E ) Results of GSEA using combined ATF - 1 gene sets identified in B across treatment groups versus tumor - bearing control ( cachexia ) transcriptomes .

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E) Results of GSEA using combined ATF-1 gene sets identified in B across treatment groups versus tumor-bearing control (cachexia) transcriptomes.


Tumor - free control ( black checkered ) , GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) . F ) Heat map of DEGs within the combined ATF - 1 gene sets ( mean z score ) . Treatment groups are as in D .

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

Tumor-free control (black checkered), GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green). F) Heat map of DEGs within the combined ATF-1 gene sets (mean z score). Treatment groups are as in D.


G ) mRNA expression of mediators of IL - 6 signaling upstream of STAT3 . Data presented as mean ± SD of per animal log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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

G) mRNA expression of mediators of IL-6 signaling upstream of STAT3. Data presented as mean±SD of per animal log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


A ) Volcano plot from RNA - seq analyses of Study 1 gastrocnemius muscles for tumor - bearing combination - treated mice versus tumor - bearing controls . Genes not differentially expressed in this comparison are indicated with blue . The remaining genes ( red and green ) are DEGs in the combination - treated versus tumor - bearing control comparison . The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR - 42 - treated tumor - bearing mice versus tumor - bearing controls , suggesting these genes are responsive to only the combination therapy . Log2 - transformed fold change ( FC ) in expression is plotted on the x - axis and - log10 transformed Benjamini - Hochburg adjusted p - values are plotted on the y - axis .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Volcano plot from RNA-seq analyses of Study 1 gastrocnemius muscles for tumor-bearing combination-treated mice versus tumor-bearing controls. Genes not differentially expressed in this comparison are indicated with blue. The remaining genes (red and green) are DEGs in the combination-treated versus tumor-bearing control comparison. The green coloring indicates the subset of these DEGs that are not also differentially expressed in the comparison of AR-42-treated tumor-bearing mice versus tumor-bearing controls, suggesting these genes are responsive to only the combination therapy. Log2-transformed fold change (FC) in expression is plotted on the x-axis and -log10 transformed Benjamini-Hochburg adjusted p-values are plotted on the y-axis.


C ) Significance values from GSEA using combined CTNNB1 gene sets . Each treatment group was compared to tumor - free control transcriptomes . * FDR < 1e - 5 determined , set to 1e - 5 for plot .

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

C) Significance values from GSEA using combined CTNNB1 gene sets. Each treatment group was compared to tumor-free control transcriptomes. *FDR < 1e-5 determined, set to 1e-5 for plot.


D ) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor - free control comparisons . GTx - 024 - treated tumor - free ( blue checkered ) , tumor - bearing control ( black ) , GTx - 024 - treated tumor - bearing ( blue ) , AR - 42 - treated tumor - bearing ( red ) and Combination - treated tumor - bearing ( green ) .

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

D) Enrichment plots from GSEA of the CTNNB1 gene set for each treatment group versus tumor-free control comparisons. GTx-024-treated tumor-free (blue checkered), tumor-bearing control (black), GTx-024-treated tumor-bearing (blue), AR-42-treated tumor-bearing (red) and Combination-treated tumor-bearing (green).


E ) mRNA expression of WNT effectors upstream of β - catenin . Data are presented as mean ± SD of log - transformed fold change ( log2FC ) values versus tumor - free controls . Groups : Tumor - free control ( n = 6 ) , tumor - free GTx - 024 treated ( n = 5 ) , tumor - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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

E) mRNA expression of WNT effectors upstream of β-catenin. Data are presented as mean±SD of log-transformed fold change (log2FC) values versus tumor-free controls. Groups: Tumor-free control (n=6), tumor-free GTx-024 treated (n=5), tumor-bearing receiving vehicle (n=4), GTx-024 (n=5), AR-42 (n=5) or Combination (n=6).


A) Neutrophils and lymphocytes count.


B ) Levels of creatine kinase ( CK ) and creatinine .

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B) Levels of creatine kinase (CK) and creatinine.


C ) Levels of procalcitonin ( PCT ) and C - reactive protein ( CRP ) .

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C) Levels of procalcitonin (PCT) and C-reactive protein (CRP).


D ) Levels of erythrocyte sedimentation rate ( ESR ) and ferritin .

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D) Levels of erythrocyte sedimentation rate (ESR) and ferritin.


E ) Levels of D - dimer and lactate dehydrogenase ( LDH ) .

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E) Levels of D-dimer and lactate dehydrogenase (LDH).


F ) Proportion of total CD3 + and CD4 + T cells .

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F) Proportion of total CD3+ and CD4+ T cells.


G ) Proportion of CD8 + T cells and CD4 + / CD8 + T cell ratio .

G) Proportion of CD8+ T cells and CD4+/CD8+ T cell ratio.


H ) Proportion of B and natural killer ( NK ) cells .

H) Proportion of B and natural killer (NK) cells.


cytokine profile of COVID - 19 patients for IL - 2 , IL - 4 ( I )

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cytokine profile of COVID-19 patients for IL-2, IL-4 (I)


cytokine profile of COVID - 19 patients for , IL - 6 , IL - 10 ( J ) and TNF - α and IFN - γ ( K ) .

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cytokine profile of COVID-19 patients for , IL-6, IL-10 (J) and TNF-α and IFN-γ (K).


Time from symptom onset to initial diagnosis ( TFSD ) .

Time from symptom onset to initial diagnosis (TFSD).


Time from symptom onset to development of pneumonia ( TFSP ) .

Time from symptom onset to development of pneumonia (TFSP).


Time from symptom onset to treatment ( TFST ) .

Time from symptom onset to treatment (TFST).


Time from development of pneumonia to recovery ( TFPR ) .

Time from development of pneumonia to recovery (TFPR).


Correlation between IL - 6 and maximal body temperature during hospitalization .

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Correlation between IL-6 and maximal body temperature during hospitalization.


Correlation between IL - 6 and C - reactive protein ( CRP ) .

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Correlation between IL-6 and C-reactive protein (CRP).


Correlation between IL - 6 and lactate dehydrogenase ( LDH ) .

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Correlation between IL-6 and lactate dehydrogenase (LDH).


Correlation between IL - 6 and D - dimer .

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Correlation between IL-6 and D-dimer.


Correlation between IL - 6 and ferritin .

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Correlation between IL-6 and ferritin.


Levels of baseline IL - 6 in patients who received glucocorticoids , human immunoglobulin , high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not . Error bars , SEM .

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Levels of baseline IL-6 in patients who received glucocorticoids, human immunoglobulin, high flow oxygen inhalation or mechanical ventilation during hospitalization versus patients who did not. Error bars, SEM.


IL - 6 levels before and after treatment in three patient groups classified according to baseline IL - 6 levels of ≥ 40 pg / mL ( n = 11 ) , ≥ 20 pg / mL ( n = 6 ) or < 20 pg / mL ( n = 9 ) ( left ) . Two representative chest computed tomography ( CT ) scans before and after treatment from each group ( right ) .

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IL-6 levels before and after treatment in three patient groups classified according to baseline IL-6 levels of ≥ 40 pg/mL (n = 11), ≥ 20 pg/mL (n = 6) or < 20 pg/mL (n = 9) (left). Two representative chest computed tomography (CT) scans before and after treatment from each group (right).


IL - 6 levels at diagnosis and after disease progression in three exacerbated patients ( left ) . Progressed radiological findings were recorded in two patients ( right ) , while radiological assessment after treatment was not performed in the third patient due to poor general condition .

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IL-6 levels at diagnosis and after disease progression in three exacerbated patients (left). Progressed radiological findings were recorded in two patients (right), while radiological assessment after treatment was not performed in the third patient due to poor general condition.


The baseline IL - 6 level was 197 . 39 pg / mL in a 69 - year - old female patient who showed high fever and dyspnea . IL - 6 decreased to 9 . 47 pg / mL after treatment ( day 8 ) , while the symptoms were not relieved . The C - reactive protein ( CRP ) rebounded and procalcitonin ( PCT ) increased together with disease exacerbation . Follow - up chest computed tomography ( CT ) assessment was not performed due to poor general condition , whereas chest X - ray showed aggravated pneumonia . Follow - up sputum culture confirmed the exacerbation was caused by bacterial infection .

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

The baseline IL-6 level was 197.39 pg/mL in a 69-year-old female patient who showed high fever and dyspnea. IL-6 decreased to 9.47 pg/mL after treatment (day 8), while the symptoms were not relieved. The C-reactive protein (CRP) rebounded and procalcitonin (PCT) increased together with disease exacerbation. Follow-up chest computed tomography (CT) assessment was not performed due to poor general condition, whereas chest X-ray showed aggravated pneumonia. Follow-up sputum culture confirmed the exacerbation was caused by bacterial infection.


A ) The chest computed tomography ( CT ) was normal five days after symptom onset . Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities . Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset .

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

A) The chest computed tomography (CT) was normal five days after symptom onset. Disease aggravation was evidenced by progressed chest CT images ten days after symptom onset displaying bilateral multiple patchy ground glass opacities. Disease alleviation was evidenced by improved chest CT images 25 days after symptom onset.


B ) Procalcitonin ( PCT ) levels stayed within normal range throughout the course of disease .

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B) Procalcitonin (PCT) levels stayed within normal range throughout the course of disease.


C ) Lymphocyte count was normal five days after symptom onset , while firstly decreasing then returning to normal range during the course of disease .

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

C) Lymphocyte count was normal five days after symptom onset, while firstly decreasing then returning to normal range during the course of disease.


Both abnormal C - reactive protein ( CRP ) ( D ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset .

O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O

Both abnormal C-reactive protein (CRP) (D) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, CRP further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.


IL - 6 ( E ) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal . In association with changes in chest CT scans , IL - 6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset .

B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-TISSUE B-EXP_ASSAY O B-CELL_TYPE B-EXP_ASSAY O O O O O O O O O B-TISSUE B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O

IL-6 (E) were detected five days after symptom onset when the chest CT and lymphocyte count were still normal. In association with changes in chest CT scans, IL-6 further increased ten days after symptom onset and returned to normal range 25 days after symptom onset.


F ) The body temperature was elevated when CRP and IL - 6 levels were rising , but returned to normal when CRP and IL - 6 . Levels were declining .

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

F) The body temperature was elevated when CRP and IL-6 levels were rising, but returned to normal when CRP and IL-6. Levels were declining.


RAW 264 . 7 mouse macrophages were treated with either oxLDL ( 50 µg / ml ) or TG ( 300 nM ) for 6 hours . Protein lysates were treated with λ Phosphatase ( PPase ) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 6 biological replicates ) .

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

RAW 264.7 mouse macrophages were treated with either oxLDL (50 µg/ml) or TG (300 nM) for 6 hours. Protein lysates were treated with λ Phosphatase (PPase) for 30 minutes and analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=6 biological replicates).


Control - or IRE1 - siRNA transfected HEK293T cells were stimulated by either PA ( 500 µM ) or TG ( 600 nM ) for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) .

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

Control- or IRE1-siRNA transfected HEK293T cells were stimulated by either PA (500 µM) or TG (600 nM) for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).


MEF cells were transfected with either empty vector , EGFP - FMRP or 3xFLAG - IRE1 plasmids then pre - treated either with vehicle ( dimethyl sulfoxide , DMSO ) or AMG - 18 ( 25 µM ; 1 hour ) followed by TG ( 600 nM ) stimulation for 4 hours . Protein lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 and β - Actin . pFMRP / FMRP fold induction is depicted above the blots ( n = 4 biological replicates ) .

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

MEF cells were transfected with either empty vector, EGFP-FMRP or 3xFLAG-IRE1 plasmids then pre-treated either with vehicle (dimethyl sulfoxide, DMSO) or AMG-18 (25 µM; 1 hour) followed by TG (600 nM) stimulation for 4 hours. Protein lysates were analyzed by western blotting using specific antibodies for pFMRP, FMRP, pIRE1, IRE1 and β-Actin. pFMRP/FMRP fold induction is depicted above the blots (n=4 biological replicates).


Purified FMRP and IRE1 kinase ( activated ) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP , IRE1 and FMRP ( n = 3 biological replicates ) and with LC - MS / MS . Identified IRE1 kinase - mediated FMRP phosphorylation sites ( bottom ) .

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

Purified FMRP and IRE1 kinase (activated) proteins were subjected to kinase assay and analyzed by western blotting using specific antibodies for ThioP, IRE1 and FMRP (n=3 biological replicates) and with LC-MS/MS. Identified IRE1 kinase-mediated FMRP phosphorylation sites (bottom).


Fmr1 + / + and Fmr1 - / - mice were injected with AAV _ PCSK9 and fed with 16 weeks of WD . Residential PM were stained with Oil Red O ( ORO ) and imaged ( n = 7 mice per group ; Scale bar = 50 µm ) . Apoe - / - mice were fed with WD ( 12 weeks ) and injected with vehicle ( DMSO ) or AMG - 18 ( 30 mg / kg / day ) in the last 4 weeks of WD . Residential PM were stained with ORO and imaged ( n = 5 mice per group ; Scale bar = 50 µm ) .

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

Fmr1+/+ and Fmr1-/- mice were injected with AAV_PCSK9 and fed with 16 weeks of WD. Residential PM were stained with Oil Red O (ORO) and imaged (n=7 mice per group; Scale bar = 50 µm).    Apoe-/- mice were fed with WD (12 weeks) and injected with vehicle (DMSO) or AMG-18 (30 mg/kg/day) in the last 4 weeks of WD. Residential PM were stained with ORO and imaged (n=5 mice per group; Scale bar = 50 µm).


RCT experiment ( I ) plasma cholesterol levels after 24 and 48 hours , ( J ) liver cholesterol levels after 48 hours , and ( K ) feces cholesterol levels after 48 hours ( n = 12 mice per group ) .

B-EXP_ASSAY O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O O B-TISSUE B-SMALL_MOLECULE O O O O O O O O B-ORGANISM O O O O

RCT experiment (I) plasma cholesterol levels after 24 and 48 hours, (J) liver cholesterol levels after 48 hours, and (K) feces cholesterol levels after 48 hours (n=12 mice per group).


A - E In vitro and in vivo efferocytosis experiments , where percentage of macrophages F4 / 80 + ( red ) that ingested apoptotic cells ( AC ) labeled with carboxyfluorescein succinimidyl ester ( CFSE ) + ( green ) were reported as % efferocytosis . ( A ) BMDMs were transfected with Fmr1 - or control - siRNA and incubated CFSE - labeled AC for the indicated hours ( n = 4 biological replicates ) . ( B ) Fmr1 + / + and Fmr1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours and then incubated with CFSE - labeled ACs for 4 hours ( n = 4 biological replicates ) . ( C ) Fmr1 + / + and Fmr1 - / - mice were fed WD ( 16 weeks ) and injected intraperitoneally with CFSE - labeled AC ( 1 . 5 hours ) , followed by PM elicitation ( n = 4 - 5 mice per group ) . ( D ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour then incubated with CFSE - labeled ACs for 4 hours ( n = 3 biological replicates ) . ( E ) C57BL / 6 mice were injected with AMG - 18 ( 30 mg / kg ) or vehicle ( DMSO ) for 8 hours , followed by intraperitoneal injection with CFSE - labeled ACs for 1 . 5 hours and PM elicitation ( n = 4 mice per group ) .

O O O O O O O O O O O O O O B-CELL_TYPE B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_TYPE O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE O O O O O O O B-ORGANISM O O O O O O O B-CELL_TYPE O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-CELL_TYPE O O O O O B-ORGANISM O O O O

A-E In vitro and in vivo efferocytosis experiments, where percentage of macrophages F4/80+ (red) that ingested apoptotic cells (AC) labeled with carboxyfluorescein succinimidyl ester (CFSE)+ (green) were reported as % efferocytosis. (A) BMDMs were transfected with Fmr1- or control-siRNA and incubated CFSE-labeled AC for the indicated hours (n=4 biological replicates). (B) Fmr1+/+ and Fmr1-/- BMDMs were treated with PA (500 µM) for 6 hours and then incubated with CFSE-labeled ACs for 4 hours (n=4 biological replicates). (C) Fmr1+/+ and Fmr1-/- mice were fed WD (16 weeks) and injected intraperitoneally with CFSE-labeled AC (1.5 hours), followed by PM elicitation (n=4-5 mice per group). (D) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour then incubated with CFSE-labeled ACs for 4 hours (n=3 biological replicates). (E) C57BL/6 mice were injected with AMG-18 (30 mg/kg) or vehicle (DMSO) for 8 hours, followed by intraperitoneal injection with CFSE-labeled ACs for 1.5 hours and PM elicitation (n=4 mice per group).


F - G In vitro continuous efferocytosis experiments , where macrophages were stained for F4 / 80 + ( red ) , AC were labeled with CFSE ( AC - 1 ; green ) or Violet ( AC2 ; violet ) . % continuous efferocytosis was determined by the ratio of F4 / 80 + , CFSE + and Violet + ( triple positive ) cells to total F4 / 80 + and CFSE + ( double positive ) cells . ( F ) Fmr1 + / + and Fmr1 - / - BMDM were incubated with AC - 1 for 2 hours , and after 2 hours interval , incubated with AC - 2 for 2 more hours ( n = 4 - 3 biological replicates ) . ( G ) BMDM were pre - treated either with vehicle ( DMSO ) or AMG - 18 ( 5 µM ) for 1 hour , incubated with CFSE - labeled AC - 1 for 2 hours , followed by incubation with Violet - labeled AC - 2 for 2 hours and PM collection ( n = 4 biological replicates ) .

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

F-G In vitro continuous efferocytosis experiments, where macrophages were stained for F4/80+ (red), AC were labeled with CFSE (AC-1; green) or Violet (AC2; violet). % continuous efferocytosis was determined by the ratio of F4/80+, CFSE+ and Violet+ (triple positive) cells to total F4/80+ and CFSE+ (double positive) cells. (F) Fmr1+/+ and Fmr1-/- BMDM were incubated with AC-1 for 2 hours, and after 2 hours interval, incubated with AC-2 for 2 more hours (n=4-3 biological replicates). (G) BMDM were pre-treated either with vehicle (DMSO) or AMG-18 (5 µM) for 1 hour, incubated with CFSE-labeled AC-1 for 2 hours, followed by incubation with Violet-labeled AC-2 for 2 hours and PM collection (n=4 biological replicates).


RNA lysates from Fmr1 + / + and Fmr1 - / - BMDM that were treated with PA ( 500 µM ; 6 hours ) were fractionated using a 10 % - 50 % sucrose gradient and separated to polysome , monosome / NTR fractions . The absorbance ( 260 nm ) of RNA was measured and plotted as a function of time ( n = 3 biological replicates ) . ( B ) The ratio of the Abca1 , Abcg1 , Mertk , Lrp1 , Cd36 , Cd47 and Rac1 mRNA in polysome to NTR fraction ( n = 3 biological replicates ) .

O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O

RNA lysates from Fmr1+/+ and Fmr1-/- BMDM that were treated with PA (500 µM; 6 hours) were fractionated using a 10%-50% sucrose gradient and separated to polysome, monosome/NTR fractions. The absorbance (260 nm) of RNA was measured and plotted as a function of time (n=3 biological replicates). (B) The ratio of the Abca1, Abcg1, Mertk, Lrp1, Cd36, Cd47 and Rac1 mRNA in polysome to NTR fraction (n=3 biological replicates).


D Fmr1 - / - MEF cells were transfected with EV , WT - FMRP or STSA - FMRP plasmids followed by PA treatment ( 500 µM ; 6 hours ) . Protein lysates were analyzed by western blotting using specific antibodies for ABCA1 , MerTK , LRP1 , pFMRP , FMRP and β - Actin and fold inductions relative to β - Actin are depicted above the blots ( n = 5 biological replicates ) .

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

D Fmr1-/- MEF cells were transfected with EV, WT-FMRP or STSA-FMRP plasmids followed by PA treatment (500 µM; 6 hours). Protein lysates were analyzed by western blotting using specific antibodies for ABCA1, MerTK, LRP1, pFMRP, FMRP and β-Actin and fold inductions relative to β-Actin are depicted above the blots (n=5 biological replicates).


Lesion area calculated from en face aorta , stained with ORO ( n = 12 - 13 mice per group ; Scale bar = 5 mm ) .

O O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O B-ORGANISM O O O O O O O O O O

Lesion area calculated from en face aorta, stained with ORO (n=12-13 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 9 mice per group ; Scale bar = 5 mm ) .

O O O O O B-TISSUE I-TISSUE O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O

Lesion area calculated from en face aorta, stained with ORO (n=9 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 5 mice per group ; Scale bar = 5 mm ) .

Lesion area calculated from en face aorta, stained with ORO (n=5 mice per group; Scale bar = 5 mm).


Lesion area calculated from en face aorta , stained with ORO ( n = 6 mice per group ; Scale bar = 5 mm ) .

Lesion area calculated from en face aorta, stained with ORO (n=6 mice per group; Scale bar = 5 mm).


( C ) Competition of sybodies and ACE2 for spike protein binding , investigated by GCI . Biotinylated spike protein was immobilized on the GCI sensor and then Sb # 15 ( 200 nM , left ) , or Sb # 68 ( 200 nM , right ) were injected alone or premixed with human ACE2 ( 100 nM ) . Sb # 0 represents a non - randomized control sybody .

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

(C) Competition of sybodies and ACE2 for spike protein binding, investigated by GCI. Biotinylated spike protein was immobilized on the GCI sensor and then Sb#15 (200 nM, left), or Sb#68 (200 nM, right) were injected alone or premixed with human ACE2 (100 nM). Sb#0 represents a non-randomized control sybody.


Neutralization assays using VSVΔG pseudotyped with wildtype SARS - CoV - 2 spike protein . Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs . ( A ) Sb # 15 , Sb # 68 , or an equimolar mixure of both sybodies . ( B ) Sb # 14 , Sb # 16 , Sb # 45 , or , as a positive control , the previously - described sybody MR3 . ( C ) Neutralization by bivalent sybody - Fc fusions . Error bars correspond to standard deviations of three biological replicates .

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

Neutralization assays using VSVΔG pseudotyped with wildtype SARS-CoV-2 spike protein. Panels show relative infectivity upon exposure to increasing concentrations of the indicated sybody constructs. (A) Sb#15, Sb#68, or an equimolar mixure of both sybodies. (B) Sb#14, Sb#16, Sb#45, or, as a positive control, the previously-described sybody MR3. (C) Neutralization by bivalent sybody-Fc fusions. Error bars correspond to standard deviations of three biological replicates.


( C ) Neutralization assay using VSVΔG pseudotyped with wildtype Spike - protein . Relative infectivity was determined in response to increasing GS4 or Tripod - GS4r concentration . The corresponding neutralization data for isolated Sb # 15 and Sb # 68 are provided as reference . ( D ) Control experiment using VSVΔG pseudotyped with VSV - G . Infectivity was not affected by Tripod - GS4r .

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

(C) Neutralization assay using VSVΔG pseudotyped with wildtype Spike-protein. Relative infectivity was determined in response to increasing GS4 or Tripod-GS4r concentration. The corresponding neutralization data for isolated Sb#15 and Sb#68 are provided as reference. (D) Control experiment using VSVΔG pseudotyped with VSV-G. Infectivity was not affected by Tripod-GS4r.


( C ) Neutralization assay using VSVΔG pseudotyped with Spike - protein containing the Q493R or P384H mutation , respectively . Relative infectivity in response to increasing binder concentrations was determined .

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

(C) Neutralization assay using VSVΔG pseudotyped with Spike-protein containing the Q493R or P384H mutation, respectively. Relative infectivity in response to increasing binder concentrations was determined.


( A ) GCI - based kinetic analysis of interactions between immobilized sybodies ( indicated above each plot ) and wild - type ( WT ) or mutant RBDs carrying the individual K417N , E484K or N501Y mutations ( left panels ) , the combined triple KEN ( Beta ) mutations ( middle panels ) , or the double L452R / T478K ( Delta ) mutations ( right panels ) . Sb # 15 , Sb # 68 and GS4 were immobilized on independent flow - cells via biotinylated Avi - tags , and the RBD variants were sequentially injected at a concentration of 200 nM .

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

(A) GCI-based kinetic analysis of interactions between immobilized sybodies (indicated above each plot) and wild-type (WT) or mutant RBDs carrying the individual K417N, E484K or N501Y mutations (left panels), the combined triple KEN (Beta) mutations (middle panels), or the double L452R/T478K (Delta) mutations (right panels). Sb#15, Sb#68 and GS4 were immobilized on independent flow-cells via biotinylated Avi-tags, and the RBD variants were sequentially injected at a concentration of 200 nM.


A . Growth curves of the library ( black ) compared to two different controls under increasing selective pressures . DSB ( double - stranded break ) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK . n = 3 for each

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

A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve


B . Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non - Target gRNA . n = 3 . C . Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non - Target gRNA . n =

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

B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3


D . Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants . Quantification was performed using LC - MS , as described in the methods section . n = 3 . A two - sample student t - test assuming unequal variances was performed to calculate statistical significance . Concentrations are reported as fold - change relative to the wild - type control

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

D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samples


E . DapF assay showing kinetics of the wild type , G210D and M260Y mutants . Assay was performed as described in the methods section . n =

O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O

E. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5


F . Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT , DapF G210D and DapF M260Y

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

F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds


C . Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

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

C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


F . Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

F. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


A Representative images of E18 . 5 coronal cortical sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmids encoding EGFP , EGFP + Cre ( Cre ) or EGFP + Cre + KCC2FL ( Cre + KCC2FL ) . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . B Quantification of the number of EGFP + neurons / ROI from embryos electroporated with constructs in ( A ) . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 . Data are presented as mean ± S . E . M . , n ( EGFP ) = 8 embryos ; n ( Cre ) = 8 embryos ; n ( Cre + KCC2FL ) = 13 embryos .

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

A Representative images of E18.5 coronal cortical sections from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmids encoding EGFP, EGFP+Cre (Cre) or EGFP+Cre+KCC2FL (Cre+KCC2FL). DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. B Quantification of the number of EGFP+ neurons/ROI from embryos electroporated with constructs in (A). Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01. Data are presented as mean ± S.E.M., n (EGFP) = 8 embryos; n (Cre) = 8 embryos; n (Cre+KCC2FL) = 13 embryos.


C Representative image of cleaved Caspase 3 ( Cas - 3 , upper panel ) and TUNEL ( lower panel ) staining in coronal sections from Kcc2lox / lox cortex at E16 . 5 electroporated with EGFP or EGFP + Cre are shown . Arrowheads point to neurons expressing Cas - 3 ( upper panel ) and TUNEL ( lower panel ) . DAPI staining ( blue ) marks cell nuclei . CP , cortical plate ; VZ , ventricular zone ; SVZ , subventricular zone ; Sp , subplate ; IZ , intermediate zone . Large scale bar : 50 µm , small scale bar : 20 µm . D Quantification of the percentage of EGFP + neurons expressing apoptotic markers at E16 . 5 from embryos electroporated with EGFP ± Cre . Statistical significance was determined using Mann - Whitney U test ( Cas - 3 ) ; and two - tailed t test ( TUNEL ) , * * * P < 0 . 001 . Data are presented as mean ± S . E . M . , n ( - Cre ) = 6 embryos ; n ( + Cre ) = 6 embryos . E The number of EGFP + Cre neurons as a percentage of neurons expressing EGFP alone . Statistical significance was determined using a two - tailed Student ' s t test . Data are presented as mean ± S . E . M . , n = 6 embryos .

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

C Representative image of cleaved Caspase 3 (Cas-3, upper panel) and TUNEL (lower panel) staining in coronal sections from Kcc2lox/lox cortex at E16.5 electroporated with EGFP or EGFP+Cre are shown. Arrowheads point to neurons expressing Cas-3 (upper panel) and TUNEL (lower panel). DAPI staining (blue) marks cell nuclei. CP, cortical plate; VZ, ventricular zone; SVZ, subventricular zone; Sp, subplate; IZ, intermediate zone. Large scale bar: 50 µm, small scale bar: 20 µm. D Quantification of the percentage of EGFP+ neurons expressing apoptotic markers at E16.5 from embryos electroporated with EGFP ± Cre. Statistical significance was determined using Mann-Whitney U test (Cas-3); and two-tailed t test (TUNEL), ***P < 0.001. Data are presented as mean ± S.E.M., n (-Cre) = 6 embryos; n (+Cre) = 6 embryos. E The number of EGFP+Cre neurons as a percentage of neurons expressing EGFP alone. Statistical significance was determined using a two-tailed Student's t test. Data are presented as mean ± S.E.M., n = 6 embryos.


A Top : Representative images of E18 . 5 coronal cortical brain sections from Kcc2lox / lox embryos electroporated in utero at E14 . 5 together with plasmid constructs encoding Cre , a fluorescent marker ( EGFP or mRFP , both pseudo - colored in green ) and one of the following constructs : KCC2ΔNTD , KCC2CTD , KCC2R952H , or cofilinS3A . DAPI staining ( blue ) marks cell nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . Bottom : schematic representation of the KCC2 constructs . B Quantification of the number of transfected neurons / ROI from embryos electroporated with constructs in ( A ) . The mean number of transfected neurons from embryos electroporated with Cre + KCC2FL taken from Fig 1B shown as dotted line . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Cre + KCC2FL . Data are presented as mean ± S . E . M . , n ( KCC2ΔNTD ) = 7 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos ; n ( Cre + cofilinS3A ) = 8 embryos .

O O O O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O B-GENEPROD O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE I-TISSUE I-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-CELL_TYPE O O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O B-CELL_TYPE O B-ORGANISM B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O

A Top: Representative images of E18.5 coronal cortical brain sections from Kcc2lox/lox embryos electroporated in utero at E14.5 together with plasmid constructs encoding Cre, a fluorescent marker (EGFP or mRFP, both pseudo-colored in green) and one of the following constructs: KCC2ΔNTD, KCC2CTD, KCC2R952H, or cofilinS3A. DAPI staining (blue) marks cell nuclei. UCP, upper cortical plate; LCP, lower cortical plate; Sp, subplate; IZ, intermediate zone. Scale bar: 50 µm. Bottom: schematic representation of the KCC2 constructs. B Quantification of the number of transfected neurons/ROI from embryos electroporated with constructs in (A). The mean number of transfected neurons from embryos electroporated with Cre+KCC2FL taken from Fig 1B shown as dotted line. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Cre+KCC2FL. Data are presented as mean ± S.E.M., n (KCC2ΔNTD) = 7 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos; n (Cre+cofilinS3A) = 8 embryos.


A Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding EGFP or EGFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . EGFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . B Number of EGFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to EGFP . Data are presented as mean ± S . E . M . , n ( EGFP + Cre ) = 8 embryos ; n ( EGFP ) = 8 embryos .

O O B-EXP_ASSAY O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O B-ORGANISM O

A Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding EGFP or EGFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. EGFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. B Number of EGFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with EGFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to EGFP. Data are presented as mean ± S.E.M., n (EGFP +Cre) = 8 embryos; n (EGFP) = 8 embryos.


C Representative image of E18 . 5 coronal brain sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos electroporated in utero at E14 . 5 with plasmid constructs encoding mRFP or mRFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . mRFP signal is shown as green pseudocolor . DAPI staining ( blue ) marks cell nuclei . D Number of mRFP + Cre neurons migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * * P < 0 . 001 to mRFP . Data are presented as mean ± S . E . M . , n ( mRFP + Cre ) = 5 embryos ; n ( mRFP ) = 5 embryos .

O O B-EXP_ASSAY O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD B-CELL_TYPE O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O O O B-ORGANISM O

C Representative image of E18.5 coronal brain sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos electroporated in utero at E14.5 with plasmid constructs encoding mRFP or mRFP+Cre (Cre). Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. mRFP signal is shown as green pseudocolor. DAPI staining (blue) marks cell nuclei. D Number of mRFP+Cre neurons migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective data from embryos electroporated with mRFP alone. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, ***P < 0.001 to mRFP. Data are presented as mean ± S.E.M., n (mRFP+Cre) = 5 embryos; n (mRFP) = 5 embryos.


E Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) from Kcc2lox / lox embryos co - electroporated in utero at E14 . 5 with plasmids encoding a fluorescent marker ( green ) together and Cre + KCC2FL , Cre + KCC2CTD , or Cre + KCC2R952H . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . F Number of transfected neurons migrating above ( II - IV , left ) and below ( V - VI / IZ - SVZ , right ) the upper border of layer V in embryos electroporated with constructs in ( E ) normalized to respective data from embryos electroporated with Cre . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 to Cre . Data are presented as mean ± S . E . M . , n ( Cre + KCC2FL ) = 13 embryos ; n ( Cre + KCC2CTD ) = 9 embryos ; n ( Cre + KCC2R952H ) = 9 embryos .

O O B-EXP_ASSAY O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O O O B-TISSUE I-TISSUE O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O O O B-GENEPROD O B-GENEPROD O O O B-ORGANISM O

E Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) from Kcc2lox/lox embryos co-electroporated in utero at E14.5 with plasmids encoding a fluorescent marker (green) together and Cre+KCC2FL, Cre+KCC2CTD, or Cre+KCC2R952H. DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. F Number of transfected neurons migrating above (II-IV, left) and below (V-VI/IZ-SVZ, right) the upper border of layer V in embryos electroporated with constructs in (E) normalized to respective data from embryos electroporated with Cre. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001 to Cre. Data are presented as mean ± S.E.M., n (Cre+KCC2FL) = 13 embryos; n (Cre+KCC2CTD) = 9 embryos; n (Cre+KCC2R952H) = 9 embryos.


A , B , C The layer thickness and neuronal number within each layer was assessed using layer - specific markers in Kcc2 - / - and Kcc2 + / + embryos at E18 . 5 . Cux1 was used to label layers II - IV ( A ) , Ctip2 to label layer V ( B ) , and Tbr1 to label layer VI ( C ) . Dashed white lines in the representative images in ( A - C ) indicate upper and lower layer boundaries . Sp , subplate . Statistical significance was determined using Kruskal - Wallis test with Dunn ' s post hoc ( neuronal numbers ) and one - way ANOVA with Holm - Sidak ' s post hoc ( layer thickness ) . Data are presented as mean ± S . E . M . , n = 11 embryos for both genotypes . Scale bar = 50 µm .

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

A, B, C The layer thickness and neuronal number within each layer was assessed using layer-specific markers in Kcc2-/-and Kcc2+/+ embryos at E18.5. Cux1 was used to label layers II-IV (A), Ctip2 to label layer V (B), and Tbr1 to label layer VI (C). Dashed white lines in the representative images in (A-C) indicate upper and lower layer boundaries. Sp, subplate. Statistical significance was determined using Kruskal-Wallis test with Dunn's post hoc (neuronal numbers) and one-way ANOVA with Holm-Sidak's post hoc (layer thickness). Data are presented as mean ± S.E.M., n = 11 embryos for both genotypes. Scale bar = 50 µm.


D Representative image of E18 . 5 coronal cortical sections stained for layer V marker Ctip2 ( red ) in Kcc2 - / - and Kcc2 + / + embryos electroporated in utero at E14 . 5 with a plasmid encoding EGFP ( green ) . DAPI staining ( blue ) marks cell nuclei . Upper boundary of layer V indicted with dotted line . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . E Neurons from the brain sections in ( D ) were quantified and the total number of EGFP + neurons in the Kcc2 - / - sections is presented as a percentage of pooled Kcc2 + / + and Kcc2 + / - values . Statistical significance was determined using one - tailed Student ' s t test , * P < 0 . 05 . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos . F Number of EGFP + neurons in Kcc2 - / - embryos migrating above ( II - IV ) and below ( V - VI / IZ - SVZ ) the upper border of layer V normalized to respective pooled data from Kcc2 + / + and Kcc2 + / - embryos . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc test , * * P < 0 . 01 to Kcc2 + / + + Kcc2 + / - . Data are presented as mean ± S . E . M . , n ( Kcc2 + / + + Kcc2 + / - ) = 14 embryos ; n ( Kcc2 - / - ) = 9 embryos .

O O B-EXP_ASSAY O O O O O B-TISSUE O O O B-TISSUE I-TISSUE O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-SUBCELLULAR O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O B-TISSUE O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O O O O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-ORGANISM O O O B-GENEPROD O O O O O O B-ORGANISM O

D Representative image of E18.5 coronal cortical sections stained for layer V marker Ctip2 (red) in Kcc2-/- and Kcc2+/+ embryos electroporated in utero at E14.5 with a plasmid encoding EGFP (green). DAPI staining (blue) marks cell nuclei. Upper boundary of layer V indicted with dotted line. Sp, subplate; IZ, intermediate zone. Scale bar = 50 µm. E Neurons from the brain sections in (D) were quantified and the total number of EGFP+ neurons in the Kcc2-/- sections is presented as a percentage of pooled Kcc2+/+ and Kcc2+/- values. Statistical significance was determined using one-tailed Student's t test, *P < 0.05. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos. F Number of EGFP+ neurons in Kcc2-/- embryos migrating above (II-IV) and below (V-VI/IZ-SVZ) the upper border of layer V normalized to respective pooled data from Kcc2+/+ and Kcc2+/- embryos. Statistical significance was determined using one-way ANOVA with Holm-Sidak's post hoc test, **P < 0.01 to Kcc2+/+ + Kcc2+/-. Data are presented as mean ± S.E.M., n (Kcc2+/+ + Kcc2+/-) = 14 embryos; n (Kcc2-/-) = 9 embryos.


( B ) Representative images of GFAP + Sox2 + BrdU + RGLs , GFAP - Sox2 + BrdU + progenitors , Tbr2 + BrdU + IPCs in the DG . Scale bar = 20 μm . ( C ) Quantification of GFAP + Sox2 + BrdU + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 022 ) , GFAP - Sox2 + BrdU + progenitors ( WT vs . KO , Student ' s t - test , p = 0 . 0046 ) , Tbr2 + BrdU + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 4 mice ; KO , n = 6 mice . ( D ) Representative images of GFAP + Mcm2 + RGLs , Tbr2 + Mcm2 + IPCs , DCX + Mcm2 + NBs in the DG . Scale bar = 20 μm . ( E ) Quantification of GFAP + Mcm2 + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 016 ) , Tbr2 + Mcm2 + IPCs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) , DCX + Mcm2 + NBs ( WT vs . KO , Student ' s t - test , p = 0 . 003 ) in the DG of WT and KO mice . WT , n = 3 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative images of GFAP+Sox2+BrdU+ RGLs, GFAP-Sox2+BrdU+ progenitors, Tbr2+BrdU+ IPCs in the DG. Scale bar = 20 μm. (C) Quantification of GFAP+Sox2+BrdU+ RGLs (WT vs. KO, Student's t-test, p = 0.022), GFAP-Sox2+BrdU+ progenitors (WT vs. KO, Student's t-test, p = 0.0046), Tbr2+BrdU+ IPCs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 4 mice; KO, n = 6 mice. (D) Representative images of GFAP+Mcm2+ RGLs, Tbr2+Mcm2+ IPCs, DCX+Mcm2+ NBs in the DG. Scale bar = 20 μm. (E) Quantification of GFAP+Mcm2+ RGLs (WT vs. KO, Student's t-test, p = 0.016), Tbr2+Mcm2+ IPCs (WT vs. KO, Student's t-test, p = 0.003), DCX+Mcm2+ NBs (WT vs. KO, Student's t-test, p = 0.003) in the DG of WT and KO mice. WT, n = 3 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( B ) Representative images used for fate mapping of tdT + cells in the DG by co - stained with GFAP ( RGL ) , Tbr2 ( IPC ) , DCX ( IN ) , NeuN ( MN ) . Scale bar = 20 μm .

O O O O O O O O O O B-GENEPROD O O O O B-TISSUE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O

(B) Representative images used for fate mapping of tdT+ cells in the DG by co-stained with GFAP (RGL), Tbr2 (IPC), DCX (IN), NeuN (MN). Scale bar = 20 μm.


( C ) Quantification of composition of tdT + cells ( RGL : WT vs . KO , Student ' s t - test , p = 0 . 026 ; IPC : WT vs . KO , Student ' s t - test , p = 0 . 028 ; IN : WT vs . KO , Student ' s t - test , p = 0 . 049 ; N : WT vs . KO , Student ' s t - test , p = 0 . 048 ) in the DG of WT and cKO mice . WT , n = 3 mice ; cKO , n = 3 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 .

O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of composition of tdT+ cells (RGL: WT vs. KO, Student's t-test, p = 0.026; IPC: WT vs. KO, Student's t-test, p = 0.028; IN: WT vs. KO, Student's t-test, p = 0.049; N: WT vs. KO, Student's t-test, p = 0.048) in the DG of WT and cKO mice. WT, n = 3 mice; cKO, n = 3 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01


( H ) Representative images of BrdU - retaining RGLs ( tdT + BrdU + ) in the DG . Scale bar = 20 μm . ( I ) Quantification of BrdU - retaining RGLs ( BrdU + tdT + GFAP + , WT vs . cKO , Student ' s t - test , p = 0 . 009 ) in the DG of WT and cKO mice . WT , n = 4 mice ; cKO , n = 3 mice .

O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O B-GENEPROD O B-SMALL_MOLECULE O O O O B-TISSUE O O O O O O O O O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-ORGANISM O

(H) Representative images of BrdU-retaining RGLs (tdT+BrdU+) in the DG. Scale bar = 20 μm. (I) Quantification of BrdU-retaining RGLs (BrdU+tdT+GFAP+, WT vs. cKO, Student's t-test, p = 0.009) in the DG of WT and cKO mice. WT, n = 4 mice; cKO, n = 3 mice.


( B ) Representative images of Nestin - GFP + GFAP + Mcm2 + RGLs in the DG . Scale bar = 50 μm . ( C ) Quantification of GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 001 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . ( D ) Percentage of GFAP + Nestin - GFP + Mcm2 + activated RGLs among total GFAP + Nestin - GFP + RGLs ( WT vs . KO , Student ' s t - test , p = 0 . 0013 ) in the DG of WT and KO mice at 6 months of age . WT , n = 5 mice ; KO , n = 4 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative images of Nestin-GFP+GFAP+Mcm2+ RGLs in the DG. Scale bar = 50 μm. (C) Quantification of GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.001) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. (D) Percentage of GFAP+Nestin-GFP+Mcm2+ activated RGLs among total GFAP+Nestin-GFP+ RGLs (WT vs. KO, Student's t-test, p = 0.0013) in the DG of WT and KO mice at 6 months of age. WT, n = 5 mice; KO, n = 4 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


( I ) Representative images of NeuN + BrdU + adult - born neurons in the DG of 6 month - old mice . Scale bar = 50 μm . ( J ) Quantification of NeuN + BrdU + adult - born neurons ( WT vs . KO , Student ' s t - test , p = 0 . 0052 ) in the DG of WT and KO mice at 7 months of age . WT , n = 4 mice ; KO , n = 3 mice . Data information : Data are presented as the mean ± SEM . * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O B-ORGANISM O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) Representative images of NeuN+BrdU+ adult-born neurons in the DG of 6 month-old mice. Scale bar = 50 μm. (J) Quantification of NeuN+BrdU+ adult-born neurons (WT vs. KO, Student's t-test, p = 0.0052) in the DG of WT and KO mice at 7 months of age. WT, n = 4 mice; KO, n = 3 mice. Data information: Data are presented as the mean ± SEM. **, p < 0.01, ***, p < 0.001.


( A ) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling . Scale bar = 50 μm . ( B ) Percentage of BrdU + aNPCs among total aNPCs isolated from WT or KO mice ( WT vs . KO , Student ' s t - test , p = 0 . 0075 ) . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(A) Representative images of aNPCs in vitro isolated from WT and KO mice and followed by a BrdU pulse labeling. Scale bar = 50 μm. (B) Percentage of BrdU+ aNPCs among total aNPCs isolated from WT or KO mice (WT vs. KO, Student's t-test, p = 0.0075). WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; **, p < 0.01, ***, p < 0.001.


( A ) DARTS analysis indicated HK1 as a molecular target of L - arginine in aNPCs . The aNPC lysates treated with L - arginine , digested with pronase and then analyzed by Western blot with anti - HK1 and GAPDH antibodies . ( B ) Quantification of HK1 expression level in aNPCs lysates treated with L - arginine and followed by digesting with pronase ( One - way ANOVA , F ( 3 , 8 ) = 3 . 61 , p = 0 . 043 ) . n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(A) DARTS analysis indicated HK1 as a molecular target of L-arginine in aNPCs. The aNPC lysates treated with L-arginine, digested with pronase and then analyzed by Western blot with anti-HK1 and GAPDH antibodies. (B) Quantification of HK1 expression level in aNPCs lysates treated with L-arginine and followed by digesting with pronase (One-way ANOVA, F(3, 8)= 3.61, p = 0.043). n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( I ) Co - IP of HK1 with VDAC1 in vehicle or L - arginine - treated cells . ( J ) Quantification of the co - IP efficacy of HK1 and VDAC interaction in vehicle or L - arginine - treated cells ( IP : HK1 , vehicle vs . L - arginine , Student ' s t - test , p < 0 . 001 ; IP : VDAC1 , vehicle vs . L - arginine , Student ' s t - test , p = 0 . 0078 ) . Vehicle , n = 3 independent experiments ; L - arginine , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(I) Co-IP of HK1 with VDAC1 in vehicle or L-arginine-treated cells. (J) Quantification of the co-IP efficacy of HK1 and VDAC interaction in vehicle or L-arginine-treated cells (IP: HK1, vehicle vs. L-arginine, Student's t-test, p < 0.001; IP: VDAC1, vehicle vs. L-arginine, Student's t-test, p = 0.0078). Vehicle, n = 3 independent experiments; L-arginine, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( C - E ) Quantification of glucose ( C , WT vs . KO , Student ' s t - test , p < 0 . 001 ) and glutamine ( E , WT vs . KO , Student ' s t - test , p = 0 . 94 ) consumption and lactate production ( D , WT vs . KO , Student ' s t - test , p = 0 . 019 ) in WT - and KO - aNPCs . WT , n = 3 independent experiments ; KO , n = 3 independent experiments . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 * * * , p < 0 . 001 .

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

(C-E) Quantification of glucose (C, WT vs. KO, Student's t-test, p < 0.001) and glutamine (E, WT vs. KO, Student's t-test, p = 0.94) consumption and lactate production (D, WT vs. KO, Student's t-test, p = 0.019) in WT- and KO-aNPCs. WT, n = 3 independent experiments; KO, n = 3 independent experiments. Data information: Data are presented as the mean ± SEM; *, p < 0.05 ***, p < 0.001.


( J ) Representative profile of mitochondria stress test showing the OCR of WT - or KO - aNPCs . WT , n = 4 independent experiments , KO , n = 4 independent experiments .

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

(J) Representative profile of mitochondria stress test showing the OCR of WT- or KO-aNPCs. WT, n = 4 independent experiments, KO, n = 4 independent experiments.


( C ) Quantification of Nestin - GFP + GFAP + RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 99 ) . WT + Vehicle , n = 4 mice ; WT + IACS , n = 4 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 3 mice . ( D ) Quantification of Nestin - GFP + GFAP + BrdU + activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( E ) Quantification of Nestin - GFP + GFAP - BrdU + progenitors ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 028 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( F ) Quantification of Nestin - GFP + Tbr2 + BrdU + IPCs ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p = 0 . 02 ) in the DG of WT and KO mice after vehicle or IACS treatment . WT + Vehicle , n = 4 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 4 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of Nestin-GFP+GFAP+ RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.99). WT + Vehicle, n = 4 mice; WT + IACS, n = 4 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 3 mice. (D) Quantification of Nestin-GFP+GFAP+BrdU+ activated RGLs in the DG of WT and KO mice after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (E) Quantification of Nestin-GFP+GFAP-BrdU+ progenitors (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.028) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 3 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 3 mice, KO + IACS, n = 3 mice. (F) Quantification of Nestin-GFP+Tbr2+BrdU+ IPCs (KO + Vehicle vs. KO + IACS, Student's t-test, p = 0.02) in the DG of WT and KO mice after vehicle or IACS treatment. WT + Vehicle, n = 4 mice; WT + IACS, n = 3 mice; KO + Vehicle, n = 4 mice, KO + IACS, n = 4 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


( H ) Representative images of NeuN + BrdU + adult - born neurons in the DG . Scale bar = 20 μm . ( I ) Quantification of NeuN + BrdU + adult - born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - test , p < 0 . 001 ) . WT + Vehicle , n = 5 mice ; WT + IACS , n = 5 mice ; KO + Vehicle , n = 5 mice , KO + IACS , n = 5 mice . Data information : Data are presented as the mean ± SEM ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-ORGANISM O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(H) Representative images of NeuN+BrdU+ adult-born neurons in the DG. Scale bar = 20 μm. (I) Quantification of NeuN+BrdU+ adult-born neurons in the DG of WT and KO mice at P90 after vehicle or IACS treatment (KO + Vehicle vs. KO + IACS, Student's t-test, p < 0.001). WT + Vehicle, n = 5 mice; WT + IACS, n = 5 mice; KO + Vehicle, n = 5 mice, KO + IACS, n = 5 mice. Data information: Data are presented as the mean ± SEM; *, p < 0.05, **, p < 0.01, ***, p < 0.001.


EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation ( A - C ) ( A , C ) The successive pellets ( 2K , 10K , 100K ) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side - by - side with the lysate of 0 . 4 x 106 producing cells : C . Images from two different representative experiments are shown . Amount of proteins recovered in 2K , 10K and 100K pellets analyzed in stain - free gel images was similar between pellets : AU ( protein amount in a given pellet / total protein amount in 2K + 10K + 100K ) for 2K , 10K and 100K pellets were 1 . 13 ± 0 . 28 , 0 , 96 ± 0 . 10 and 0 . 91 ± 0 . 33 , respectively in control cells ( A ) , and 1 . 22 ± 0 . 29 , 0 , 84 ± 0 . 35 and 0 . 94 ± 0 . 30 , respectively in infected cells ( C ) ( mean ± SD ) .

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

EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation (A-C) (A, C) The successive pellets (2K, 10K, 100K) recovered from the CCM of 20 x 106 cells were analyzed by Western blot for the indicated proteins side-by-side with the lysate of 0.4 x 106 producing cells: C. Images from two different representative experiments are shown. Amount of proteins recovered in 2K, 10K and 100K pellets analyzed in stain-free gel images was similar between pellets: AU (protein amount in a given pellet/total protein amount in 2K+10K+100K) for 2K, 10K and 100K pellets were 1.13±0.28, 0,96±0.10 and 0.91±0.33, respectively in control cells (A), and 1.22±0.29, 0,84±0.35 and 0.94±0.30, respectively in infected cells (C) (mean±SD).


EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD ) . ( B ) Whole - mount EM analysis showing representative images and size distribution of particles in 2K , 10K and 100K pellets ( Scale bar , 200 nm ) . The diameter ( nm ) of particles was determined with ImageJ for five different experiments . 387 , 363 and 385 particles were counted for 2K , 10K and 100K pellets , respectivelly .

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

EVs were isolated from CCM of uninfected Jurkat cells by differential ultracentrifugation SD). (B) Whole-mount EM analysis showing representative images and size distribution of particles in 2K, 10K and 100K pellets (Scale bar, 200 nm). The diameter (nm) of particles was determined with ImageJ for five different experiments. 387, 363 and 385 particles were counted for 2K, 10K and 100K pellets, respectivelly.


( A - D ) EVs were isolated from CCM of uninfected or HIV - 1 - infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient ( D ) . ( D ) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non - infected and infected Jurkat cells and analysed by Western blot for the indicated proteins .

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

(A-D) EVs were isolated from CCM of uninfected or HIV-1-infected Jurkat cells by differential ultracentrifugation and sEVs contained in the 100K pellet were further separated by velocity iodixanol gradient (D). (D) 12 fractions were recovered from iodixanol gradients of the 100K pellets obtained from CCM of non-infected and infected Jurkat cells and analysed by Western blot for the indicated proteins.


( A ) Proteomic profiles of different proteins , showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells . Proteins with very similar profiles ( represented by the same colours ) are likely part of the same EV subtypes . Each profile consists of three independent data triplets ( F1 - F2 - F3A , F1 - F2 - F3B and F1 - F2 - F3C ) .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O 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) Proteomic profiles of different proteins, showing the relative abundance distribution across the 3x3 subfractions obtained from Jurkat cells. Proteins with very similar profiles (represented by the same colours) are likely part of the same EV subtypes. Each profile consists of three independent data triplets (F1- F2- F3A, F1- F2- F3B and F1-F2-F3C).


( C ) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1 - F2 - F3 subfractions as compared to expression in the total cell proteome . Solid horizontal lines indicate medians and dashed line indicate quartiles ( n = 3 ) . Mitochondria and ER markers are de - enriched , and plasma membrane markers are enriched in EV fractions , with progressively pronounced effects from F1 to F3 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O O O

(C) Violin plots showing enrichment of the protein markers of intracellular organelles across the F1-F2-F3 subfractions as compared to expression in the total cell proteome. Solid horizontal lines indicate medians and dashed line indicate quartiles (n=3). Mitochondria and ER markers are de-enriched, and plasma membrane markers are enriched in EV fractions, with progressively pronounced effects from F1 to F3.


EVs were purified by SEC from supernatants of Jurkat cells . ( A ) EVs were subjected to immuno - isolation with beads coupled to antibodies against CD81 or CD63 . Bead - associated ( Pull - down : PD ) vesicles and those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot analysis with antibodies specific for CD63 , Syntenin - 1 , CD81 , ADAM10 and CD3G . A representative blot ( top panel ) and quantification ( mean ± SD ) of the proportion of signal in FT as compared with total ( PD + FT ) from four independent experiments ( bottom panel ) are shown .

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

EVs were purified by SEC from supernatants of Jurkat cells. (A) EVs were subjected to immuno-isolation with beads coupled to antibodies against CD81 or CD63. Bead-associated (Pull-down: PD) vesicles and those left behind (Flow-Through: FT) were loaded on a gel for Western blot analysis with antibodies specific for CD63, Syntenin-1, CD81, ADAM10 and CD3G. A representative blot (top panel) and quantification (mean ± SD) of the proportion of signal in FT as compared with total (PD+FT) from four independent experiments (bottom panel) are shown.


( B ) Representative images of double - immunogold labelling of EVs purified by SEC from Jurkat cells . Left panel , CD81 / CD63 ; right panel , CD81 / CD3E . Arrows show CD63 staining in CD81 + CD63 + EVs .

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

(B) Representative images of double-immunogold labelling of EVs purified by SEC from Jurkat cells. Left panel, CD81/CD63; right panel, CD81/CD3E. Arrows show CD63 staining in CD81+CD63+ EVs.


EVs were purified by SEC from supernatants of Jurkat cells . ( C , D ) Multiplex bead - based flow cytometry assay for detection of EV surface markers . Antibody - coated capture beads were incubated with 2 x 109 particles . Captured EVs were detected with either APC - labeled anti - CD81 , anti - CD63 or anti - CD3E . ( C ) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads ( log10 scale ) . shown . ( D ) Heat - map representation of the median APC fluorescence values for the different bead populations detected with anti - CD63 or anti - CD3E antibodies relative to the values detected with anti - CD81 .

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EVs were purified by SEC from supernatants of Jurkat cells. (C,D) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with 2 x 109 particles. Captured EVs were detected with either APC-labeled anti-CD81, anti-CD63 or anti-CD3E. (C) Median APC fluorescence values for the different bead populations is shown as the ratio to the median APC fluorescence of control beads (log10 scale). shown. (D) Heat-map representation of the median APC fluorescence values for the different bead populations detected with anti-CD63 or anti-CD3E antibodies relative to the values detected with anti-CD81.


( B ) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions . Each profile consists of two independent data triplets ( F1 - F2 - F3A and F1 - F2 - F3B ) . Abundance profiles of the same groups of proteins showed the same clustering behaviour , although in this second dataset , the proportion of proteins recovered in the F2 was more variable . These differences , however , did not affect the results of the NNP or movement analysis .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Proteomic profiles of different proteins showing the relative abundance distribution across the six subfractions. Each profile consists of two independent data triplets (F1- F2- F3A and F1- F2- F3B). Abundance profiles of the same groups of proteins showed the same clustering behaviour, although in this second dataset, the proportion of proteins recovered in the F2 was more variable. These differences, however, did not affect the results of the NNP or movement analysis.


( C ) Unbiased identification of significant translocation events triggered by HIV - 1 infection . Each protein is scored for magnitude of translocation ( M score , x - axis ) and reproducibility of translocation direction ( R score , y - axis ) across the two replicates . MR plot analysis reveals significant translocations in the top right quadrant . Proteins with M > 1 and R > 0 . 9 are candidate hits for changing localization ( estimated FDR = 8 % ) .

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

(C) Unbiased identification of significant translocation events triggered by HIV-1 infection. Each protein is scored for magnitude of translocation (M score, x-axis) and reproducibility of translocation direction (R score, y-axis) across the two replicates. MR plot analysis reveals significant translocations in the top right quadrant. Proteins with M>1 and R>0.9 are candidate hits for changing localization (estimated FDR = 8%).


( A , B ) ( A ) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected ( red boxes ) Jurkat cells . The successive pellets ( 2K , 10K , 100K ) were analyzed by Western blot , using antibodies against SPN , MOV10 , SERINC3 and p24 ( HIV - 1 protein ) . The 2K , 10K , and 100K pellets obtained from 20 x 106 cells were loaded on the gel . Representative images ( A ) and quantifications in three to five independent experiments ( B ) are shown . For each pellet , arbitrary units ( AU ) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet . In cell lysates from the same experiments , AU ( given protein / total amount of protein ) for SPN and MOV10 were 1 . 29 ± 0 . 15 and 0 . 84 ± 0 . 33 times the levels in control cells , respectively ( Mean ± SD ) .

O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O 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) (A) Analysis of the three candidate markers in EV pellets isolated from CCM of control and infected (red boxes) Jurkat cells. The successive pellets (2K, 10K, 100K) were analyzed by Western blot, using antibodies against SPN, MOV10, SERINC3 and p24 (HIV-1 protein). The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (A) and quantifications in three to five independent experiments (B) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity for the analysed protein in the given pellet to the total amount of proteins in the same pellet. In cell lysates from the same experiments, AU (given protein/total amount of protein) for SPN and MOV10 were 1.29±0.15 and 0.84±0.33 times the levels in control cells, respectively (Mean±SD).


( C ) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV - 1 infected ( red boxes ) cells . Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN . SERINC3 was recovered in the middle fractions . ( T = top ; B = bottom ) . ( D , E ) Quantification of MOV10 , SPN , SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV - 1 infected samples . AU = band intensity in a given pellet / sum ( band intensity in all six pellets ) . ( F ) Quantification of SERINC3 signals in the different fractions from control and infected samples . AU = band intensity in a given pellet / sum ( band intensity in all 12 pellets ) . Lines represent the medians of three independent experiments .

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

(C) WB showing the distribution of the same proteins in six fractions recovered after velocity gradient separation of vesicles contained in the 100K pellets of control or HIV-1 infected (red boxes) cells. Viral p24 protein was recovered in the bottom fractions of infected samples together with MOV10 and SPN. SERINC3 was recovered in the middle fractions. (T = top; B = bottom). (D, E) Quantification of MOV10, SPN, SERINC3 and p24 signals in 4 to 7 independent Western blots from HIV-1 infected samples. AU = band intensity in a given pellet / sum (band intensity in all six pellets). (F) Quantification of SERINC3 signals in the different fractions from control and infected samples. AU = band intensity in a given pellet / sum (band intensity in all 12 pellets). Lines represent the medians of three independent experiments.