poly ( I : C ) for 24 h . Identical concentrations were used in later experiments unless otherwise stated . qPCR analysis for the expression of MLKL . C U937 cells were treated with PBS , IFN - α , IFN - β , IFN - γ , LPS , or poly ( I : C ) for 24 h . qPCR analysis for the expression of miR - 324 - 5p . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . B U937 cells were treated with IFN - α , IFN - β , IFN - γ , LPS , or poly ( I : C ) for 24 h . and western blotting analysis of MLKL and β - actin ( left ) . Quantification of MLKL normalized to β - actin levels ( right ) . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . D , E U937 cells were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were treated with IFN - β ( D ) or IFN - γ ( E ) for 24 h . Western blotting analysis of MLKL and β - actin . F , G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN - β ( F ) or IFN - γ ( G ) treatment . After 24 h , qPCR analysis for the expression of miR - 324 - 5p . After 48 h , western blotting analysis of p - STAT1 , STAT1 , MLKL and β - actin . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . H , I U937 cells were transfected with NC , or STAT1 siRNA oliogs ( siSTAT1 ) . After 48 h , cells were treated with IFN - β ( Η ) or IFN - γ ( Ι ) . qPCR analysis for the expression of miR - 324 - 5p . Western blotting analysis of STAT1 , MLKL and β - actin . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . J HT - 29 cells were treated with IFN - γ for 24 h . The cell lysate was collected for ChIP analysis . STAT1 binding to miR - 324 - 5p promoter DNA region was determined by Chip - qPCR . The amount of precipitated DNA was calculated as percent input . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown .

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 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 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 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 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

Figure 5A U937 cells were treated with 100 ng/ml IFN-α, 100 ng/ml IFN-β, 100 ng/ml IFN-γ, 100 ng/ml LPS, or 25 μg/ml poly(I:C) for 24 h. Identical concentrations were used in later experiments unless otherwise stated. qPCR analysis for the expression of MLKL. C U937 cells were treated with PBS, IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. qPCR analysis for the expression of miR-324-5p. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.B U937 cells were treated with IFN-α, IFN-β, IFN-γ, LPS, or poly(I:C) for 24 h. and western blotting analysis of MLKL and β-actin (left). Quantification of MLKL normalized to β-actin levels (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D, E U937 cells were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were treated with IFN-β (D) or IFN-γ (E) for 24 h. Western blotting analysis of MLKL and β-actin.F, G U937 cells were incubated with 300 nM ruxolitinib for 2 h prior to IFN-β (F) or IFN-γ (G) treatment. After 24 h, qPCR analysis for the expression of miR-324-5p. After 48 h, western blotting analysis of p-STAT1, STAT1, MLKL and β-actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.H, I U937 cells were transfected with NC, or STAT1 siRNA oliogs (siSTAT1). After 48 h, cells were treated with IFN-β (Η) or IFN-γ (Ι). qPCR analysis for the expression of miR-324-5p. Western blotting analysis of STAT1, MLKL and β-actin. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.J HT-29 cells were treated with IFN-γ for 24 h. The cell lysate was collected for ChIP analysis. STAT1 binding to miR-324-5p promoter DNA region was determined by Chip-qPCR. The amount of precipitated DNA was calculated as percent input. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


Figure 6A U937 cells were transfected with NC , siMLKL - 3 ' UTR , or miR - 324 - 5p . After 48 h , cells were exposed to 100 ng / ml IFN - γ plus 20 μM z - VAD for 24 h . Identical concentrations were used in later experiments unless otherwise stated . Cell viability was determined by measuring ATP levels . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . B qPCR analysis for the expression of MLKL ( left ) and miR - 324 - 5p ( middle ) in PBMC - derived macrophages that were treated with PBS or IFN - γ for 24 h . The corresponding western blotting analysis of MLKL and β - actin ( right ) . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . C , D PBMC - derived macrophages ( C ) and HT - 29 cells ( D ) were transfected with NC , siMLKL , or miR - 324 - 5p . After 48 h , cells were exposed to S + Z or IFN - γ + S + Z for 24 h . Cell viability was determined by measuring ATP levels ( left ) . The corresponding western blotting analysis of MLKL and β - actin ( right ) . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . E qPCR analysis for the expression of miR - 324 - 5p in miR - 324 - 5p knockout HT - 29 cells ( miR - 324 - 5p - / - cells ) . Altered miR - 324 - 5p DNA sequences were shown in miR - 324 - 5p - / - clone 19 # and 36 # . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . F MiR - 324 - 5p + / + HT - 29 , miR - 324 - 5p - / - - 19 # , and miR - 324 - 5p - / - - 36 # cells were treated with T + S + Z for the indicated times . Cell viability was determined by measuring ATP levels . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . G , H MiR - 324 - 5p + / + HT - 29 , and miR - 324 - 5p - / - HT - 29 cells were treated with IFN - β ( G ) or IFN - γ ( H ) plus 20 μM z - VAD for 24 h . Cell viability was determined by measuring ATP levels . Data information : Data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown .

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Figure 6A U937 cells were transfected with NC, siMLKL-3'UTR, or miR-324-5p. After 48 h, cells were exposed to 100 ng/ml IFN-γ plus 20 μM z-VAD for 24 h. Identical concentrations were used in later experiments unless otherwise stated. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.B qPCR analysis for the expression of MLKL (left) and miR-324-5p (middle) in PBMC-derived macrophages that were treated with PBS or IFN-γ for 24 h. The corresponding western blotting analysis of MLKL and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.C, D PBMC-derived macrophages (C) and HT-29 cells (D) were transfected with NC, siMLKL, or miR-324-5p. After 48 h, cells were exposed to S+Z or IFN-γ+S+Z for 24 h. Cell viability was determined by measuring ATP levels (left). The corresponding western blotting analysis of MLKL and β-actin (right). Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.E qPCR analysis for the expression of miR-324-5p in miR-324-5p knockout HT-29 cells (miR-324-5p-/- cells) . Altered miR-324-5p DNA sequences were shown in miR-324-5p-/- clone 19# and 36#. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.F MiR-324-5p+/+ HT-29, miR-324-5p-/- -19# , and miR-324-5p-/- -36# cells were treated with T+S+Z for the indicated times. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.G, H MiR-324-5p+/+ HT-29, and miR-324-5p-/- HT-29 cells were treated with IFN-β (G) or IFN-γ (H) plus 20 μM z-VAD for 24 h. Cell viability was determined by measuring ATP levels. Data information: Data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


Figure 7A qPCR analysis for the expression of IFN - β ( left ) and IFN - γ ( right ) in PBMC - derived macrophages that were infected with IAV ( H1N1 strain PR8 ) at an multiplicity of infection ( MOI ) of 0 . 2 for 24 h . B qPCR analysis for the expression of MLKL ( left ) and miR - 324 - 5p ( right ) in PBMC - derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV ( MOI = 0 . 2 ) for 24 h . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . C PBMC - derived macrophages were transfected with NC , siMLKL , or miR - 324 - 5p . After 48h , cells were treated with S + Z in the presence or absence of IAV ( MOI = 0 . 2 ) for 24 h . Cell viability was determined by measuring ATP levels . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown . D qPCR analysis for the expression of M gene of IAV in PBMC - derived macrophages that were transfected with NC , siMLKL or miR - 324 - 5p for 48h , followed by the infection with IAV ( MOI = 0 . 2 ) for 24 h . Data information : data are represented as the means ± SD of three biological replicates . Statistical analyses were performed using unpaired Student ' s t - test . All experiments were performed at least three times , and representative data are shown .

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Figure 7A qPCR analysis for the expression of IFN-β (left) and IFN-γ (right) in PBMC-derived macrophages that were infected with IAV (H1N1 strain PR8) at an multiplicity of infection (MOI) of 0.2 for 24 h. B qPCR analysis for the expression of MLKL (left) and miR-324-5p (right) in PBMC-derived macrophages that were treated with the 300nM ruxolitinib for 2 h prior to the infection with IAV (MOI=0.2) for 24 h. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.C PBMC-derived macrophages were transfected with NC, siMLKL, or miR-324-5p. After 48h, cells were treated with S+Z in the presence or absence of IAV (MOI=0.2) for 24 h. Cell viability was determined by measuring ATP levels. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.D qPCR analysis for the expression of M gene of IAV in PBMC-derived macrophages that were transfected with NC, siMLKL or miR-324-5p for 48h, followed by the infection with IAV (MOI=0.2) for 24 h. Data information: data are represented as the means ± SD of three biological replicates. Statistical analyses were performed using unpaired Student's t-test. All experiments were performed at least three times, and representative data are shown.


Figure 3The rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( b ) Northern blotting experiment detecting GlmZ ( top panel ) and 5S rRNA ( bottom panel ; loading control ) to assess the degree of GlmZ cleavage triggered by the RapZ variants . Processed GlmZ is indicated with an asterisk . Strains Z8 ( wild - type , lanes 1 , 2 ) and Z28 ( ∆ rapZ , all other lanes were used . Strain Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( c ) GlmS protein ( MW = 66 . 9 kDa ) levels in the same cultures as revealed by SDS - PAGE of total protein extracts followed by Coomassie blue staining . Strains Z8 ( wild - type , lanes 1 , 2 ) and Z28 ( ∆ rapZ , all other lanes were used . Strain Z28 carried plasmid pBAD33 ( lanes 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter ( induction with arabinose , repression by glucose ) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in ( d ) β - Galactosidase activities produced by the same cultures and replicates ( mean values and standard deviations are shown ) . Cells carry a glmS ' - lacZ fusion on the chromosome , which requires base - pairing with sRNA GlmZ for high expression . Strains Z8 ( wild - type columns 1 , 2 ) and Z28 all other columns ) were used . Strain Z28 carried plasmid pBAD33 columns 3 , 4 ; empty vector control = VC ) or derivatives triggering synthesis of the indicated RapZ

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Figure 3The rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (b) Northern blotting experiment detecting GlmZ (top panel) and 5S rRNA (bottom panel; loading control) to assess the degree of GlmZ cleavage triggered by the RapZ variants. Processed GlmZ is indicated with an asterisk. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (c) GlmS protein (MW = 66.9 kDa) levels in the same cultures as revealed by SDS-PAGE of total protein extracts followed by Coomassie blue staining. Strains Z8 (wild-type, lanes 1, 2) and Z28 (∆rapZ, all other lanes were used. Strain Z28 carried plasmid pBAD33 (lanes 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variantsThe rapZ mutants were expressed from plasmids under control of the PBAD promoter (induction with arabinose, repression by glucose) and tested for the ability to complement a strain lacking the chromosomal rapZ gene in (d) β-Galactosidase activities produced by the same cultures and replicates (mean values and standard deviations are shown). Cells carry a glmS'-lacZ fusion on the chromosome, which requires base-pairing with sRNA GlmZ for high expression. Strains Z8 (wild-type columns 1, 2) and Z28 all other columns) were used. Strain Z28 carried plasmid pBAD33 columns 3, 4; empty vector control = VC) or derivatives triggering synthesis of the indicated RapZ variants


Figure 2D Bax / BAK1 protein levels were detected by western blotting . G , Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated . H Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from HEK293T cells transfected with different constructs as indicated . I Immublotting results with IP or input ( whole - cell lysates ) from mouse midbrain , SH - SY5Y , HEK293T or HeLa cells as indicated . The α - ctrl represents that the magnetic beads used in IP were not pre - incubated with any antibody .

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Figure 2D Bax/BAK1 protein levels were detected by western blotting.G, Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.H Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from HEK293T cells transfected with different constructs as indicated.I Immublotting results with IP or input (whole-cell lysates) from mouse midbrain, SH-SY5Y, HEK293T or HeLa cells as indicated. The α-ctrl represents that the magnetic beads used in IP were not pre-incubated with any antibody.


Figure 3B Results of immunoblotting with immunoprecipitates ( IP ) or whole - cell lysates from cells transfected with Bcl - 2 and TMEM175 or its mutants TMEM175 - V145A , TMEM175 - R377V , as indicated . C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector , TMEM175 , TMEM175 - V145A or TMEM175 - R377V overexpressing HEK293T cells . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests ( C and is indicated with NS for not significant ( P > 0 . 05 ) , * * for p < 0 . 01 , and * * * for p < 0 . 001 . n value means the number of biological replicates made for each data point .

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Figure 3B Results of immunoblotting with immunoprecipitates (IP) or whole-cell lysates from cells transfected with Bcl-2 and TMEM175 or its mutants TMEM175-V145A, TMEM175-R377V, as indicated. C Statistics of the relative TMEM175 protein levels normalized to GAPDH in vector, TMEM175, TMEM175-V145A or TMEM175-R377V overexpressing HEK293T cells. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests (C and is indicated with NS for not significant (P > 0.05), ** for p < 0.01, and *** for p < 0.001. n value means the number of biological replicates made for each data point.


Figure 4K ( Upper ) schematic diagram showing the strategy of TMEM175 - KO in HEK293T cells . 92 bp in exon 4 and intron 4 were removed from the tmem175 locus . ( Lower ) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western - blotting . L CM - H2DCFDA ( green ) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre - treatment with vehicle or 10 μM HA14 - 1 for 20 min . Scale bars = 10 μm . M Statistics of CM - H2DCFDA fluorescence intensity of ( L ) . The n values represent the cell numbers counted within the same experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - tests M ) and is indicated with NS for not significant ( P > 0 . 05 ) , * for p < 0 . 05 , * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panel M , n value means the number of technical replicates .

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Figure 4K (Upper) schematic diagram showing the strategy of TMEM175-KO in HEK293T cells. 92 bp in exon 4 and intron 4 were removed from the tmem175 locus. (Lower) TMEM175 levels in the WT or TMEM175 KO cells were detected by Western-blotting.L CM-H2DCFDA (green) indicating cellular ROS levels of WT or TMEM175 KO HEK293T cells pre-treatment with vehicle or 10 μM HA14-1 for 20 min. Scale bars = 10 μm. M Statistics of CM-H2DCFDA fluorescence intensity of (L). The n values represent the cell numbers counted within the same experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-tests M) and is indicated with NS for not significant (P > 0.05), * for p < 0.05, ** for p < 0.01, and *** for p < 0.001. In panel M, n value means the number of technical replicates.


Figure 5F Fluorescence of G3BP1 excited with 488 nm in control ( upper ) and TMEM175 - overexpressing ( lower ) cells . Scale bars = 10 μm . G Statistics of the numbers of puncta per cell ( left ) and the percentage of cells with puncta in ( F ) . We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels G n value means the number of technical replicates . H Co - localization of mitochondrial indicator TOM20 - mCherry and lysosomal indicator LysoTracker in WT ( upper row ) or TMEM175 KO ( lower row ) HEK293T cells . Co - localization of mitochondrion and lysosome is observed as a yellow colour in the merged image ( marked by white arrows ) . Scale bars = 10 μm . I The number of merged puncta per cell in ( H ) . We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view . n value represents the number of different fields of view randomly selected in the same imaging experiment . Data information : The data are presented as the mean ± SEM . Statistical significance was calculated with two - sided student ' s t - test , and is indicated with * * for p < 0 . 01 , and * * * for p < 0 . 001 . In panels I , n value means the number of technical replicates .

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Figure 5F Fluorescence of G3BP1 excited with 488 nm in control (upper) and TMEM175-overexpressing (lower) cells. Scale bars = 10 μm. G Statistics of the numbers of puncta per cell (left) and the percentage of cells with puncta in (F). We counted 547 cells in Vector group from 37 different fields of view and 332 cells in TMEM175 group from 76 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels G n value means the number of technical replicates.H Co-localization of mitochondrial indicator TOM20-mCherry and lysosomal indicator LysoTracker in WT (upper row) or TMEM175 KO (lower row) HEK293T cells. Co-localization of mitochondrion and lysosome is observed as a yellow colour in the merged image (marked by white arrows). Scale bars = 10 μm. I The number of merged puncta per cell in (H). We counted 139 cells in WT group from 38 different fields of view and 148 cells in TMEM175 KO group from 42 different fields of view. n value represents the number of different fields of view randomly selected in the same imaging experiment. Data information: The data are presented as the mean ± SEM. Statistical significance was calculated with two-sided student's t-test, and is indicated with ** for p < 0.01, and *** for p < 0.001. In panels I, n value means the number of technical replicates.