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

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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 log - transformed fold change ( log2FC ) values versus - free controls . Groups : - free control ( n = 6 ) , - free GTx - 024 treated ( n = 5 ) , - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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

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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 - free control transcriptomes . * FDR < 1e - 5 determined , set to 1e - 5 for plot .

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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 CTNNB1 gene set for each treatment group versus - free control comparisons . GTx - 024 - treated - free ( blue checkered ) , - bearing control ( black ) , GTx - 024 - treated - bearing ( blue ) , AR - 42 - treated - bearing ( red ) Combination - treated - bearing ( green ) .

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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 - free controls . Groups : - free control ( n = 6 ) , - free GTx - 024 treated ( n = 5 ) , - bearing receiving vehicle ( n = 4 ) , GTx - 024 ( n = 5 ) , AR - 42 ( n = 5 ) or Combination ( n = 6 ) .

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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 ) of creatine kinase ( CK ) creatinine .

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


C ) of procalcitonin ( PCT ) C - reactive ( CRP ) .

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


D ) of erythrocyte sedimentation rate ( ESR ) ferritin .

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


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

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


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


G ) Proportion of CD8 + T CD4 + / CD8 + T .

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


H ) Proportion of B natural killer ( NK ) .

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


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

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


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

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 maximal during hospitalization .

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


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

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


Correlation between IL - 6 lactate dehydrogenase ( LDH ) .

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


Correlation between IL-6 and D-dimer.


Correlation between IL-6 and ferritin.


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

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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 before after treatment in three groups classified according to baseline IL - 6 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 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 at diagnosis after disease progression in three exacerbated ( left ) . Progressed radiological findings were recorded in two ( right ) , while radiological assessment after treatment was not performed in third 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.


baseline IL - 6 level was 197 . 39 pg / mL in a 69 - year - old who showed high fever dyspnea . IL - 6 decreased to 9 . 47 pg / mL after treatment ( day 8 ) , while symptoms were not relieved . C - reactive ( CRP ) rebounded 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 exacerbation was caused by infection .

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

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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 ) stayed within normal range throughout course of disease .

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


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

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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 ( CRP ) ( D ) were detected five days after symptom onset when chest CT lymphocyte were still normal . In association with changes in chest CT scans , CRP further increased ten days after symptom onset returned to normal range 25 days after symptom onset .

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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 chest CT lymphocyte were still normal . In association with changes in chest CT scans , IL - 6 further increased ten days after symptom onset returned to normal range 25 days after symptom onset .

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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 ) was elevated when CRP IL - 6 were rising , but returned to normal when CRP IL - 6 . were declining .

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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 . lysates were treated with λ Phosphatase ( PPase ) for 30 minutes analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 β - Actin . pFMRP / FMRP fold induction is depicted above blots ( n = 6 biological replicates ) .

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

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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 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 . lysates were analyzed by western blotting using specific antibodies for pFMRP , FMRP , pIRE1 , IRE1 β - Actin . pFMRP / FMRP fold induction is depicted above blots ( n = 4 biological replicates ) .

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

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

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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 after 24 48 hours , ( J ) liver cholesterol after 48 hours , ( K ) feces cholesterol after 48 hours ( n = 12 mice per group ) .

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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 in vivo efferocytosis experiments , where of macrophages F4 / 80 + ( red ) that ingested apoptotic ( AC ) labeled with carboxyfluorescein succinimidyl ester ( CFSE ) + ( green ) were reported as % efferocytosis . ( A ) BMDMs were transfected with Fmr1 - or control - siRNA incubated CFSE - labeled AC for indicated hours ( n = 4 biological replicates ) . ( B ) Fmr1 + / + Fmr1 - / - BMDMs were treated with PA ( 500 µM ) for 6 hours then incubated with CFSE - labeled ACs for 4 hours ( n = 4 biological replicates ) . ( C ) Fmr1 + / + Fmr1 - / - mice were fed WD ( 16 ) 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 PM elicitation ( n = 4 mice per group ) .

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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 of F4 / 80 + , CFSE + Violet + ( triple positive ) to total F4 / 80 + CFSE + ( double positive ) . ( F ) Fmr1 + / + Fmr1 - / - BMDM were incubated with AC - 1 for 2 hours , 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 PM collection ( n = 4 biological replicates ) .

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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).


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

O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR O O O 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 were transfected with EV , WT - FMRP or STSA - FMRP plasmids followed by PA treatment ( 500 µM ; 6 hours ) . lysates were analyzed by western blotting using specific antibodies for ABCA1 , MerTK , LRP1 , pFMRP , FMRP β - Actin fold inductions relative to β - Actin are depicted above blots ( n = 5 biological replicates ) .

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

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

O O O O O O O O O O O O O O O O O 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 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=9 mice per group; Scale bar = 5 mm).


Lesion 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 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 ACE2 for spike binding , investigated by GCI . Biotinylated spike was immobilized on GCI sensor 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 B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

(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 . Panels show relative infectivity upon exposure to increasing concentrations of 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 , previously - described sybody MR3 . ( C ) Neutralization by bivalent sybody - Fc fusions . Error bars correspond to standard deviations of three biological replicates .

O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 using VSVΔG pseudotyped with wildtype Spike - . Relative infectivity was determined in response to increasing GS4 or Tripod - GS4r . corresponding neutralization data for isolated Sb # 15 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 O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 unrelated gene galK . n = 3 for each

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

O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR 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 of intracellular lysine in wild type reconstructed DapF mutants . was performed using LC - MS , as described in methods section . n = 3 . A two - sample student t - assuming unequal variances was performed to calculate statistical significance . Concentrations are reported as fold - change relative to wild - type control

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

D. 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 showing kinetics of wild type , G210D M260Y mutants . was performed as described in methods section . n =

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

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 dapF lysA genes on a WT , DapF G210D DapF M260Y

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 of intracellular lysine in wild type reconstructed LysR S36R mutant . was performed using LC - MS , as described in methods section . n = 2 . Concentrations are reported as fold - change relative to wild - type control

O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of intracellular lysine in wild type reconstructed ArgP E246 mutant . was performed using LC - MS , as described in methods section . n = 2 . Concentrations are reported as fold - change relative to 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 of E18 . 5 coronal cortical sections from Kcc2lox / lox electroporated in utero at E14 . 5 with plasmids encoding EGFP , EGFP + Cre ( Cre ) or EGFP + Cre + KCC2FL ( Cre + KCC2FL ) . DAPI ( blue ) marks nuclei . UCP , upper cortical plate ; LCP , lower cortical plate ; Sp , subplate ; IZ , intermediate zone . Scale bar : 50 µm . B of number of EGFP + neurons / ROI from electroporated with constructs in ( A ) . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc , * * P < 0 . 01 . Data are presented as mean ± S . E . M . , n ( EGFP ) = 8 ; n ( Cre ) = 8 ; n ( Cre + KCC2FL ) = 13 .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 Caspase 3 ( Cas - 3 , upper panel ) TUNEL ( lower panel ) 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 ) TUNEL ( lower panel ) . DAPI ( blue ) marks 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 of of EGFP + neurons expressing apoptotic markers at E16 . 5 from electroporated with EGFP ± Cre . Statistical significance was determined using Mann - Whitney U ( Cas - 3 ) ; two - t ( TUNEL ) , * * * P < 0 . 001 . Data are presented as mean ± S . E . M . , n ( - Cre ) = 6 ; n ( + Cre ) = 6 . E number of EGFP + Cre neurons as a of neurons expressing EGFP alone . Statistical significance was determined using a two - Student ' s t . Data are presented as mean ± S . E . M . , n = 6 .

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

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

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

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 electroporated in utero at E14 . 5 with plasmid constructs encoding mRFP or mRFP + Cre ( Cre ) . Upper boundary of layer V indicted with dotted . Sp , subplate ; IZ , intermediate zone . Scale bar = 50 µm . mRFP signal is shown as green pseudocolor . DAPI ( blue ) marks nuclei . D Number of mRFP + Cre neurons migrating above ( II - IV ) below ( V - VI / IZ - SVZ ) upper border of layer V normalized to respective data from electroporated with mRFP alone . Statistical significance was determined using one - way ANOVA with Holm - Sidak ' s post hoc , * * * P < 0 . 001 to mRFP . Data are presented as mean ± S . E . M . , n ( mRFP + Cre ) = 5 ; n ( mRFP ) = 5 .

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

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

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

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

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

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

(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 used for fate mapping of tdT + in 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 O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O 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 ) of composition of tdT + ( RGL : WT vs . KO , Student ' s t - , p = 0 . 026 ; IPC : WT vs . KO , Student ' s t - , p = 0 . 028 ; IN : WT vs . KO , Student ' s t - , p = 0 . 049 ; N : WT vs . KO , Student ' s t - , p = 0 . 048 ) in DG of WT cKO mice . WT , n = 3 mice ; cKO , n = 3 mice . Data information : Data are presented as mean ± ; * , p < 0 . 05 , * * , p < 0 .

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

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

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

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 indicated HK1 as a molecular target of L - arginine in aNPCs . aNPC lysates treated with L - arginine , digested with pronase then analyzed by Western blot with anti - HK1 GAPDH antibodies . ( B ) of HK1 expression level in aNPCs lysates treated with L - arginine 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 mean ± ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

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

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

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 showing OCR of WT - or KO - aNPCs . WT , n = 4 independent experiments , KO , n = 4 independent experiments .

O O O O O O O O O O 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 ) of Nestin - GFP + GFAP + RGLs in DG of WT KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - , p = 0 . 99 ) . WT + Vehicle , n = 4 mice ; WT + IACS , n = 4 mice ; KO + Vehicle , n = 4 mice , KO + IACS , n = 3 mice . ( D ) of Nestin - GFP + GFAP + BrdU + activated RGLs in DG of WT KO mice after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - , p < 0 . 001 ) . WT + Vehicle , n = 3 mice ; WT + IACS , n = 3 mice ; KO + Vehicle , n = 3 mice , KO + IACS , n = 3 mice . ( E ) of Nestin - GFP + GFAP - BrdU + progenitors ( KO + Vehicle vs . KO + IACS , Student ' s t - , p = 0 . 028 ) in DG of WT 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 ) of Nestin - GFP + Tbr2 + BrdU + IPCs ( KO + Vehicle vs . KO + IACS , Student ' s t - , p = 0 . 02 ) in DG of WT 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 mean ± ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

(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 of NeuN + BrdU + adult - born neurons in DG . Scale bar = 20 μm . ( I ) of NeuN + BrdU + adult - born neurons in DG of WT KO mice at P90 after vehicle or IACS treatment ( KO + Vehicle vs . KO + IACS , Student ' s t - , 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 mean ± ; * , p < 0 . 05 , * * , p < 0 . 01 , * * * , p < 0 . 001 .

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 by differential ultracentrifugation SD ) . ( B ) Whole - mount EM showing representative distribution of particles in 2K , 10K 100K pellets ( Scale bar , 200 nm ) . diameter ( nm ) of particles was determined with ImageJ for five different experiments . 387 , 363 385 particles were counted for 2K , 10K 100K pellets , respectivelly .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 by differential ultracentrifugation sEVs contained in 100K pellet were further separated by velocity iodixanol gradient ( D ) . ( D ) 12 fractions were recovered from iodixanol gradients of 100K pellets obtained from CCM of non - infected infected Jurkat analysed by Western blot for indicated .

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 markers of intracellular organelles across F1 - F2 - F3 subfractions as compared to expression in total proteome . Solid horizontal lines indicate medians dashed indicate quartiles ( n = 3 ) . Mitochondria ER markers are de - enriched , plasma 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 O O O O O O O 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 . ( A ) EVs were subjected to immuno - isolation with beads coupled to antibodies against CD81 or CD63 . Bead - associated ( Pull - down : PD ) vesicles those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot with antibodies specific for CD63 , Syntenin - 1 , CD81 , ADAM10 CD3G . A representative blot ( top panel ) ( mean ± SD ) of proportion of signal in FT as compared with total ( PD + FT ) from four independent experiments ( bottom panel ) are shown .

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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 of double - immunogold labelling of EVs purified by SEC from Jurkat . Left panel , CD81 / CD63 ; right panel , CD81 / CD3E . Arrows show CD63 in CD81 + CD63 + EVs .

O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O 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 . ( C , D ) Multiplex bead - flow cytometry 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 values for different bead populations is shown as to median APC of control beads ( log10 scale ) . shown . ( D ) Heat - map representation of median APC values for different bead populations detected with anti - CD63 or anti - CD3E antibodies relative to 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 showing relative abundance distribution across six subfractions . Each profile consists of two independent data triplets ( F1 - F2 - F3A F1 - F2 - F3B ) . Abundance profiles of same groups of showed same clustering behaviour , although in this second dataset , proportion of recovered in F2 was more variable . These differences , however , did not affect results of NNP or movement .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 is scored for magnitude of translocation ( M score , x - axis ) reproducibility of translocation direction ( R score , y - axis ) across two replicates . MR plot reveals significant translocations in top right quadrant . with M > 1 R > 0 . 9 are candidate hits for changing localization ( estimated FDR = 8 % ) .

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

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

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(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.


( G , H ) EVs from supernatant of HIV - infected Jurkat were subjected to immuno - isolation with beads coupled to antibody against SPN . Bead - associated ( Pull - down : PD ) vesicles those left behind ( Flow - Through : FT ) were loaded on a gel for Western blot with antibodies specific for SPN , MOV10 , CD63 , SERINC3 viral p24 . A representative image ( G ) ( H ) Mean ± SD of proportion of signal in PD as compared with total ( PD + FT ) in three independent experiments are shown .

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(G, H) EVs from supernatant of HIV-infected Jurkat cells were subjected to immuno-isolation with beads coupled to antibody against SPN. 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 SPN, MOV10, CD63, SERINC3 and viral p24. A representative image (G) and quantification (H) Mean ± SD of the proportion of signal in PD as compared with total (PD+FT) in three independent experiments are shown.


( I ) EVs remaining in FT , or EVs secreted by same number of but not subjected to immuno - isolation ( input ) were used to infect a reporter . Mean ± SD of six independent experiments is shown .

O O O O O O O O O O O O O O O O O O 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) EVs remaining in the FT, or EVs secreted by the same number of cells but not subjected to immuno-isolation (input) were used to infect a reporter cell line. Mean± SD of six independent experiments is shown.


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 ( A ) GFP + Ghost X4R5 obtained after treatment with supernatant from infected SERINC3 KD . Results are expressed as a to number of GFP + after treatment with supernatant from infected control . . Mean ± SD of three independent experiments is shown .

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Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 (A) GFP+ Ghost X4R5 cells obtained after treatment with supernatant from infected SERINC3 KD cells. Results are expressed as a ratio to the number of GFP+ cells after treatment with supernatant from infected control cells. . Mean ± SD of three independent experiments is shown.


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( B ) Number of particles secreted were quantified by NTA . Results are expressed as a to number of particles secreted by uninfected luciferase shRNA .

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Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (B) Number of particles secreted were quantified by NTA. Results are expressed as a ratio to the number of particles secreted by uninfected luciferase shRNA cells.


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( C ) 100K pellets from infected were subjected to iodixanol velocity gradient separation . Six fractions were recovered analyzed by Western blot for presence of SPN , SERINC3 p24 . ( T = top ; B = bottom ) . ( D ) of SPN p24 signals in different fractions of two independent iodixanol fractionations ( exp 1 : open symbols , exp 2 : closed symbols ) . AU = band in a given pellet / sum ( band in 12 pellets ) .

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

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (C) 100K pellets from infected cells were subjected to iodixanol velocity gradient separation. Six fractions were recovered and analyzed by Western blot for the presence of SPN, SERINC3 and p24. (T = top; B = bottom). (D) Quantification of SPN and p24 signals in the different fractions of two independent iodixanol fractionations (exp 1: open symbols, exp 2: closed symbols). AU = band intensity in a given pellet / sum (band intensity in all 12 pellets).


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K obtained from CCM of control SERINC3 KD either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed by Western blot with antibodies against SPN , p24 . 2K , 10K , 100K pellets obtained from 20 x 106 were loaded on gel . Representative ( E )

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

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K obtained from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed by Western blot with antibodies against SPN, and p24. The 2K, 10K, and 100K pellets obtained from 20 x 106 cells were loaded on the gel. Representative images (E)


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . 2K , 10K , 100K from CCM of control SERINC3 KD either uninfected of infected with NL4 - 3 EGFP - Nef + were analyzed of SPN p24 signals ( F ) are shown . For each pellet , arbitrary units ( AU ) represent of signal in given pellet to total amount of in same given pellet relative to total amount in 2K + 10K + 100K from control .

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

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). 2K, 10K, 100K from CCM of control cells and SERINC3 KD cells either uninfected of infected with NL4-3 EGFP-Nef+ were analyzed and quantification of SPN and p24 signals (F) are shown. For each pellet, arbitrary units (AU) represent the ratio of signal intensity in the given pellet to the total amount of protein in the same given pellet and relative to the total amount in 2K+10K+100K from control cells.


Jurkat were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3 , followed or not by infection with NL4 - 3 EGFP - Nef + HIV - 1 ( red boxes ) . ( G ) Multiplex bead - flow cytometry for detection of EV surface markers . Antibody - coated capture beads were incubated with EV samples . Captured EVs were detected with a mixture of APC - labeled anti - CD9 , anti - CD63 , anti - CD81 . of median APC values for bead populations after background isotype antibody correction . Results were relativized to values obtained with control sample for each experiment . Dotted represents values obtained for EVs isolated from control CM .

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

Jurkat cells were infected with lentivirus expressing control luciferase shRNA or shRNAs targeting SERINC3, followed or not by infection with NL4-3 EGFP-Nef+ HIV-1 (red boxes). (G) Multiplex bead-based flow cytometry assay for detection of EV surface markers. Antibody-coated capture beads were incubated with EV samples. Captured EVs were detected with a mixture of APC-labeled anti-CD9, anti-CD63, and anti-CD81. Quantification of the median APC fluorescence values for all bead populations after background and isotype antibody correction. Results were relativized to the values obtained with control sample for each experiment. Dotted line represents values obtained for EVs isolated from control cell CM.


A CAFs isolation from human pancreatic tumour resections ( left panel ) in vitro primo - culture ( right panel ) .

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A CAFs isolation from human pancreatic tumour resections (left panel) and in vitro primo-culture (right panel).


B Isolated CAFs PaSC characterization in vitro by immunofluorescence using anti - vimentin ( left panel ) or anti - α - SMA ( right panel ) antibody ( one representative field of n = 3 ) .

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B Isolated CAFs and PaSC characterization in vitro by immunofluorescence using anti-vimentin (left panel) or anti-α-SMA (right panel) antibody (one representative field of n = 3).


D , E Panc - 1 viability was assessed by MTT . Results ( mean ± SD ) are presented for each treatment ( CM from PaSC , or from CAF , or from CAF ± SOM230 , SOM230 ) as a of respective gemcitabine - untreated ( = 100 % ) ( n = 4 ; from left to right : * * P = 0 . 007 , # # P = 0 . 006 in D ; * P = 0 . 032 , # P = 0 . 041 , # P = 0 . 047 in E ) .

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D, E Panc-1 cell viability was assessed by MTT. Results (mean ± SD) are presented for each treatment (CM from PaSC, or from CAF, or from CAF ± SOM230, SOM230) as a percentage of the respective gemcitabine-untreated cells (= 100%) (n = 4; from left to right: **P = 0.007, ##P = 0.006 in D; *P = 0.032, #P = 0.041, #P = 0.047 in E).


F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti - caspase - 3 or anti - PARP antibody ( representative of n = 3 ) .

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F Apoptosis induced by gemcitabine was evaluated by Western blot using an anti-cleaved caspase-3 or anti-PARP antibody (representative of n = 3).


G Panc - 1 was analysed by flow cytometry . Percentages ( mean ± SD ) of annexin V - positive are indicated ( n = 3 ; from left to right : * * P = 0 . 005 , # # P = 0 . 004 , * * P = 0 . 006 ) .

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G Panc-1 was analysed by flow cytometry. Percentages (mean ± SD) of annexin V-positive cells are indicated (n = 3; from left to right: **P = 0.005, ##P = 0.004, **P = 0.006).


Immunoblotting of extracts from PaSCs or CAFs treated ( + ) or not with SOM230 ( 10 − 7 M ) for 48 h , using anti - puromycin antibody ( representative of n = 3 ) .

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Immunoblotting of protein extracts from PaSCs or CAFs treated (+) or not with SOM230 (10−7 M) for 48 h, using the anti-puromycin antibody (representative of n = 3).


Polysomes profiles of CAFs treated or not with SOM230 for 48 h ( representative of n = 3 ) .

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Polysomes profiles of CAFs treated or not with SOM230 for 48 h (representative of n = 3).


Immunoblotting using an anti - P - Akt , anti - P - S6 or anti - β - actin ( loading control ) antibody of extracts from PaSCs or CAFs treated ( + ) with SOM230 for 30 min ( representative of n = 3 ) .

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Immunoblotting using an anti-P-Akt, anti-P-S6 or anti-β-actin (loading control) antibody of protein extracts from PaSCs or CAFs treated (+) with SOM230 for 30 min (representative of n = 3).