Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . D Path persistence ( calculated as the ratio between the effective length and the total length of the cell trajectory ) of migrating DCs . P ‐ values were determined with a t ‐ test analysis .

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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.D Path persistence (calculated as the ratio between the effective length and the total length of the cell trajectory) of migrating DCs. P‐values were determined with a t‐test analysis.


Analysis of shScramble ( gray , n = 2 , 400 cells ) ‐ , shIP3R ( 1 , 3 ) A ( blue , n = 1 , 611 cells ) ‐ , and shIP3R ( 1 , 3 ) C ( green , n = 1 , 454 cells ) ‐ expressing DCs migrating in the device described in Supplementary Fig S3I , stained with Hoechst and imaged every 2 min during 24 h at 10 × magnification . Values were obtained from three independent experiments . Cell trajectories were obtained by tracking their nucleus . E Bars showing the proportion of the effective length displacement ( L ) of DCs . A Fisher ' s exact test ( P ‐ value 2 . 2 × 10 − 16 ) was used for statistics .

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

Analysis of shScramble (gray, n = 2,400 cells)‐, shIP3R(1,3)A (blue, n = 1,611 cells)‐, and shIP3R(1,3)C (green, n = 1,454 cells)‐expressing DCs migrating in the device described in Supplementary Fig S3I, stained with Hoechst and imaged every 2 min during 24 h at 10× magnification. Values were obtained from three independent experiments. Cell trajectories were obtained by tracking their nucleus.E Bars showing the proportion of the effective length displacement (L) of DCs. A Fisher's exact test (P‐value 2.2 × 10−16) was used for statistics.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs ( shIP3R ( 1 , 3 ) A ′ , shIP3R ( 2 , 3 ) B ′ , and shIP3R ( 1 , 3 ) C ′ ) . Actin was used as a control .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs transduced with the shScramble or shRNAs for IP3Rs (shIP3R(1,3)A′, shIP3R(2,3)B′, and shIP3R(1,3)C′). Actin was used as a control.


B Inverted sequential epifluorescence images ( 20 × ) showing myosin IIA ‐ GFP localization of a representative shScramble ( left ) ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DC ( right ) migrating in micro ‐ channels . Myosin IIA ‐ GFP knock ‐ in DCs were imaged every 1 min ( 1 out of 2 images is shown ) . Asterisks highlight changes of direction during locomotion .

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B Inverted sequential epifluorescence images (20×) showing myosin IIA‐GFP localization of a representative shScramble (left)‐ and shIP3R(1,3)C‐expressing DC (right) migrating in micro‐channels. Myosin IIA‐GFP knock‐in DCs were imaged every 1 min (1 out of 2 images is shown). Asterisks highlight changes of direction during locomotion.


C , D Myosin IIA ‐ GFP localization ( red ) versus velocity fluctuations ( blue , ΔV / V0 ) in a representative shScramble ‐ transduced DC ( C ) or shIP3R ( 1 , 3 ) C ‐ silenced ( D ) migrating in micro ‐ channels . DCs were recorded as in ( B ) . E Quantification of the interval between curves ( highlighted in D ) obtained for myosin IIA positioning and velocity from shScramble ‐ and shIP3R ( 1 , 3 ) C ‐ expressing DCs . Cells were recorded as in ( B ) ( n = 20 cells from two experiments ) . The Mann - Whitney test was applied for statistical analysis .

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C, D Myosin IIA‐GFP localization (red) versus velocity fluctuations (blue, ΔV/V0) in a representative shScramble‐transduced DC (C) or shIP3R(1,3)C‐silenced (D) migrating in micro‐channels. DCs were recorded as in (B).E Quantification of the interval between curves (highlighted in D) obtained for myosin IIA positioning and velocity from shScramble‐ and shIP3R(1,3)C‐expressing DCs. Cells were recorded as in (B) (n = 20 cells from two experiments). The Mann-Whitney test was applied for statistical analysis.


A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor , ML7 .

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A Immunoblot showing the intracellular levels of phosphorylated MLC in DCs treated overnight with increasing amounts of the MLCK inhibitor, ML7.


B , C Analysis of immature DCs migrating in micro ‐ channels in the presence of ML7 or DMSO as a control ( n > 170 cells from two independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( B ) . Whiskers show 10 - 90 percentiles plus the median . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( C ) in direction of DCs migrating in micro ‐ channels . Mean plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C Analysis of immature DCs migrating in micro‐channels in the presence of ML7 or DMSO as a control (n > 170 cells from two independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (B). Whiskers show 10-90 percentiles plus the median. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (C) in direction of DCs migrating in micro‐channels. Mean plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


D , E Analysis of shScramble ( gray ) ‐ and shIP3R ( 1 , 3 ) C ( green ) ‐ expressing DCs migrating in micro ‐ channels in the presence of 20 μM ML7 ( n > 70 cells per condition from 3 independent experiments ) . Velocity fluctuations ( ΔV / V0 ) of immature DCs migrating in micro ‐ channels ( D ) . Boxes illustrate 10 - 90 percentiles of values , and whiskers represent the range of values . P ‐ values were calculated using a Kruskal - Wallis test . Frequency of changes ( E ) in direction observed in DCs migrating in micro ‐ channels . Means plus SD are shown . A Kruskal - Wallis test was applied for statistical analysis .

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

D, E Analysis of shScramble (gray)‐ and shIP3R(1,3)C (green)‐expressing DCs migrating in micro‐channels in the presence of 20 μM ML7 (n > 70 cells per condition from 3 independent experiments). Velocity fluctuations (ΔV/V0) of immature DCs migrating in micro‐channels (D). Boxes illustrate 10-90 percentiles of values, and whiskers represent the range of values. P‐values were calculated using a Kruskal-Wallis test. Frequency of changes (E) in direction observed in DCs migrating in micro‐channels. Means plus SD are shown. A Kruskal-Wallis test was applied for statistical analysis.


Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA _ KO mutants versus WT . Of 759 predicted miRNA target genes , 728 ( 96 % ) were detectable in the ribosome profiling data and appear in the plot . Data information : In A , student ' s t - test was applied to assess the significance for the increase in ribosome occupancy of the 728 / 759 miRNA targets ( p - value < 1e51 for every mutant ) .

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Cumulative distribution function of differential ribosome occupancy as detected by ribosome profiling in miRNA_KO mutants versus WT. Of 759 predicted miRNA target genes, 728 (96%) were detectable in the ribosome profiling data and appear in the plot. Data information: In A, student's t-test was applied to assess the significance for the increase in ribosome occupancy of the 728/759 miRNA targets (p-value<1e51 for every mutant).


Top : Immunoblot analysis of CIC in WT , Drosha _ KO , Dicer _ KO and Ago2 & 1 _ KO mESCs . TUBULIN was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to TUBULIN and relative to the WT sample in three biological replicates . Data information : Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN . Values are relative to WT , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O 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-MEASURED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: Immunoblot analysis of CIC in WT, Drosha_KO, Dicer_KO and Ago2&1_KO mESCs. TUBULIN was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to TUBULIN and relative to the WT sample in three biological replicates. Data information: Bar graph in Fig 2F shows mean intensity of CIC signal ± SD normalized to TUBULIN. Values are relative to WT, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05.


MA plot of the DGE analysis in miR - 290 - 295 _ KO mESCs . Data information : In A , significant genes in MA plot were determined using an adjusted p - value of 0 . 1 .

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MA plot of the DGE analysis in miR-290-295_KO mESCs. Data information: In A, significant genes in MA plot were determined using an adjusted p-value of 0.1.


F , G . Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR - 290 - 295 . In addition to the data shown and described in Fig 1D , E , a second AGO2 - binding dataset , based on cross - linking immunoprecipitation and sequencing ( CLIP - seq ) , has been integrated . Response elements for miR - 290 - 295 cluster members are denoted in red and labeled .

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F, G. Integrated data for Tfap4 gene shows multiple lines of evidence for its regulation by miR-290-295. In addition to the data shown and described in Fig 1D, E, a second AGO2-binding dataset, based on cross-linking immunoprecipitation and sequencing (CLIP-seq), has been integrated. Response elements for miR-290-295 cluster members are denoted in red and labeled.


Top : Immunoblot analysis of TFAP4 in WT , Drosha _ KO , Dicer _ KO , Ago2 & 1 _ KO and miR - 290 - 295 _ KO mESCs . LAMINB1 was used as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of CIC intensity , normalized to LAMINB1 and relative to the WT sample in three biological replicates . Data information : In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

O O O O O B-MEASURED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O 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-MEASURED_VAR O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: Immunoblot analysis of TFAP4 in WT, Drosha_KO, Dicer_KO, Ago2&1_KO and miR-290-295_KO mESCs. LAMINB1 was used as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of CIC intensity, normalized to LAMINB1 and relative to the WT sample in three biological replicates. Data information: In H bar graphs show mean intensity of TFAP4 signal ± SD normalized to LAMINB1 Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Immunoblot analysis of TFAP4 after transfection of miRNA mimics ( miR - 291a - 5p , miR - 291a - 3p and miR - 291a - 5p + miR - 291a - 3p combined ) in miR - 290 - 295 _ KO mESCs . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of three biological replicates . Bottom : Bar graph showing quantification of TFAP4 intensity , normalized to Coomassie and relative to the WT sample in three biological replicates . Data information : In I , bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie . Values are relative to WT or the negative control mimic , which was set to 1 . P - values were calculated using a student ' s t - test comparing each value to the WT . * p - value < 0 . 05 , * * p - value < 0 . 01 , * * * * p - value < 0 . 0001 .

O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Top: Immunoblot analysis of TFAP4 after transfection of miRNA mimics (miR-291a-5p, miR-291a-3p and miR-291a-5p+miR-291a-3p combined) in miR-290-295_KO mESCs. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of three biological replicates. Bottom: Bar graph showing quantification of TFAP4 intensity, normalized to Coomassie and relative to the WT sample in three biological replicates. Data information: In I, bar graphs show mean intensity of TFAP4 signal ± SD normalized to Coomassie. Values are relative to WT or the negative control mimic, which was set to 1. P-values were calculated using a student's t-test comparing each value to the WT. *p-value<0.05, **p-value<0.01, ****p-value<0.0001.


Top : Mature sequences of 5p and 3p miRNAs of the miR - 290 - 295 family and the mature sequence of the human miR - 302c - 3p . Colors indicate identical seed sequences . Bottom : AGO2 CLIP - seq shows a peak at the miR - 302c - 3p binding site on human TFAP4 .

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Top: Mature sequences of 5p and 3p miRNAs of the miR-290-295 family and the mature sequence of the human miR-302c-3p. Colors indicate identical seed sequences. Bottom: AGO2 CLIP-seq shows a peak at the miR-302c-3p binding site on human TFAP4.


Immunoblot validation of siPOOL - mediated knock down of Tfap4 . TFAP4 levels were compared between untreated WT versus miR - 290 - 295 _ KO cells treated with a negative control and a Tfap4 - targeted siPOOL . Immunoblots were stained with Coomassie blue dye as a loading control . Blot is a representative image of two biological replicates .

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Immunoblot validation of siPOOL-mediated knock down of Tfap4. TFAP4 levels were compared between untreated WT versus miR-290-295_KO cells treated with a negative control and a Tfap4-targeted siPOOL. Immunoblots were stained with Coomassie blue dye as a loading control. Blot is a representative image of two biological replicates.


Scatterplot of DEG in miR - 290 - 295 _ KO control versus siPOOL - Tfap4 . SiPOOL experiments were performed in miR - 290 - 295 _ KO cells and DE was assessed relative to a negative control siPOOL transfection . Only genes that are statistically significantly differentially expressed in miR - 290 - 295 _ KO are shown . Genes predicted to be targeted by miR - 290 - 295 are marked in blue . Genes are defined as rescued ( orange ) if the log2FoldChange - ratio between miR - 290 - 295 _ KO control and siPOOL - Tfap4 is in the range [ - 2 , - 1 / 2 ] . Tfap4 is marked by a red dot .

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Scatterplot of DEG in miR-290-295_KO control versus siPOOL-Tfap4. SiPOOL experiments were performed in miR-290-295_KO cells and DE was assessed relative to a negative control siPOOL transfection. Only genes that are statistically significantly differentially expressed in miR-290-295_KO are shown. Genes predicted to be targeted by miR-290-295 are marked in blue. Genes are defined as rescued (orange) if the log2FoldChange-ratio between miR-290-295_KO control and siPOOL-Tfap4 is in the range [-2, -1/2]. Tfap4 is marked by a red dot.


Representative images of NANOG ( red ) and OCT4 ( green ) immunofluorescence staining in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs . Scale bar = 20 μm . Quantification of NANOG fluorescence signal intensity in WT , Drosha _ KO and Drosha & Tfap4 _ KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4 _ KO Data information : In E , box plots show nuclear intensity of NANOG in ~ 300 cells per genotype . P - values were generated using ordinary one - way ANOVA . * * * * p - value < 0 . 0001 . In panel E , the box plot ' s central band represents the median relative intensity for each genotype . The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values .

O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of NANOG (red) and OCT4 (green) immunofluorescence staining in WT, Drosha_KO and Drosha & Tfap4_KO mESCs. Scale bar = 20 μm. Quantification of NANOG fluorescence signal intensity in WT, Drosha_KO and Drosha & Tfap4_KO mESCs show no significant rescue of NANOG intensity in Drosha & Tfap4_KO Data information: In E, box plots show nuclear intensity of NANOG in ~300 cells per genotype. P-values were generated using ordinary one-way ANOVA. ****p-value<0.0001. In panel E, the box plot's central band represents the median relative intensity for each genotype. The boxes represent the interquartile range and the whiskers represent the maximum and minimum relative intensity values.


( D ) Two - color dSTORM imaging of RIM1 / 2 and gephyrin in cryosections . From left to right : rendered images of RIM1 / 2 and gephyrin clusters showing the aligned SSDs ( arrows ) ; gephyrin SSDs segmented by watershed outlined with different colors ; RIM1 / 2 SSDs outlined with different colors ; binary SSDs of gephyrin and RIM1 / 2 . Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images . Scale bar : 200 nm . RIM1 / 2 was labelled with Alexa 647 , gephyrin ( mAb7a ) with Cy3B in these experiments .

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(D) Two-color dSTORM imaging of RIM1/2 and gephyrin in cryosections. From left to right: rendered images of RIM1/2 and gephyrin clusters showing the aligned SSDs (arrows); gephyrin SSDs segmented by watershed outlined with different colors; RIM1/2 SSDs outlined with different colors; binary SSDs of gephyrin and RIM1/2. Inhibitory synapses were identified by the gephyrin clusters in the epifluorescence images. Scale bar: 200 nm. RIM1/2 was labelled with Alexa 647, gephyrin (mAb7a) with Cy3B in these experiments.


( A - C ) Alignment of SSDs of pre - synaptic RIM1 / 2 and SSDs of post - synaptic gephyrin , GlyRs , and GABAARs , respectively . ( A1 - C1 ) Distances were measured between the intensity peaks of paired SSDs in rendered dual - color dSTORM images ( yellow line , values given in mean ± SD ) . RIM1 / 2 was labelled with Alexa 647 and gephyrin ( mAb7a ) with Cy3B in ( A1 ) . RIM1 / 2 was labelled with Cy3B , while GlyRs or GABAARs were labelled with Alexa 647 in ( B1 ) and ( C1 ) . ( A2 - C2 ) Trans - synaptic alignment of SSDs of gephyrin , GlyRs , GABAARs and RIM1 / 2 . SSDs were segmented by H - watershed . Data are plotted as mean ± SD . Number of synapses : n = 48 ( A1 , A2 ) from three independent experiments ( no treatment ) , n = 30 ( B1 , B2 ) from two experiments with TTX treatment , n = 16 ( C1 , C2 ) from two experiments with TTX . Only synapses with side view profiles were included . All images are adjusted to the same scale . Scale bar : 200 nm .

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(A-C) Alignment of SSDs of pre-synaptic RIM1/2 and SSDs of post-synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1-C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual-color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labelled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labelled with Cy3B, while GlyRs or GABAARs were labelled with Alexa 647 in (B1) and (C1). (A2-C2) Trans-synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs and RIM1/2. SSDs were segmented by H-watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.


( A - B ) Representative dSTORM images of GlyRs and GABAARs at synapses in side view ( A ) or en face view ( B ) . The top row of images shows the pointillist representation of the detections , the second row the rendered images and the bottom row the corresponding segmented SSDs . GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments . Scale bar : 200 nm .

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(A-B) Representative dSTORM images of GlyRs and GABAARs at synapses in side view (A) or en face view (B). The top row of images shows the pointillist representation of the detections, the second row the rendered images and the bottom row the corresponding segmented SSDs. GlyRs were labelled with Alexa 647 and GABAARs with Cy3B in these experiments. Scale bar: 200 nm.


( A ) Triple immuno - labelling of gephyrin , GlyRs and GABAARs at mixed inhibitory synapses ( arrow ) imaged with conventional fluorescence microscopy . Scale bar : 5 µm .

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(A) Triple immuno-labelling of gephyrin, GlyRs and GABAARs at mixed inhibitory synapses (arrow) imaged with conventional fluorescence microscopy. Scale bar: 5 µm.


Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses . ( A ) Synaptic receptor cluster rendered from detections of both GABAARs ( labelled with Alexa 647 ) and GlyRs ( labelled with Cy3B ) . ( B ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with binary synaptic mask ( white ) .

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Characterization of the nanoscale organization of GABAARs and GlyRs at mixed inhibitory synapses. (A) Synaptic receptor cluster rendered from detections of both GABAARs (labelled with Alexa 647) and GlyRs (labelled with Cy3B). (B) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with binary synaptic mask (white).


Sub - synaptic characterization of GABAAR and GlyR domains . ( F ) Watershed segmentation of sub - synaptic domains ( SSDs ) . Each SSD is shown in different colors . ( G ) Detections of GABAARs ( red dots ) and GlyRs ( blue dots ) overlaid with SSDs ( white ) .

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Sub-synaptic characterization of GABAAR and GlyR domains. (F) Watershed segmentation of sub-synaptic domains (SSDs). Each SSD is shown in different colors. (G) Detections of GABAARs (red dots) and GlyRs (blue dots) overlaid with SSDs (white).


( A ) The number of receptor SSDs , segmented from the combined receptor clusters of GABAARs and GlyRs , was decreased after 4 - AP treatment ( MW test , p < 0 . 0001 ) . ( B ) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only ( MW , * * * * p < 0 . 0001 ) . ( C ) The number of GlyR SSDs ( based on GlyR detections ) were only marginally reduced ( MW , * * p < 0 . 01 ) . Data information : Number of synapses : n = 452 in TTX , n = 583 in 4 - AP condition , from three independent experiments . Mean ± SEM .

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(A) The number of receptor SSDs, segmented from the combined receptor clusters of GABAARs and GlyRs, was decreased after 4-AP treatment (MW test, p < 0.0001). (B) This was mostly due to the loss of GABAAR SSDs that were segmented from the rendered images of GABAAR detections only (MW, ****p < 0.0001). (C) The number of GlyR SSDs (based on GlyR detections) were only marginally reduced (MW, **p < 0.01). Data information: Number of synapses: n = 452 in TTX, n = 583 in 4-AP condition, from three independent experiments. Mean ± SEM.


( A - C ) Reduced immuno - reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4 - AP treatment , revealed by conventional fluorescence microscopy ( KS test , p < 0 . 001 in B1 and p < 0 . 0001 in B2 , p < 0 . 0001 in C ) . Total gephyrin was probed with polyclonal rabbit primary antibody ( rbGPHN ) , and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody ( m7a ) . Number of synapses : n = 4040 in TTX and n = 3818 in 4 - AP conditions from two independent experiments . Scale bar : 2 µm .

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(A-C) Reduced immuno-reactivity of S270 phosphorylated gephyrin but not total gephyrin levels after 4-AP treatment, revealed by conventional fluorescence microscopy (KS test, p < 0.001 in B1 and p < 0.0001 in B2, p < 0.0001 in C). Total gephyrin was probed with polyclonal rabbit primary antibody (rbGPHN), and pS270 phosphorylated gephyrin with monoclonal mouse primary antibody (m7a). Number of synapses: n = 4040 in TTX and n = 3818 in 4-AP conditions from two independent experiments. Scale bar: 2 µm.


( D - F ) Reduced numbers of pS270 gephyrin ( m7a ) detections were recorded by dSTORM for the entire synaptic area ( D , KS test , p < 0 . 01 ) . The number of detections of pS270 gephyrin per SSD ( E , KS , p = 0 . 18 ) and the number of SSDs ( F , MW , p = 0 . 11 ) were not changed by 4 - AP treatment . Gephyrin was probed with mAb7a antibody and Alexa 647 dye . Number of synapses : n = 810 in TTX and n = 727 in 4 - AP conditions from three independent experiments ; ( F ) mean ± SEM . Scale bar : 100 nm .

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(D-F) Reduced numbers of pS270 gephyrin (m7a) detections were recorded by dSTORM for the entire synaptic area (D, KS test, p < 0.01). The number of detections of pS270 gephyrin per SSD (E, KS, p = 0.18) and the number of SSDs (F, MW, p = 0.11) were not changed by 4-AP treatment. Gephyrin was probed with mAb7a antibody and Alexa 647 dye. Number of synapses: n = 810 in TTX and n = 727 in 4-AP conditions from three independent experiments; (F) mean ± SEM. Scale bar: 100 nm.


A - C ) A ) Bilateral bowing deformity and short femora and tibiae with atypical growth plates , and B ) short and bowed humerus at age 4 . 5 years , C ) vertebral fractures ( arrows ) on lateral imaging at age 9 years .

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A-C) A) Bilateral bowing deformity and short femora and tibiae with atypical growth plates, and B) short and bowed humerus at age 4.5 years, C) vertebral fractures (arrows) on lateral imaging at age 9 years.


( A - H ) Transiliac bone sample of the patient at age of 10 years viewed in the light microscope . A - E ) Goldner stained sections , F - H ) : Giemsa stained sections . A ) Overview of the entire sample shows scarcity of mineralized bone ( stained in green ) . Black arrowheads point towards the very thin cortices ( right side , only a fractured cortex is visible ) . B ) Trabecular features are mostly small and isolated . C ) Black arrows point toward three bone resorbing osteoclasts leading to the disconnection of two trabecular features . D ) Cortical bone viewed under polarized light : white arrows point towards collagen fibers arranged in a parallel concentric way around osteon next to a region where collagen fibers are randomly arranged , as typical for woven bone ( white asterisk ) . E ) Detail from cortical bone showing roundish cells on the periosteal side surrounded by a mainly red , thus , poorly mineralized matrix ( white stars ) . F ) , G ) Details from the cortex in Giemsa stained section show areas with unmineralized cartilage ( black arrows ) and mineralized woven bone and or mineralized cartilage ( dark purple , black asterisks ) , mineralized bone is stained pink . H ) Trabecular feature with mineralized cartilage inclusion stained deep purple ( arrowhead ) , surrounded by bone tissue stained pink . Note many osteoblasts on the trabecular surface ( black arrows )

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(A-H) Transiliac bone sample of the patient at age of 10 years viewed in the light microscope. A-E) Goldner stained sections, F-H): Giemsa stained sections. A) Overview of the entire sample shows scarcity of mineralized bone (stained in green). Black arrowheads point towards the very thin cortices (right side, only a fractured cortex is visible). B) Trabecular features are mostly small and isolated. C) Black arrows point toward three bone resorbing osteoclasts leading to the disconnection of two trabecular features. D) Cortical bone viewed under polarized light: white arrows point towards collagen fibers arranged in a parallel concentric way around osteon next to a region where collagen fibers are randomly arranged, as typical for woven bone (white asterisk). E) Detail from cortical bone showing roundish cells on the periosteal side surrounded by a mainly red, thus, poorly mineralized matrix (white stars). F), G) Details from the cortex in Giemsa stained section show areas with unmineralized cartilage (black arrows) and mineralized woven bone and or mineralized cartilage (dark purple, black asterisks), mineralized bone is stained pink. H) Trabecular feature with mineralized cartilage inclusion stained deep purple (arrowhead), surrounded by bone tissue stained pink. Note many osteoblasts on the trabecular surface (black arrows)


A ) Family pedigree with SEC16B homozygosity in the patient and heterozygosity in the mother and one brother . B ) Validation of the SEC16B mutation by Sanger sequencing . Sanger sequencing of gDNA of the patient , brother , mother and healthy control . Magnification of the electropherogram shows the position c . 424 and the base change CT .

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A) Family pedigree with SEC16B homozygosity in the patient and heterozygosity in the mother and one brother. B) Validation of the SEC16B mutation by Sanger sequencing. Sanger sequencing of gDNA of the patient, brother, mother and healthy control. Magnification of the electropherogram shows the position c.424 and the base change CT.


C - F ) C ) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting . KDEL , Calnexin , BIP and PDI , all ER - resident proteins were used as ER markers . E ) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology , Unit 3 . 9 from healthy control cells and patient derived fibroblasts . Subsequently , COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting . GM130 ( Golgi - resident protein ) was used as marker for Golgi complex . All cells were treated with ascorbic acid 0 . 05 mg / ml for 20 hours before the Golgi complex was isolated . D , F ) Quantification of the bands that was done with Bio - Rad Image Lab 6 . 1 software to calculate the expression levels . COL1A1 and COL1A2 band intensities were either normalized to ( D ) calnexin or ( F ) GM130 . The normalized intensities were reported as fold change patient to control cells . Columns represent the mean of three ( D ) or two ( F ) independent experiments ( raw values : 0 . 27 and 0 . 53 for COL1A1 and 0 . 29 , 0 . 21 for COL1A2 ) respectively . Biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two - tailed t

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C-F) C) COL1A1 and COL1A2 expression in the ER of SEC16B patient and control fibroblasts assessed by western blotting. KDEL, Calnexin, BIP and PDI, all ER-resident proteins were used as ER markers. E) Golgi complex was isolated according to the method described in Current Protocols in Cell Biology, Unit 3.9 from healthy control cells and patient derived fibroblasts. Subsequently, COL1A1 and COL1A2 expression in the Golgi complex of SEC16B patient and control fibroblasts were determined by western blotting. GM130 (Golgi-resident protein) was used as marker for Golgi complex. All cells were treated with ascorbic acid 0.05 mg/ml for 20 hours before the Golgi complex was isolated. D, F) Quantification of the bands that was done with Bio-Rad Image Lab 6.1 software to calculate the expression levels. COL1A1 and COL1A2 band intensities were either normalized to (D) calnexin or (F) GM130. The normalized intensities were reported as fold change patient to control cells. Columns represent the mean of three (D) or two (F) independent experiments (raw values: 0.27 and 0.53 for COL1A1 and 0.29, 0.21 for COL1A2) respectively. Biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two-tailed t test


A ) Patient and control fibroblasts were plated on glass cover slips . After 24 hours , cells were fixed and stained for SEC31 to label ERES . The scale bars are 10 µm . B ) Quantification of the number of ERES per area from cells processed . The values are from four independent experiments , biological replicates . 71 cell per experiment were acquired . Bars are means ± standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using a paired two - tailed t - test .

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A) Patient and control fibroblasts were plated on glass cover slips. After 24 hours, cells were fixed and stained for SEC31 to label ERES. The scale bars are 10 µm. B) Quantification of the number of ERES per area from cells processed. The values are from four independent experiments, biological replicates. 71 cell per experiment were acquired. Bars are means ± standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using a paired two-tailed t-test.


C ) Patient and control fibroblasts were electroporated with plasmids encoding mCherry - tagged Mannosidase II ( ManII ) as part of a RUSH construct . After 24 hours , cells were fixed ( T0 ) or treated with 40 mM biotin for 20 minutes ( T20 ) followed by fixation and immunostaining for GM130 to label the Golgi complex . The scale bars are 10 µm . D ) quantification of 59 to 81 cells from three independent RUSH experiments ( biological replicates ) . Biological replicates were performed . Central band represents the median . Whiskers are minimum and maximum values . Outliers are defined as greater than 1 . 5 fold of upper quartiles . * * highly significant ( p < 0 . 005 ) differences obtained by unpaired two tailed t -

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C) Patient and control fibroblasts were electroporated with plasmids encoding mCherry-tagged Mannosidase II (ManII) as part of a RUSH construct. After 24 hours, cells were fixed (T0) or treated with 40 mM biotin for 20 minutes (T20) followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm. D) quantification of 59 to 81 cells from three independent RUSH experiments (biological replicates). Biological replicates were performed. Central band represents the median. Whiskers are minimum and maximum values. Outliers are defined as greater than 1.5 fold of upper quartiles.** highly significant (p < 0.005) differences obtained by unpaired two tailed t-test


A ) Micrograph of thin - section electron microscopy for fibroblasts from SEC16B mutant patient cells and healthy control cells . Arrows point to rER , arrowheads to MVBs / MLBs , star indicates autophagosome . Scale bar is 1 µm . Summary of the number of the autophagosomes .

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A) Micrograph of thin-section electron microscopy for fibroblasts from SEC16B mutant patient cells and healthy control cells. Arrows point to rER, arrowheads to MVBs/MLBs, star indicates autophagosome. Scale bar is 1 µm. Summary of the number of the autophagosomes.


C ) Western blotting anaylsis of autophagy marker LC3B . D ) Quantification of the bands . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two tailed t - test .

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C) Western blotting anaylsis of autophagy marker LC3B. D) Quantification of the bands. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.


E ) 24 hours after plating of patient and control fibroblasts , cells were treated with DMSO , 10mM thapsigargin , or 1 mg / ml tunicamycin for 6 hours . Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading .

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E) 24 hours after plating of patient and control fibroblasts, cells were treated with DMSO, 10mM thapsigargin, or 1 mg/ml tunicamycin for 6 hours. Cells were lysed and immunoblotted for XBP1s as a marker for ER stress and for vinculin to ensure equal loading.


F ) Western blotting anaylsis of apoptosis marker cleaved caspase 3 show enhanced expression in patient cells when compared to healthy control . A representative experiment out of three independent experiments is shown . Biological replicates were performed . G ) Quantification of the bands . All band intensities were normalized to those of Actin and reported as fold change patient to control cells . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * significant ( p < 0 . 05 ) differences obtained by using unpaired two tailed t - test .

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F) Western blotting anaylsis of apoptosis marker cleaved caspase 3 show enhanced expression in patient cells when compared to healthy control. A representative experiment out of three independent experiments is shown. Biological replicates were performed. G) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); *significant (p < 0.05) differences obtained by using unpaired two tailed t-test.


Patient cells were either transfected with pCDNA3 empty vector ( empty vector ) or pcDNA3 which encodes wt SEC16B ( TF ) . Cells were then selected with puromycin and single colonies were picked up and expanded . Established single colonies were used . B ) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 ( I ) and rough ER marker KDEL . The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector , it is not accumulated in the ER of the patient cells transfected with SEC16B wt . Scale bar is 25

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Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. B) Immunofluorescence labeling of the control and the patient fibroblasts for COL1A1 marker proα1 (I) and rough ER marker KDEL. The staining showed that while COL1A1 is accumulated in the ER of patient cells transfected with empty vector, it is not accumulated in the ER of the patient cells transfected with SEC16B wt. Scale bar is 25 µm


Patient cells were either transfected with pCDNA3 empty vector ( empty vector ) or pcDNA3 which encodes wt SEC16B ( TF ) . Cells were then selected with puromycin and single colonies were picked up and expanded . Established single colonies were used . D ) LC3B showed reduced expression in the TF SEC16B mutant cells ( similar to expression in healthy control Fig . 5B ) when compared to empty vector transfected patient cells . E ) Quantification of the bands . All band intensities were normalized to those of Actin and reported as fold change patient to control cells . Columns represent the mean of three independent experiments , biological replicates were performed ; bars , standard error of the mean ( SEM ) ; * * highly significant ( p < 0 . 001 differences obtained by using unpaired two tailed t - test .

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Patient cells were either transfected with pCDNA3 empty vector (empty vector) or pcDNA3 which encodes wt SEC16B (TF). Cells were then selected with puromycin and single colonies were picked up and expanded. Established single colonies were used. D) LC3B showed reduced expression in the TF SEC16B mutant cells (similar to expression in healthy control Fig. 5B) when compared to empty vector transfected patient cells. E) Quantification of the bands. All band intensities were normalized to those of Actin and reported as fold change patient to control cells. Columns represent the mean of three independent experiments, biological replicates were performed; bars, standard error of the mean (SEM); **highly significant (p < 0.001 differences obtained by using unpaired two tailed t-test.


A ) HeLa cells were transfected with plasmids encoding GFP - tagged SEC16B - R142W . After 24 hours , cells were fixed and immunostained against SEC13 to label ERES . Green color shows SEC16B - R142W expression , red colors shows SEC13 expression and yellow color shows the overlap of the two colors . The scale bars are 10 µm .

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A) HeLa cells were transfected with plasmids encoding GFP-tagged SEC16B-R142W. After 24 hours, cells were fixed and immunostained against SEC13 to label ERES. Green color shows SEC16B-R142W expression, red colors shows SEC13 expression and yellow color shows the overlap of the two colors. The scale bars are 10 µm.


D ) HepG2 cells were transfected with the indicated siRNA . After 48h , cells were transfected with plasmids encoding signal sequence GFP ( ssGFP ) as part of a RUSH construct . After 24 hours , cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex . The scale bars are 10 µm .

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D) HepG2 cells were transfected with the indicated siRNA. After 48h, cells were transfected with plasmids encoding signal sequence GFP (ssGFP) as part of a RUSH construct. After 24 hours, cells were treated with 40 mM biotin for 15 minutes followed by fixation and immunostaining for GM130 to label the Golgi complex. The scale bars are 10 µm.


Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein of the huCoV - 19 / WH01 ( B ) and Beta variants ( D )

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

Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein of the huCoV-19/WH01 (B) and Beta variants (D)


Ability of the SARS - CoV - 2 DNA vaccine OC - 2 . 4 to induce antibodies against S protein the ability to neutralize these viruses in vitro ( C and E ) .

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

Ability of the SARS-CoV-2 DNA vaccine OC-2.4 to induce antibodies against S protein the ability to neutralize these viruses in vitro (C and E).


Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime and boost antibodies to S protein ( huCoV - 19 / WH01 ) following priming with a recombinant S protein in adjuvant ( F )

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Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime and boost antibodies to S protein (huCoV-19/WH01) following priming with a recombinant S protein in adjuvant (F)


Also shown is the ability of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro ( G ) .

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

Also shown is the ability of the universal SARS-CoV-2 DNA vaccine OC-2.4 to prime the ability of these antibodies to neutralize the Delta and Omicron variants in vitro (G).


Finally , three doses of the universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 induce antibodies that cross react with S proteins from the huCoV - 19 / WH01 , Beta , and Delta variants in mice ( H ) ,

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

Finally, three doses of the universal SARS-CoV-2 DNA vaccine OC-2.4 induce antibodies that cross react with S proteins from the huCoV-19/WH01, Beta, and Delta variants in mice (H),


four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro ( I ) .

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four 840 µg doses of the same vaccine induces antibodies in female Zeeland White rabbits that neutralize both the Delta and Omicron variants in vitro (I).


A - D ) T cell responses as determined by ELISpot in Balb / c mice immunized three times with either spike protein in QS21 adjuvant ( rS / QS21 ; A ) , 50µg universal SARS - CoV - 2 vaccine OC - 2 . 4 ( B ) , primed with rS / QS21 and boosted with two doses of OC - 2 . 4 ( C ) , or empty pVAX plasmid ( D ) , using in vivo electroporation . Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ producing ( spot forming ) cells ( SFCs ) per million splenocytes . Each color indicate an individual mice .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O

A-D) T cell responses as determined by ELISpot in Balb/c mice immunized three times with either spike protein in QS21 adjuvant (rS/QS21; A), 50µg universal SARS-CoV-2 vaccine OC-2.4 (B), primed with rS/QS21 and boosted with two doses of OC-2.4 (C), or empty pVAX plasmid (D), using in vivo electroporation. Spleens were harvested and individual mice were analyzed for the presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ producing (spot forming) cells (SFCs) per million splenocytes. Each color indicate an individual mice.


E - H ) Also shown is analysis of T cell responses in New Zeeland White rabbits , which were immunized four times with either vehicle only ( E ) , or 84µg ( F ) or 840 µg ( G ) universal SARS - CoV - 2 DNA vaccine OC - 2 . 4 using in vivo electroporation . Peripheral blood mononuclear cells ( PBMCs ) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens . Data has been given as the number of IFNγ SFCs per million PBMCs . The dotted line indicates the 50 SFC cut off . Also shown ( H ) is the cumulated IFNγ SFCs per million PBMCs to the RBD , M and N peptide pools added together for each group of immunized rabbits ( 84µg DNA , 840µg DNA , and vehicle only ) at each time point ( day ) . The numbers on the x - axis of graphs A ) to D ) indicates the number of the respective peptide pool ( WH1 variant ) covering a part of the indicated protein .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H) Also shown is analysis of T cell responses in New Zeeland White rabbits, which were immunized four times with either vehicle only (E), or 84µg (F) or 840 µg (G) universal SARS-CoV-2 DNA vaccine OC-2.4 using in vivo electroporation. Peripheral blood mononuclear cells (PBMCs) were harvested and analysed for presence of IFNγ producing T cells using the indicated antigens. Data has been given as the number of IFNγ SFCs per million PBMCs. The dotted line indicates the 50 SFC cut off. Also shown (H) is the cumulated IFNγ SFCs per million PBMCs to the RBD, M and N peptide pools added together for each group of immunized rabbits (84µg DNA, 840µg DNA, and vehicle only) at each time point (day). The numbers on the x-axis of graphs A) to D) indicates the number of the respective peptide pool (WH1 variant) covering a part of the indicated protein.


Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues ( b )

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Three doses of respective vaccine fully or partially protected the mice against severe disease as determined by histological scoring of brochial and alveolar lung tissues (b)


Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss ( c ) .

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

Three doses of respective vaccine fully or partially protected the mice against severe disease as percent weight loss (c).


D ) Three doses of respective vaccine also protect against viral replication in the nose , lungs and spleen . Values have been given as cycle times ( CT ) , where lower values indicate a higher viral load . Statistical comparisons in the graph are shown with lines with one asterisc indicating p < 0 , 05 and two asterics p < 0 ,

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D) Three doses of respective vaccine also protect against viral replication in the nose, lungs and spleen. Values have been given as cycle times (CT), where lower values indicate a higher viral load. Statistical comparisons in the graph are shown with lines with one asterisc indicating p<0,05 and two asterics p<0,01


( A ) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter . Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis .

(A) Human myeloid cells and lymphoid cells were purified from PBMCs using a cell sorter. Total RNA was isolated from these primary cells and primary AECs to perform chip hybridization and microarray analysis.


The profile of EphA2 expression in different cells is indicated . ( B ) Murine B cells and T cells were purified from spleens using a cell sorter , total RNA was isolated from murine B cells , T cells , BMDCs , BMDMs , AMs and AECs . Shown is mean ± SD . Data represent three independent biological replicates .

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The profile of EphA2 expression in different cells is indicated. (B) Murine B cells and T cells were purified from spleens using a cell sorter, total RNA was isolated from murine B cells, T cells, BMDCs, BMDMs, AMs and AECs. Shown is mean ± SD. Data represent three independent biological replicates.


The profile of EphA2 expression in different cells is indicated . ( C ) Immunoblot analysis of EphA2 and β - actin in BMDCs , BMDMs , AMs , and AECs .

O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O

The profile of EphA2 expression in different cells is indicated. (C) Immunoblot analysis of EphA2 and β-actin in BMDCs, BMDMs, AMs, and AECs.


( A - D ) ELISA of IL - 1β , IL - 18 , IFN - β and IL - 6 in supernatant of AEC cells , which were stimulated for 16 h with RNA virus reovirus ( MOI = 5 ) or DNA virus HSV - 1 ( MOI = 5 ) . n = 3 primary samples . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . Mock indicated unstimulated cells . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . NS means no statistical difference . Data represent three independent biological replicates .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) ELISA of IL-1β, IL-18, IFN-β and IL-6 in supernatant of AEC cells, which were stimulated for 16 h with RNA virus reovirus (MOI=5) or DNA virus HSV-1 (MOI=5). n = 3 primary samples. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.


( E ) Immunoblot analysis of IL - 1β and caspase - 1 in AECs cell lysate or cell supernatant , β - actin served as a loading control throughout . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . Mock indicated unstimulated cells . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . NS means no statistical difference . Data represent three independent biological replicates .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Immunoblot analysis of IL-1β and caspase-1 in AECs cell lysate or cell supernatant, β-actin served as a loading control throughout. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. Mock indicated unstimulated cells. **P <0.01 and ***P<0.001 (unpaired t test). NS means no statistical difference. Data represent three independent biological replicates.


( A - B ) ELISA of IL - 1β and IL - 18 in BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-B) ELISA of IL-1β and IL-18 in BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


Cell number of BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

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Cell number of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


Cell number and category of BALF samples were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice ( n = 3 per strain ) on day 1 and day 3 after intranasal infection with reovirus ( 4 × 104 PFU per mouse ) . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell number and category of BALF samples were from wild-type (WT) and EphA2-kockout (KO) mice (n=3 per strain) on day 1 and day 3 after intranasal infection with reovirus (4×104 PFU per mouse). Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


( F ) Hematoxylin and eosin ( H & E ) - staining of lung sections were from EphA2 + / + and EphA2 − / − mice intranasally infected with reovirus ( 4 × 104 PFU per mouse ) on day 0 , day 1 and day 3 . Scale bars represent 200μm .

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(F) Hematoxylin and eosin (H&E)-staining of lung sections were from EphA2+/+ and EphA2−/− mice intranasally infected with reovirus (4×104 PFU per mouse) on day 0, day 1 and day 3. Scale bars represent 200μm.


( G ) Immunoblot analysis of EphA2 and IL - 1β in lung homogenates were from wild - type ( WT ) and EphA2 - kockout ( KO ) mice on day 1 and day 3 of intranasal infection with reovirus ( 4 × 104 PFU per mouse ) , β - actin served as a loading control throughout .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunoblot analysis of EphA2 and IL-1β in lung homogenates were from wild-type (WT) and EphA2-kockout (KO) mice on day 1 and day 3 of intranasal infection with reovirus (4×104 PFU per mouse), β-actin served as a loading control throughout.


Quantitative RT - PCR analysis of mRNA encoding various cytokines ( IL - 4 , IL - 5 , IL - 13 in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR analysis of mRNA encoding various cytokines (IL-4, IL-5, IL-13 in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


Quantitative RT - PCR analysis of mRNA encoding various cytokines IL - 33 ) in lung samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 4 per strain ) sensitized with ova ; results presented relative to β - actin . Shown is mean ± SD . ( C ) ELISA analysis of IL - 33 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantitative RT-PCR analysis of mRNA encoding various cytokines IL-33) in lung samples from wild-type (WT) and EphA2-knockout (KO) mice (n=4 per strain) sensitized with ova; results presented relative to β-actin. Shown is mean ± SD. (C) ELISA analysis of IL-33 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( D - G ) ELISA analysis of IL - 4 , IL - 13 , IL - 1β and IL - 18 in lung tissue homogenate samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-G) ELISA analysis of IL-4, IL-13, IL-1β and IL-18 in lung tissue homogenate samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( H ) Cell number and category of BALF samples from wild - type ( WT ) and EphA2 - knockout ( KO ) mice ( n = 3 per strain ) sensitized with ova . Shown is mean ± SD . Data information : Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three or four independent biological replicates .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Cell number and category of BALF samples from wild-type (WT) and EphA2-knockout (KO) mice (n=3 per strain) sensitized with ova. Shown is mean ± SD. Data information: Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. *P<0.05, **P <0.01 and ***P<0.001 (unpaired t test). Data represent three or four independent biological replicates.


( I ) H & E staining and PAS staining of lung sections from wild - type ( WT ) and EphA2 - knockout ( KO ) mice sensitized with ova . Scale bars represent 200μm .

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(I) H&E staining and PAS staining of lung sections from wild-type (WT) and EphA2-knockout (KO) mice sensitized with ova. Scale bars represent 200μm.


( A ) Immunoblot analysis of proteins precipitated with anti - EphA2 or IgG ( control ) were from whole - cell lysates of AECs from wild - type mice stimulated with reovirus ( MOI = 5 ) for 3 h . Input , 10 % of the AECs lysate . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot analysis of proteins precipitated with anti-EphA2 or IgG (control) were from whole-cell lysates of AECs from wild-type mice stimulated with reovirus (MOI=5) for 3 h. Input, 10% of the AECs lysate. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( C ) The indicated plasmids of HA - tagged full - length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged NLRP3 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc Antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) The indicated plasmids of HA-tagged full-length EphA2 and EphA2 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged NLRP3, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc Antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( D ) The indicated plasmids of HA - tagged full - length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc - tagged EphA2 , and then cell lysates were immunoprecipitated with anti - Myc antibody , followed by western blot analysis with anti - HA antibody and anti - Myc antibody . Data information : The position of protein markers ( shown in kDa ) is indicated on the right - hand side . Data are representative of three independent biological replicates .

(D) The indicated plasmids of HA-tagged full-length NLRP3 and NLRP3 truncation mutants were respectively transfected into HEK293T cells along with Myc-tagged EphA2, and then cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with anti-HA antibody and anti-Myc antibody. Data information: The position of protein markers (shown in kDa) is indicated on the right-hand side. Data are representative of three independent biological replicates.


( E ) Colocalization of endogenous EphA2 and NLRP3 in AEC cells . AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis . EphA2 was stained with rabbit anti - EphA2 ( 1 : 500 ) , followed by Alexa Fluor 594 goat anti - rabbit secondary antibody ( red ) , while NLRP3 was stained with mouse anti - NLRP3 ( 1 : 500 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) served as the nuclei marker ( blue ) . Scale bars represent 20μm . The Pearson ' s correlation coefficient and the Mander ' s overlap coefficient in the upper red box are 0 . 5 , and 0 . 95 respectively , and the lower red box 0 . 78 , and 0 . 94 respectively .

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(E) Colocalization of endogenous EphA2 and NLRP3 in AEC cells. AEC cells were unstimulated or stimulated with reovirus for 2h before confocal microscopy analysis. EphA2 was stained with rabbit anti-EphA2 (1:500), followed by Alexa Fluor 594 goat anti-rabbit secondary antibody (red), while NLRP3 was stained with mouse anti-NLRP3 (1:500), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI (4′,6-diamidino-2-phenylindole) served as the nuclei marker (blue). Scale bars represent 20μm. The Pearson's correlation coefficient and the Mander's overlap coefficient in the upper red box are 0.5, and 0.95 respectively, and the lower red box 0.78, and 0.94 respectively.


( A ) The indicated plasmids of Myc - tagged NLRP3 and GFP - tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - GFP and anti - Myc .

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(A) The indicated plasmids of Myc-tagged NLRP3 and GFP-tagged EphA2 were transfected into HEK293T cells and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-GFP and anti-Myc.


( B ) Immunoblot analysis of p - Tyr of NLRP3 precipitated with anti - NLRP3 antibody in AECs without ( Ctrl ) or with LPS stimulation or reovirus ( Reo ) infection .

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(B) Immunoblot analysis of p-Tyr of NLRP3 precipitated with anti-NLRP3 antibody in AECs without (Ctrl) or with LPS stimulation or reovirus (Reo) infection.


( C ) The indicated plasmids of Myc - tagged wild - type NLRP3 or its mutants Y132F , Y164F , Y251F , Y570F and Y589F were respectively transfected into HEK293T cells along with GFP - tagged EphA2 , and then cell lysates were immunoprecipitated with the anti - Myc antibody , followed by western blot analysis with the anti - p - Tyr antibody , anti - HA antibody and anti - Myc antibody .

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(C) The indicated plasmids of Myc-tagged wild-type NLRP3 or its mutants Y132F, Y164F, Y251F, Y570F and Y589F were respectively transfected into HEK293T cells along with GFP-tagged EphA2, and then cell lysates were immunoprecipitated with the anti-Myc antibody, followed by western blot analysis with the anti-p-Tyr antibody, anti-HA antibody and anti-Myc antibody.


( D ) Immunoblot analysis of EphA2 , ASC , NLRP3 , caspase - 1 and IL - 1β in HEK293T cells were co - transfected with 200ng EphA2 , 40ng ASC , 40ng caspase - 1 and 200ng IL - 1β , vector or wild NLPR3 or Y132F as indicated . The position of protein markers ( shown in kDa ) is indicated on the right - hand side .

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(D) Immunoblot analysis of EphA2, ASC, NLRP3, caspase-1 and IL-1β in HEK293T cells were co-transfected with 200ng EphA2, 40ng ASC, 40ng caspase-1 and 200ng IL-1β, vector or wild NLPR3 or Y132F as indicated. The position of protein markers (shown in kDa) is indicated on the right-hand side.


( E ) Densitometry analysis * P < 0 . 05 ( unpaired t test ) . NS means no statistical difference . Shown is mean ± SD . Data represent three independent biological replicates .

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(E) Densitometry analysis *P<0.05 (unpaired t test). NS means no statistical difference. Shown is mean ± SD. Data represent three independent biological replicates.


( A - B ) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection ( MOI ) of 20 for 3h . AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control . ASC was stained with mouse anti - ASC ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - mouse secondary antibody ( green ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

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(A-B) Confocal microscopy analysis of AEC cells uninfected or infected with reovirus at multiplicity of infection (MOI) of 20 for 3h. AECs primed with LPS and stimulated with ATP for 30 min severed as a positive control. ASC was stained with mouse anti-ASC (1:200), followed by Alexa Fluor 488 goat anti-mouse secondary antibody (green). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


( C - D ) Confocal microscopy analysis of HEK293T cells transfected with flag - ASC and Myc - tagged vector or Myc - tagged NLRP3 or Myc - tagged NLRP3 Y132F . Cells were stained with anti - flag ( 1 : 200 ) , anti - Myc ( 1 : 200 ) , followed by Alexa Fluor 488 goat anti - rabbit secondary antibody ( green ) or Alexa Fluor 594 goat anti - mouse secondary antibody ( red ) . DAPI served as the nuclei marker ( blue ) . Data information : Scale bars represent 50μm . Each symbol represents an independent experiment ; small horizontal lines indicate the average of triplicates . * * P < 0 . 01 and * * * P < 0 . 001 ( unpaired t test ) . Data represent three independent biological replicates .

O 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-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-D) Confocal microscopy analysis of HEK293T cells transfected with flag-ASC and Myc-tagged vector or Myc-tagged NLRP3 or Myc-tagged NLRP3 Y132F. Cells were stained with anti-flag (1:200), anti-Myc (1:200), followed by Alexa Fluor 488 goat anti-rabbit secondary antibody (green) or Alexa Fluor 594 goat anti-mouse secondary antibody (red). DAPI served as the nuclei marker (blue). Data information: Scale bars represent 50μm. Each symbol represents an independent experiment; small horizontal lines indicate the average of triplicates. **P <0.01 and ***P<0.001 (unpaired t test). Data represent three independent biological replicates.


A - B . SH - SY5Y cells and primary cortical neurons were treated with 1 μM α - syn fibrils ( A ) or monomers ( B ) for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 compared to PBS , unpaired t - test .

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A-B. SH-SY5Y cells and primary cortical neurons were treated with 1 μM α-syn fibrils (A) or monomers (B) for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01 compared to PBS, unpaired t-test.


C . Frontal cortices of A53T α - syn heterozygous transgenic mice and control mice at the age of 9 months ( old ) or 2 months ( young ) were lysed and Western blot was performed with the indicated antibodies ( n = 3 ) . * * * P < 0 . 001 compared to WT , unpaired t - test .

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C. Frontal cortices of A53T α-syn heterozygous transgenic mice and control mice at the age of 9 months (old) or 2 months (young) were lysed and Western blot was performed with the indicated antibodies (n=3). *** P < 0.001 compared to WT, unpaired t-test.


FcγRIIB KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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FcγRIIB KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 1 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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SHP-1 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SHP - 2 KD SH - SY5Y cells were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

SHP-2 KD SH-SY5Y cells were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells with 20 μM NSC87877 , 10 μM SSG and 1 μM PHPS - 1 were treated with 1 μM α - syn fibrils for 30 min . The cells were lysed and Western blot was performed with the indicated antibodies . Images are representative of three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * * P < 0 . 01 , * P < 0 . 05 compared to PBS , one - way ANOVA .

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SH-SY5Y cells with 20 μM NSC87877, 10 μM SSG and 1 μM PHPS-1 were treated with 1 μM α-syn fibrils for 30 min. The cells were lysed and Western blot was performed with the indicated antibodies. Images are representative of three independent experiments (n=3). *** P < 0.001, ** P < 0.01, * P < 0.05 compared to PBS, one-way ANOVA.


SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


NT , c - src KD # 1 and # 2 SH - SY5Y cells were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells , one - way

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NT, c-src KD #1 and #2 SH-SY5Y cells were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells, one-way ANOVA


, D . primary cortical neurons were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 for 12 h . The cells in the lower chamber were fixed and immunostained with anti - EGFP antibody . The intensity was analyzed . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 against PBS , one - way ANOVA .

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, D. primary cortical neurons were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1 for 12 h. The cells in the lower chamber were fixed and immunostained with anti-EGFP antibody. The intensity was analyzed. Values were derived from four independent experiments (n=4). *** P < 0.001 against PBS, one-way ANOVA.


, E . primary cortical neurons downregulating c - src were cocultured with differentiated A53T α - syn - EGFP OE SH - SY5Y cells for 12 h , and then fixed and immunostained with anti - EGFP antibody . Values were derived from four independent experiments ( n = 4 ) . * * * P < 0 . 001 compared to NT cells one - way ANOVA unpaired t -

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, E. primary cortical neurons downregulating c-src were cocultured with differentiated A53T α-syn-EGFP OE SH-SY5Y cells for 12 h, and then fixed and immunostained with anti-EGFP antibody. Values were derived from four independent experiments (n=4). *** P < 0.001 compared to NT cells one-way ANOVA unpaired t-test


SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


G . FcγRIIB KD SH - SY5Y cells were transfected with plasmids of WT c - src , Y529F c - src ( kinase active ) , or K297M c - src ( kinase dead ) tagged with EGFP . After 1 day , the cells were cocultured with differentiated α - syn OE SH - SY5Y cells for 12 h and then fixed and immunostained with anti - α - syn antibody . The intensity was analyzed . Values were derived from three independent experiments ( n = 3 ) . * * * P < 0 . 001 , * P < 0 . 05 compared to mock positive cells , one - way ANOVA .

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G. FcγRIIB KD SH-SY5Y cells were transfected with plasmids of WT c-src, Y529F c-src (kinase active), or K297M c-src (kinase dead) tagged with EGFP. After 1 day, the cells were cocultured with differentiated α-syn OE SH-SY5Y cells for 12 h and then fixed and immunostained with anti-α-syn antibody. The intensity was analyzed. Values were derived from three independent experiments (n=3). *** P < 0.001, * P < 0.05 compared to mock positive cells, one-way ANOVA.


B . A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured in the presence of 5 μM saracatinib ( sara ) or 10 μM SKI - 1 , respectively , with 50 μM RA for 5 days .

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B. A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured in the presence of 5 μM saracatinib (sara) or 10 μM SKI-1, respectively, with 50 μM RA for 5 days.


C . NT or c - src KD # 1 or # 2 / A53T α - syn - EGFP and A53T α - syn - mCherry OE SH - SY5Y cells were cocultured with 50 μM RA for 5 days . The samples were observed under confocal microscopy , and the number of cells containing double fluorescence labeled puncta were analyzed . Values were derived from five independent experiments ( n = 5 ) . * * * P < 0 . 001 compared to PBS or NT cells , one - way ANOVA .

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C. NT or c-src KD #1 or #2 /A53T α-syn-EGFP and A53T α-syn-mCherry OE SH-SY5Y cells were cocultured with 50 μM RA for 5 days. The samples were observed under confocal microscopy, and the number of cells containing double fluorescence labeled puncta were analyzed. Values were derived from five independent experiments (n=5). *** P < 0.001 compared to PBS or NT cells, one-way ANOVA.