Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [ 17 ] . Fluorescent transferrin was added for 45 min to the cells . Cells were analyzed after washing . The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for ( B ) . The encircled area in MERGE is shown 2 - fold enlarged on the right hand side of the figure Quantification of ( A ) . The fluorescence intensities of transferrin within a 2 μm - radius circle surrounding the CEP164 signal were quantified . An ImageJ macro was used to measure the intensity as described in methods . Data were calculated from a total of 120 cells pooled from two independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001

B-MEASURED_VAR 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 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 B-MEASURED_VAR 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

Transferrin uptake was measured with serum starvation or 4 h after serum starvation as described [17]. Fluorescent transferrin was added for 45 min to the cells. Cells were analyzed after washing. The red circle around the mother centriole marker CEP164 indicates the fluorescence intensity that was determined for (B). The encircled area in MERGE is shown 2-fold enlarged on the right hand side of the figure Quantification of (A). The fluorescence intensities of transferrin within a 2 μm-radius circle surrounding the CEP164 signal were quantified. An ImageJ macro was used to measure the intensity as described in methods. Data were calculated from a total of 120 cells pooled from two independent experiments. Mean ± SD. **** P≤ 0.0001


hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm - radius of the basal body ( red cicle ) . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure Quantification of C . Data was calculated from a total of 180 cells pooled from three independent experiments . Mean ± SD . * P ≤ 0 . 05 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001

B-CONTROLLED_VAR O O O O O O B-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

hCDC14APD cells showed an increased localization of myosin Va containing vesicles within the 1 μm-radius of the basal body (red cicle). The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure Quantification of C. Data was calculated from a total of 180 cells pooled from three independent experiments. Mean ± SD. * P≤ 0.05, *** P≤ 0.001, **** P≤ 0.0001


Arp2 localization in REP1 WT , hCDC14APD and DBN1 KO cells . Signal intensity was measured 30 min after serum starvation . The encircled area in MERGE is shown 4 - fold enlarged on the right hand side of the figure

B-MEASURED_VAR 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

Arp2 localization in REP1 WT, hCDC14APD and DBN1 KO cells. Signal intensity was measured 30 min after serum starvation. The encircled area in MERGE is shown 4-fold enlarged on the right hand side of the figure


Quantification of E . Sub - centrosomal ( 1 μm - radius ; red circle in E ) Arp2 was measured by quantifying the intensity of Arp2 in WT , hCDC14APD and DBN1 KO cells . Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area ( 1 μm - radius ) compared to WT cells after 30 min of serum starvation . Data were calculated from a total of 120 cells pooled from three independent experiments . Mean ± SD . * * * * P ≤ 0 . 0001

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

Quantification of E. Sub-centrosomal (1 μm-radius; red circle in E) Arp2 was measured by quantifying the intensity of Arp2 in WT, hCDC14APD and DBN1 KO cells. Both hCDC14APD and DBN1 KO cells contain significantly higher Arp2 in the pericentrosomal area (1 μm-radius) compared to WT cells after 30 min of serum starvation. Data were calculated from a total of 120 cells pooled from three independent experiments. Mean ± SD. **** P≤ 0.0001


Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells ( Tet Control ) with DBN1 KO cells expressing different DBN1 versions ( WT , S142A , S142D ) . Data were calculated from a total of 200 cells each pooled from two independent experiments . Mean ± SD . ns : not specific

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

Pericentrosomal ARP2 enrichment was comparable in the RPE1 DBN1 KO cells (Tet Control) with DBN1 KO cells expressing different DBN1 versions (WT, S142A, S142D). Data were calculated from a total of 200 cells each pooled from two independent experiments. Mean ± SD. ns: not specific


( A ) CXCL10 assay characteristics . Standard curves of the short , long and total CXCL10Simoa assays are shown . For each curve , limit of detection ( LOD ) defined as blank + 3SD , are shown as horizontal lines . The LOD was 1 . 7pg / ml for long and short CXCL10 assays and 0 . 22pg / ml for total CXCL10 . Standard curves were fitted using the 4 Parameter Logistic non - linear regression model . Samples reporting a signal below the LOD were replaced with 1pg / ml for short and long CXCL10 .

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

(A) CXCL10 assay characteristics. Standard curves of the short, long and total CXCL10Simoa assays are shown. For each curve, limit of detection (LOD) defined as blank+3SD, are shown as horizontal lines. The LOD was 1.7pg/ml for long and short CXCL10 assays and 0.22pg/ml for total CXCL10. Standard curves were fitted using the 4 Parameter Logistic non-linear regression model. Samples reporting a signal below the LOD were replaced with 1pg/ml for short and long CXCL10.


Plasma from healthy individuals receiving ( B ) placebo ( n = 9 ) or ( C ) sitagliptin ( n = 27 ) were analyzed by Simoa . Blood samples were collected at screening visit ( SV ) and day 0 ( D0 ) before treatment , at day 3 ( D3 ) , day 14 ( W2 ) and day 28 ( W4 ) under treatment and 5 weeks after treatment interruption ( W9 ) . ( C ) Plasma from chronic HCVpatients receiving sitagliptin ( n = 3 ) were collected before ( D0 ) and weekly during sitagliptin treatment ( W1 W3 ) . Antagonist CXCL103 - 77 ( short CXCL10 , in blue ) , agonist CXCL101 - 77 ( long CXCL10 , in red ) and total CXCL10 ( in black ) levels are shown . Each dot represents a donor and bars are at the median . Grey areas highlight the period under placebo or sitagliptin treatment . Statistical analysis of ( B ) and ( C ) was performed using non parametric Friedman ' s test , ns : non significant , * * : P0 . 01 , * * * * : P0 . 001 . For ( C ) , additional size effect analysis was performed and Cohen ' s d values are reported . No statistical analysis was performed in ( D ) due to sample size , nd : non - determined .

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

Plasma from healthy individuals receiving (B) placebo (n=9) or (C) sitagliptin (n=27) were analyzed by Simoa. Blood samples were collected at screening visit (SV) and day 0 (D0) before treatment, at day 3 (D3), day 14 (W2) and day 28 (W4) under treatment and 5 weeks after treatment interruption (W9). (C) Plasma from chronic HCVpatients receiving sitagliptin (n=3) were collected before (D0) and weekly during sitagliptin treatment (W1 W3). Antagonist CXCL103-77 (short CXCL10, in blue), agonist CXCL101-77 (long CXCL10, in red) and total CXCL10 (in black) levels are shown. Each dot represents a donor and bars are at the median. Grey areas highlight the period under placebo or sitagliptin treatment. Statistical analysis of (B) and (C) was performed using non parametric Friedman's test, ns: non significant, **: P0.01, ****: P0.001. For (C), additional size effect analysis was performed and Cohen's d values are reported. No statistical analysis was performed in (D) due to sample size, nd: non-determined.


( 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 .

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

(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 .

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

(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 .

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

(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 .

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

(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 ) .

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

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 ) .

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

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 .

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

(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 .

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

(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 .

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 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 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

(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.


B . Flow cytometry analyses of recipient blood 2 months post - transplant . Bi . Transplanted cells detected by GFP fluorescence . Bii . CD11b lebelling shows a large proportion of monocytes expressing GFP .

O 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

B. Flow cytometry analyses of recipient blood 2 months post-transplant. Bi. Transplanted cells detected by GFP fluorescence. Bii. CD11b lebelling shows a large proportion of monocytes expressing GFP.


C . Demyelinating lesions in chimeric mice at 3 dpl . Ci . Demyelination was induced by injecting LPC in the spinal cord white matter . Cii - Civ . Co - immunolabeling for GFP , MBP , and a mixture of CD11b / CD68 , showing graft - derived macrophages in the lesion ( identified by lack of MBP staining ) . White lines indicate lesion borders . Scale bar 20 µm . MBP - myelin basic protein ; GFP - green fluorescent 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O B-MEASURED_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 B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O

C. Demyelinating lesions in chimeric mice at 3 dpl. Ci. Demyelination was induced by injecting LPC in the spinal cord white matter. Cii-Civ. Co-immunolabeling for GFP, MBP, and a mixture of CD11b/CD68, showing graft-derived macrophages in the lesion (identified by lack of MBP staining). White lines indicate lesion borders. Scale bar 20 µm. MBP-myelin basic protein; GFP-green fluorescent protein.


Transduction of HSCs . B . Lower numbers of GFP + cells , verified using flow cytometry , after transduction with Sema3F vector , * * * p < 0 . 0001 , Student t test , n = 8 independent experiments .

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

Transduction of HSCs. B. Lower numbers of GFP+ cells, verified using flow cytometry, after transduction with Sema3F vector, ***p<0.0001, Student t test, n=8 independent experiments.


Transduction of HSCs . C . Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F - transduced cells only .

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 B-CONTROLLED_VAR O O O O O

Transduction of HSCs. C. Western blot showing GFP expression in cells transduced with both vectors and Sema3F expression in the supernatant of Sema3F-transduced cells only.


E . Fold increase in cells that crossed the membrane insert compared to NT . * * * p ‹ 0 . 0001 . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . n = 1 - 2 normalized replicates from 3 - 5 independent experiments . Mean ± SEM . * * * p < 0 . 0001 . R . = recombinant . Super = supernatant . Anti - Nrp2 = Neuropilin2 function blocking antibody .

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

E. Fold increase in cells that crossed the membrane insert compared to NT. ***p‹0.0001. One-way ANOVA followed by Bonferroni's Multiple Comparison test. n= 1-2 normalized replicates from 3-5 independent experiments. Mean±SEM. ***p<0.0001. R.=recombinant. Super=supernatant. Anti-Nrp2=Neuropilin2 function blocking antibody.


Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . A . Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα − GFP mice ( n = 3 - 4 independent experiments ) .

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 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

Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. A. Microarray analyses results for Nrp2 expression by OPCs purified using FACS from the brains of 2 months versus 12 months old PDGFRα−GFP mice (n=3-4 independent experiments).


Expression of Nrp2 gene by OPCs is maintained in middle - age ( 12 month ( m ) old ) mice . B . RNA sequencing results for Nrp2 expression by oligodendroglia - enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice . ( n = 3 - 4 independent experiments ) .

Expression of Nrp2 gene by OPCs is maintained in middle-age (12 month (m) old) mice. B. RNA sequencing results for Nrp2 expression by oligodendroglia-enriched preparations isolated from the spinal cords of 2 month versus 12 month old mice. (n=3-4 independent experiments).


C - G . Sema 3F expression in demyelinating lesions at 4 dpl ( onset of OPC recruitment ) is decreased in middle - aged ( 12 m old ) and old ( 18m old ) mice compared to young mice ( 2m old ) . C - E . Colabellings for Sema3F and Iba1 ( macrophage marker ) in demyelinating lesions at 4 dpl in 2m ( C ) , 12m ( D ) , and 18m old mice ( E ) . Arrowheads point to double - labelled cells . F . Quantification of Sema3F expressing cells in the lesion . G . Percentage of Iba1 + cells positive for Sema3F . NWM - normal white matter . dpl - days post lesion . n = 4 - 8 mice / group .

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

C-G. Sema 3F expression in demyelinating lesions at 4 dpl (onset of OPC recruitment) is decreased in middle-aged (12 m old) and old (18m old) mice compared to young mice (2m old). C-E. Colabellings for Sema3F and Iba1 (macrophage marker) in demyelinating lesions at 4 dpl in 2m (C), 12m (D),and 18m old mice (E). Arrowheads point to double-labelled cells. F. Quantification of Sema3F expressing cells in the lesion. G. Percentage of Iba1+ cells positive for Sema3F. NWM-normal white matter. dpl-days post lesion. n=4-8 mice/group.


H . Transwell chamber assay was used to evaluate migration of young versus middle - age OPCs in response to recombinant Sema3F and supernatant from PGK - GFP - T2A - Sema3F transduced cells . OPCs of both ages migrate in response to recombinant Sema3F and PGK - GFP - T2A - Sema3F - transduced cell supernatant . n = 1 - 2 normalized replicates from 2 - 5 independent experiments . Mean ± SEM . One - way ANOVA followed by Bonferroni ' s Multiple Comparison test . * p < 0 . 05 , * * p < 0 . 01 . Scale bar 50 µm .

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

H. Transwell chamber assay was used to evaluate migration of young versus middle-age OPCs in response to recombinant Sema3F and supernatant from PGK-GFP-T2A-Sema3F transduced cells. OPCs of both ages migrate in response to recombinant Sema3F and PGK-GFP-T2A-Sema3F-transduced cell supernatant. n= 1-2 normalized replicates from 2-5 independent experiments. Mean±SEM. One-way ANOVA followed by Bonferroni's Multiple Comparison test. *p<0.05, ** p<0.01. Scale bar 50 µm.


B . Percentage of transplanted ( female ) cells in the blood . n = 18 - 19 mice / group .

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

B. Percentage of transplanted (female) cells in the blood. n=18-19 mice/group.


C . Percentage of transduced ( GFP + ) cells in the blood . Mean ± SEM . * * * p < 0 . 0001 . n = 22 - 25 mice / group .

O O O O O O O O O O O O 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. Percentage of transduced (GFP+) cells in the blood . Mean±SEM. ***p<0.0001. n=22-25 mice/group.


D . Percentages of GFP + cells labeled with CD11b , CD19 , and CD3 . The proportion of GFP + cells labeled with CD11b is increased in Sema3F mice ( p = 0 . 01 ) , while that of CD19 - labeled cells is decreased ( p = 0 . 0003 ) . Mean ± SEM . Unpaired Student t - test . n = 9 mice / group . * p < 0 . 05 , * * * p < 0 . 001 .

O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O B-MEASURED_VAR O O 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 O O O O O O O O O O O O O O O O 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. Percentages of GFP+ cells labeled with CD11b, CD19, and CD3. The proportion of GFP+ cells labeled with CD11b is increased in Sema3F mice (p=0.01), while that of CD19-labeled cells is decreased (p=0.0003). Mean±SEM. Unpaired Student t-test. n=9 mice/group. *p<0.05,***p<0.001.


E . Percentages of total blood cells expressing CD11b , CD19 , and CD3 . Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice . n = 9 mice / group . Mean ± SEM .

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

E. Percentages of total blood cells expressing CD11b, CD19, and CD3. Red dashed lines indicate the percentage expression of these antigens in blood cells of control mice. n=9 mice/group. Mean±SEM.


A - F . Co - labeling for GFP and MBP on spinal cord sections of GFP ( A - C ) and Sema3F - GFP chimeras ( D - F ) sacrificed at 7 ( A , D ) , 10 ( B , E ) , and 60 ( C , F ) days post lesion ( dpl ) . Absence of MBP labeling in A - B , D - E indicates the lesion ( delimited by yellow lines ) . Areas faintly labeled with MBP in C - F likely represent remyelinated areas .

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 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 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

A-F. Co-labeling for GFP and MBP on spinal cord sections of GFP (A-C) and Sema3F-GFP chimeras (D-F) sacrificed at 7 (A,D), 10 (B,E), and 60 (C,F) days post lesion (dpl). Absence of MBP labeling in A-B,D-E indicates the lesion (delimited by yellow lines). Areas faintly labeled with MBP in C-F likely represent remyelinated areas.


G . Quantification of GFP + cells . Kruskal - Wallis test p = 0 . 02 . No significant differences between GFP and Sema3F at any of the time points ( Dunn ' s post test ; Mann - Whitney test ) . Decrease in GFP + cells between 7 and 60 dpl for both GFP and Sema3F mice 2m .

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 B-CONTROLLED_VAR O O O

G. Quantification of GFP+ cells. Kruskal-Wallis test p=0.02. No significant differences between GFP and Sema3F at any of the time points (Dunn's post test; Mann-Whitney test). Decrease in GFP+ cells between 7 and 60 dpl for both GFP and Sema3F mice 2m.


H - M . Co - labeling for GFP / MBP / Iba1 ( macrophage / microglia marker ) shows colocalization of GFP with Iba1 . Arrows indicate double - labeled cells .

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

H-M. Co-labeling for GFP/MBP/Iba1 (macrophage/microglia marker) shows colocalization of GFP with Iba1. Arrows indicate double-labeled cells.


N - P Co - labeling for GFP and T cell marker CD3 . Arrows indicate CD3 - labeled cells .

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

N-P Co-labeling for GFP and T cell marker CD3. Arrows indicate CD3-labeled cells.


Q - S . Co - labeling for GFP and B cell marker CD45R / B220 . Arrows indicate CD45R / B220 - labeled cells .

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

Q-S. Co-labeling for GFP and B cell marker CD45R/B220. Arrows indicate CD45R/B220-labeled cells.


T . Percentages of GFP + cells that are Iba1 + , CD3 + , and CD45R / B220 + . The absolute majority of GFP + cells are Iba1 + .

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

T. Percentages of GFP+ cells that are Iba1+, CD3+, and CD45R/B220+. The absolute majority of GFP+ cells are Iba1+.


U . Quantification of Iba1 + , CD3 + , and B220 + cells . For all 3 populations , no significant differences in cell numbers were observed between GFP and Sema3F mice , and a general decrease was observed at 60 dpl compared to earlier time points .

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

U. Quantification of Iba1+, CD3+, and B220+ cells. For all 3 populations, no significant differences in cell numbers were observed between GFP and Sema3F mice, and a general decrease was observed at 60 dpl compared to earlier time points.


V - Aa . Co - labeling for GFP and microglial marker P2Y12 . Purple arrows indicate P2Y12 + cells . Exclusion between the two markers is obvious in both groups . Ab . Quantification of P2Y12 + cells . No significant changes are observed between the groups or across the time .

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

V-Aa. Co-labeling for GFP and microglial marker P2Y12. Purple arrows indicate P2Y12+ cells. Exclusion between the two markers is obvious in both groups. Ab. Quantification of P2Y12+ cells. No significant changes are observed between the groups or across the time.


B - G . Lesions at 7 dpl in GFP ( B , E ) and Sema3F ( C , F ) mice . B - C . Co - labeling for PDGFRα , MBP , and DAPI . Arrows indicate OPCs in the lesion . Bi and Ci - insets of white dotted squares in B and C . D . Higher numbers of PDGFRα + ( OPCs ) cells in Sema3F mice at 7 dpl ( Mann Whitney test : p = 0 . 0031 , n = 6 - 8 mice / group ) . E - F . Co - labeling for Olig2 and MBP . E ' - F ' . Co - labeling for Olig2 and GFP of the lesions shown in E - F . 3D reconstructions . Proximity of OPCs and Sema 3F - carrying cells . E ' i - E ' iii and F ' i - F ' iii - insets of E ' - F ' . G . Higher numbers of Olig2 + ( oligodendroglial ) cells in Sema3F - GFP mice at 7 dpl ( Mann Whitney test : p = 0 . 0047 , n = 5 - 8 mice / group ) .

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

B-G. Lesions at 7 dpl in GFP (B,E) and Sema3F (C,F) mice. B-C. Co-labeling for PDGFRα, MBP, and DAPI. Arrows indicate OPCs in the lesion. Bi and Ci-insets of white dotted squares in B and C. D. Higher numbers of PDGFRα+ (OPCs) cells in Sema3F mice at 7 dpl (Mann Whitney test: p=0.0031, n=6-8 mice/group). E-F. Co-labeling for Olig2 and MBP. E'-F'. Co-labeling for Olig2 and GFP of the lesions shown in E-F. 3D reconstructions. Proximity of OPCs and Sema 3F-carrying cells. E'i-E'iii and F'i-F'iii-insets of E'-F'. G. Higher numbers of Olig2+ (oligodendroglial) cells in Sema3F-GFP mice at 7 dpl (Mann Whitney test: p=0.0047, n=5-8 mice/group).


H - I . Immunolabelling for activated OPC marker Nkx2 . 2 . J . Numbers of Nkx2 . 2 + cells are increased in lesions of Sema3F - GFP mice at 7dpl ( Mann Whitney test : p = 0 . 014 , n = 6 - 7 mice / group ) . Arrows indicate Nkx2 . 2 + cells in close proximity of GFP + cells .

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

H-I. Immunolabelling for activated OPC marker Nkx2.2. J. Numbers of Nkx2.2+ cells are increased in lesions of Sema3F-GFP mice at 7dpl (Mann Whitney test: p=0.014, n=6-7 mice/group). Arrows indicate Nkx2.2+ cells in close proximity of GFP+ cells.


K - P . colabelling for Nkx2 . 2 and proliferation marker Ki67 . Arrows indicate double labeled cells . Q . Numbers of Nkx2 . 2 + Ki67 + cells are increased at 7 dpl in Sema3F - GFP mice . Mean ± SEM . n = 6 - 7 mice / group . R . The proportion of Nkx2 . 2 + cells co - expressing Ki67 is unchanged . Mean ± SEM . n = 6 - 7 mice / group .

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

K-P. colabelling for Nkx2.2 and proliferation marker Ki67. Arrows indicate double labeled cells. Q. Numbers of Nkx2.2+Ki67+ cells are increased at 7 dpl in Sema3F-GFP mice. Mean±SEM. n=6-7 mice/group. R. The proportion of Nkx2.2+ cells co-expressing Ki67 is unchanged. Mean±SEM. n=6-7 mice/group.


S - X . Colabelling for Olig2 and cleaved caspase 3 , marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2 - . Arrows indicate cleaved caspase 3 + cells . Y . Numbers of cleaved caspase 3 + cells are the same in two groups . Mean ± SEM . n = 6 mice / group .

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

S-X. Colabelling for Olig2 and cleaved caspase 3, marker of apoptosis indicates occasional apoptotic cells in both groups of mice that are Olig2-. Arrows indicate cleaved caspase 3+ cells.Y. Numbers of cleaved caspase 3+ cells are the same in two groups. Mean±SEM. n=6 mice/group.


A - B . Labelling for APC / CC1 , a marker of oligodendrocytes . Dotted lines indicate the lesion border . APC + cells are scarce in the lesions of GFP mice ( A ) . B . APC + cells in the lesion of Sema3F mice . C . Quantification of APC + cells ( Mann Whitney test ; p = 0 . 0061 for 7 dpl , p = 0 . 0081 for 10 dpl ; n = 6 - 7 mice / group ) .

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

A-B. Labelling for APC/CC1, a marker of oligodendrocytes. Dotted lines indicate the lesion border. APC+ cells are scarce in the lesions of GFP mice (A). B. APC+ cells in the lesion of Sema3F mice. C. Quantification of APC+ cells (Mann Whitney test; p=0.0061 for 7 dpl, p=0.0081 for 10 dpl; n= 6-7 mice/group).


D - E . EM images of the lesion in GFP ( D ) and Sema3F ( E ) chimeras at 14 dpl . Thin myelin sheaths in Sema 3F mice ( yellow arrows ) . F . Higher power image of a new myelin sheath in a Sema3F mouse . Fi . Inset of yellow square in F . G . Quantification of remyelinated axons ( Mann Whitney test : p = 0 . 03 , n = 6 mice / group ) . * p < 0 . 05 . H . In Sema3F mice , oligodendroglial cells ( O ) and early remyelination ( * ) are frequently observed in macrophage ( M ) vicinity . Hi . Inset of yellow dashed square in H .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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

D-E. EM images of the lesion in GFP (D) and Sema3F (E) chimeras at 14 dpl. Thin myelin sheaths in Sema 3F mice (yellow arrows). F. Higher power image of a new myelin sheath in a Sema3F mouse. Fi. Inset of yellow square in F. G. Quantification of remyelinated axons (Mann Whitney test: p=0.03, n=6 mice/group). *p<0.05. H. In Sema3F mice, oligodendroglial cells (O) and early remyelination (*) are frequently observed in macrophage (M) vicinity. Hi. Inset of yellow dashed square in H.


( B ) Representative images of immunostaining demonstrate that FKRP - iPSCs express specific pluripotency - associated markers , including NANOG , OCT4 , Tra - 1 - 60 and SSEA4 . Data information : Scale bars , 50 μm .

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

(B) Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60 and SSEA4. Data information: Scale bars, 50 μm.


( C ) FKRP - iPSCs have a normal karyotype . Data information : Scale bars , 50 μm .

(C) FKRP-iPSCs have a normal karyotype. Data information: Scale bars, 50 μm.


( D ) In vitro differentiation of FKRP - iPSCs to cells representing ectoderm ( β - III Tubulin , Tuj1 ) , mesoderm ( SMA , smooth muscle actin ) and endoderm ( AFP , α - fetoprotein ) . Data information : Scale bars , 50 μm .

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

(D) In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin) and endoderm (AFP, α-fetoprotein). Data information: Scale bars, 50 μm.


( C ) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones ( 5D17 , 5D23 and 3B17 ) , compared with their parental FKRPA455D - iPSCs . Selection cassette excision sites are identified in the corrected - iPSC lines .

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

(C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines.


( D ) CRISPR / Cas9 corrected - iPSC lines show a normal karyotype .

(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.


( A and B ) Representative images of NSCs derived from FKRP - and corrected - iPSC lines expressing SOX1 , SOX2 and nestin . ( C and D ) Quantification of percentage of SOX1 + ( C ) and SOX2 + ( D ) cells in culture . The efficiency of neural induction is more than 99 % in FKRP - and corrected - iPSC ( 5D17 , 5D23 and 3B17 ) lines . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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

(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin. (C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( E and F ) FKRP - and corrected NSC lines can be further differentiated to cortical neural progenitor cells , expressing PAX6 , OTX2 and vimentin . ( G - I ) Quantification of percentage of PAX6 + ( G ) and OTX2 + ( H ) cells in culture . About 91 - 98 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express PAX6 ( G ) . About 93 - 96 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express OTX2 ( H ) . Of the OTX2 + population , about 60 - 67 % cells are also Ki67 + cycling progenitors ( I ) . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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

(E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin. (G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( J and K ) Glutamatergic projection neurons derived from FKRP and corrected ( 5D17 , 5D23 and 3B17 ) progenitor cells . The vast majority of neurons contain vGlut1 + punctae in their neurites ( labelled by Tuj1 ) . Right panels are enlarged images from the insets of left panels . Data information : Scale bars , 50 μm .

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 B-MEASURED_VAR 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

(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


( A ) Molecular weight of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in the mouse brain ( ~ 120 kDa ) and lower than that in the mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that the β - Actin antibody does not cross - react with the muscle sample .

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

(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.


( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * * p < 0 . 01 )

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

(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)


( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * p < 0 . 05 ) .

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

(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


( C ) Dose response curve . H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed , stained and imaged as described above . The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non - linear regression curve was fitted for the 3 compounds showing the highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve and EC50 values were calculated using GraphPad Prism 7 software . In images shown , green represents glycosylated α - dystroglycan and blue Hoechst staining . Scale bar , 50 µm .

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

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.


( E ) Flow cytometry analysis confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 cells , compared with untreated cells . There was no significant difference in the percentage of IIH6 - positive cells . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - test ; NS , not significant ; * * p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).


( A ) FKRP - NSCs treated with 4BPPNit showed increased IIH6 reactivity , compared with untreated control cells . Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR - corrected NSCs . Under the same exposure time , the signal intensity of IIH6 reactivity is saturated in corrected - NSCs . Data information : Intensities of glycosylation are normalized to GAPDH protein expression .

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

(A) FKRP-NSCs treated with 4BPPNit showed increased IIH6 reactivity, compared with untreated control cells. Note that the augmented IIH6 reactivity by 4BPPNit is much weaker than the that in CRISPR-corrected NSCs. Under the same exposure time, the signal intensity of IIH6 reactivity is saturated in corrected-NSCs. Data information: Intensities of glycosylation are normalized to GAPDH protein expression.


( B ) Representative immunoblots with the IIH6 antibody using FKRP - and corrected - NSCs treated with 4BPPNit for 24 , 48 and 72 h , and DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time .

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

(B) Representative immunoblots with the IIH6 antibody using FKRP- and corrected-NSCs treated with 4BPPNit for 24, 48 and 72 h, and DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time.


( C ) Quantification of α - dystroglycan glycosylation ( IIH6 ) , compared with DMSO only controls . Data information : Intensities of glycosylation are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of α-dystroglycan glycosylation (IIH6), compared with DMSO only controls. Data information: Intensities of glycosylation are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( D ) Representative immunoblots of laminin - binding analysis using FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( E ) Quantification of laminin - binding activities , compared with DMSO only controls . Data information : Intensities of laminin - binding activity are normalized to GAPDH protein expression . Note that the left and right panels of the immunoblots are not equivalent exposure time . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(D) Representative immunoblots of laminin-binding analysis using FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (E) Quantification of laminin-binding activities, compared with DMSO only controls. Data information: Intensities of laminin-binding activity are normalized to GAPDH protein expression. Note that the left and right panels of the immunoblots are not equivalent exposure time. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( F ) Representative immunoblots with the AF6868 antibody detecting both α - dystroglycan core protein and β - dystroglycan in FKRP - and corrected - NSCs treated with 4BPPNit and DMSO only controls . ( G ) Quantification of α - dystroglycan core protein and β - dystroglycan expression , compared with DMSO only controls . Data information : Intensities of protein expression are normalized to GAPDH protein expression . Values indicate mean ± s . d . ( n = 3 or 4 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

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

(F) Representative immunoblots with the AF6868 antibody detecting both α-dystroglycan core protein and β-dystroglycan in FKRP- and corrected-NSCs treated with 4BPPNit and DMSO only controls. (G) Quantification of α-dystroglycan core protein and β-dystroglycan expression, compared with DMSO only controls.Data information: Intensities of protein expression are normalized to GAPDH protein expression. Values indicate mean ± s.d. (n= 3 or 4 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( A ) DAG1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 .

O O O B-MEASURED_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

(A) DAG1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01


( B ) FKRP gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

O O O B-MEASURED_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

(B) FKRP gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


( C ) LARGE1 gene expression analysis in FKRP - and corrected - NSCs treated with 4BPPNit , compared with DMSO only controls . Data information : Gene expression levels are normalized to ACTB gene expression . Values indicate mean ± s . e . m . ( n = 6 biological replicates ; One - way ANOVA ; NS , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ) .

(C) LARGE1 gene expression analysis in FKRP- and corrected-NSCs treated with 4BPPNit, compared with DMSO only controls. Data information: Gene expression levels are normalized to ACTB gene expression. Values indicate mean ± s.e.m. (n= 6 biological replicates; One-way ANOVA; NS, not significant; * p<0.05; ** p<0.01).


B - D . Mice were analyzed by Kaplan - Meier survival plot ( B ) , the percentage of body weight ( C ) , and the clinical scores ( D ) of WT and GPx8 - / - mice with colitis induced by 4 % DSS for 6 days ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( E ) Images and statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . , 5 - 6 mice were used for each experiment . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections . The infiltrated immune cells at the transmural extensions were indicated by arrowhead . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( G ) Colon sections were stained with markers for macrophages ( F4 / 80 ) and anti - GPx8 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Colons treated with 4 % DSS for 5 days were collected on day 14 . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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

Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing ( n = 5 per group ) and are presented by a heat map , ( I ) , and principal coordinates analysis , ( J ) .

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

Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J).


Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . ( K ) α - diversity indicated bacteria species in each mouse depicted by richness ( total operational taxonomic units , OTUs ) , the Chao1 richness estimator ( Chao1 ) , and the abundance - based coverage estimator ( ACE ) metrics . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 O O O

Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. (K) α-diversity indicated bacteria species in each mouse depicted by richness (total operational taxonomic units, OTUs), the Chao1 richness estimator (Chao1), and the abundance-based coverage estimator (ACE) metrics. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


B . Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages ( F4 / 80 ) .

O O O 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

B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . ( C ) The percentage of body weight . ( D ) The clinical scores of mice . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( E ) Statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( G ) Colon sections stained with anti - F4 / 80 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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 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

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (G) Colon sections stained with anti-F4/80. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . D ) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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 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 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

After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). D) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


A . Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency . BMDMs were primed for 6 h with 0 . 5 μg / ml LPS and then transfected with LPS ( TF LPS ) for 8 h , stimulated with ATP ( 5 mM ) for 3 h , MSU ( 150 μg / mL ) for 8 h , or nigericin ( 5 μM ) for 3 h . Activation of inflammasome was demonstrated by secreted IL - 1β . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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

A. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


B . GPx8 deficiency enhanced noncanonical inflammasome activation . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 O

B. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


C . Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs . BMDMs were primed for 6 h with 1 μg / ml poly ( I : C ) and then transfected with LPS for 8 h . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 O O O O O O O O

C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( D ) Activation of noncanonical inflammasome in BMDMs with empty vector ( EV ) or hGPx8 was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (D) Activation of noncanonical inflammasome in BMDMs with empty vector (EV) or hGPx8 was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( E ) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats .

O O O O O O O O B-CONTROLLED_VAR 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 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

Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (E) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.


F . GPx8 deficiency enhanced caspase - 11 cleavage and activation . Levels of processed caspase - 1 and - 11 in the supernatants ( Sup ) and full - length caspase - 1 , - 11 and GAPDH in the cell lysates ( Cell ext ) were determined by immunoblotting . Fold changes of protein expression were normalized with internal controls and are indicated below the blots .

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

F. GPx8 deficiency enhanced caspase-11 cleavage and activation. Levels of processed caspase-1 and -11 in the supernatants (Sup) and full-length caspase-1, -11 and GAPDH in the cell lysates (Cell ext) were determined by immunoblotting. Fold changes of protein expression were normalized with internal controls and are indicated below the blots.


G . RNA expression of caspase - 11 was not altered by GPx8 deficiency . Caspase - 11 RNA expression of BMDMs isolated from GPx8 - / - mice was measured after LPS priming . Data information : ata are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats .

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

G. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming. Data information: ata are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.


H . GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo . Colon samples from 5 WT or GPx8 - / - mice treated with DSS for 5 days were collected on day 7 . Levels of full - length and processed caspase - 1 , - 11 and gasdermin D ( GSDMD ) in tissue lysates were individually analyzed by immunoblots .

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

H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( I ) Survival of mice primed with ultra pure LPS ( E . coli O55 : B5 , 400 μg / kg ) and re - challenged 6 h later with LPS ( 40 mg / kg ) ( n = 8 per group ) .

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

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group).


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( J ) Survival of mice primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 10 mg / kg ) ( n = 9 per group ) .

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

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group).


GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( K ) IL - 1β production levels in mice sera were measured 2 h after being primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 1 mg / kg ) ( n = 8 - 9 per group ) . In ( K ) , data are presented as the mean ± SEM . NS , no significance . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group). In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


A . Interaction of GPx8 with human caspase - 4 ( Casp4 ) or mouse caspase - 11 ( Casp11 ) was confirmed by co - IP assays . 293T cells were co - transfected with vectors expressing FLAG tagged Casp4 or Casp11 , in addition to GPx8 expressing vectors . Cell lysate containing Casp4 - or Casp11 - FLAG proteins was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 and anti - FLAG Ab ( left panel ) . GPx8 and its interacting proteins were precipitated by anti - GPx8 Ab and analyzed by the indicated Abs ( right panel ) .

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

A. Interaction of GPx8 with human caspase-4 (Casp4) or mouse caspase-11 (Casp11) was confirmed by co-IP assays. 293T cells were co-transfected with vectors expressing FLAG tagged Casp4 or Casp11, in addition to GPx8 expressing vectors. Cell lysate containing Casp4- or Casp11-FLAG proteins was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 and anti-FLAG Ab (left panel). GPx8 and its interacting proteins were precipitated by anti-GPx8 Ab and analyzed by the indicated Abs (right panel).


B . Co - localization of Myc - tagged GPx8 ( red ) and endogenous caspase - 4 ( green ) in THP - 1 cells .

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

B. Co-localization of Myc-tagged GPx8 (red) and endogenous caspase-4 (green) in THP-1 cells.


C . Interaction between GPx8 and caspase - 4 in THP - 1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay ( PLA ) ( red dots , labeled by arrowheads ) . GPx8 - Myc stably expressing THP - 1 were used to detect the interaction between GPx8 - Myc and endogenous caspase - 4 . Untreated cells ( Ctrl ) or cells primed with LPS ( primed LPS ) were incubated with anti - Myc and anti - caspase - 4 Abs , according to the manufacturer ' s instructions , and the nucleus was stained with DAPI ( blue ) . Results were quantified by counting at least 5 different fields with an average of 300 cells . Reactions without primary Abs were used as the control for the PLA assay ( PLA Ctrl ) . Data information : All data are representative of at least 3 independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 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 O O O O O O O O O O B-MEASURED_VAR 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( D ) Complexes of GPx8 and caspase - 4 were demonstrated by co - IP assays . Cysteine - dependent covalent interactions of GPx8 and caspase - 4 ( C258S ) were analyzed under non - reducing and reducing conditions . Disulfide - linked complexes of Gpx8 and caspase - 4 are indicated .

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

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( E ) Interactions of GPx8 and caspase - 4 in response to H2O2 treatment were demonstrated by co - IP assays of lysates prepared from 293T cells treated with H2O2 ( 200 μM ) .

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

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).


293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( F ) C79S , C108S , and C2S2 mutants of GPx8 abolished most of the binding to caspase - 4 by co - IP assays .

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

293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (F) C79S, C108S, and C2S2 mutants of GPx8 abolished most of the binding to caspase-4 by co-IP assays.


C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . THP - 1 cells stably expressing empty vector ( EV ) , WT , or mutant GPx8 proteins were confirmed by immunoblotting ( G ) .

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-CONTROLLED_VAR O O O O O O O O O

C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. THP-1 cells stably expressing empty vector (EV), WT, or mutant GPx8 proteins were confirmed by immunoblotting (G).


C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . ( H and I ) Cells were treated with 12 - O - tetradecanoylphorbol 13 - actate ( PMA ) ( 0 . 1 μM ) and differentiated for 3 days then primed and transfected with LPS . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( H ) and LDH release ( I ) . Data information : All data are representative of at least 3 independent experiments . In H and I , data are presented as the mean ± SD , n = 4 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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 B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


J . E . coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase - 4 ( C258S ) to form a disulfide - linked complex ( oxidized GPx8 : oGPx8 ; reduced GPx8 : rGPx8 ) . Lanes 2 and 3 , GPx8 pretreated with 1 and 5 mM H2O2 , respectively . Lanes 4 and 5 , Gpx8 pretreated with 50 and 200 mM DTT , respectively . Disulfide - linked complexes of recombinant Gpx8 and caspase - 4 ( C258S ) are indicated .

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

J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.


K . GPx8 inhibited the activation of caspase - 4 by decreasing the oligomerization of caspase - 4 . Oxidized GPx8 and caspase - 4 ( C258S ) were co - incubated for 30 min . Different amounts of LPS were added to the complex and incubated for another 30 min . Oligomerized caspase - 4 was analyzed by native blue PAGE according to the manufacturer ' s instructions . Mock : without GPx8 ; oxidized GPx8 : oGPx8 ; oxidized GPx8C79S : oC79S . Data information : All data are representative of at least 3 independent experiments . In K , quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers / total proteins . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


A . Casp4C118S mutant partially abolished the covalent disulfide - link with GPx8 . Interacting GPx8 were co - immunoprecipitated with FLAG - tagged caspase - 4 ( WT , C118S , C329S mutants ) and analyzed under reducing ( + DTT ) or non - reducing ( - DTT ) conditions . Disulfide - linked complexes of GPx8 and caspase - 4 are indicated . Fold change of complexes was normalized with internal controls and indicated below blots .

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

A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.