Figure 2B Cell growth was determined in the absence and presence of 0 . 5 mM homocysteine or 0 . 5 mM methionine in the culture media of NE4C cells transfected with pcDNA3 . 1 vector or pcDNA3 . 1 - Flag - Bcl - 2 plasmids ( n = 3 ) . 5μg plasmids were transfected in each 1 × 106 cells . The cell index responds to changes in cell number and cell adhesion . Cleaved caspase 3 ( c - caspase3 ) levels were detected to determine apoptosis levels ( right ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . C , D Apoptotic cells were detected by flow cytometry . The apoptotic rates of untreated , MARS ( C ) - , and MARS2 ( D ) - over - expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . E Cells were treated with different methionine concentrations as indicated . Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 . F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS / MARS2 knockdown NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * * P ≤ 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Cell growth was determined in the absence and presence of 0.5 mM homocysteine or 0.5 mM methionine in the culture media of NE4C cells transfected with pcDNA3.1 vector or pcDNA3.1-Flag-Bcl-2 plasmids (n=3). 5μg plasmids were transfected in each 1×106 cells. The cell index responds to changes in cell number and cell adhesion. Cleaved caspase 3 (c-caspase3) levels were detected to determine apoptosis levels (right). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test.C, D Apoptotic cells were detected by flow cytometry. The apoptotic rates of untreated, MARS (C)-, and MARS2 (D)-over-expressing NE4C cells when cultured in the absence and presence of 20 μM homocysteine were normalized to the untreated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.E Cells were treated with different methionine concentrations as indicated. Apoptotic rates were detected by flow cytometry and normalized to those of the untreated cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.F Apoptotic rates were determined in the absence and presence of 20 μM homocysteine in the culture media of control and MARS/MARS2 knockdown NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, ***P ≤ 0.001.


Figure 3HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector , MARS ( A ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay . HTL and ROS ( superoxide ) levels were compared ( n = 4 ) between NE4C cells transfected with the empty vector or MARS2 ( B ) plasmids . Data were normalized against that of vector - transfected cells . HTL levels were determined by LC - MS , and ROS levels were determined by DHE staining assay . C MARS was knocked down by small hairpin RNAs in NE4C cells . MARS knockdown efficiencies were confirmed by western blot . HTL and superoxide levels were quantified ( n = 4 ) relative to vector - transfected NE4C cells . Cells were cultured in 20 μM Hcy - containing media . D Relative superoxide levels were determined by DHE staining assay and compared ( n = 4 ) between NE4C cells with and without MARS knockdown by shRNA . Cells were cultured in 20 μM Hcy - containing media . MARS2 knockdown efficiencies were confirmed by western blot . E NE4C cells with and without MARS knockdown were treated with either Hcy ( 20 μM ) or Met ( 75 μM ) , and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells ( n = 4 ) . F The cellular ROS ( superoxide ) levels were determined ( n = 4 ) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h . The ROS levels were normalized to those of untreated NE4C cells . G Superoxide levels in response to Hcy treatment ( 20 μM , 4 h ) in MARS - expressing and control cells were determined relative to the untreated NE4C cells . H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated . N - Hcy levels in HTL - treated and control cells were detected by western blot . I Protein N - Hcy levels in response to MARS knockdown ( by shRNA ) and over - expression were detected by western blot . J , K Untreated and MARS / MARS2 knockdown NE4C cells were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , and the N - Hcy ( J ) levels ( n = 4 ) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells . Error bars indicate SEM . Data were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J , K Untreated and MARS / MARS2 knockdown NE4C cells were treated with Hcy ( 20 μM ) or HTL ( 10 μM ) , and superoxide ( K ) levels ( n = 4 ) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells . Error bars indicate SEM . Data were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector, MARS (A) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.HTL and ROS (superoxide) levels were compared (n=4) between NE4C cells transfected with the empty vector or MARS2 (B) plasmids. Data were normalized against that of vector-transfected cells. HTL levels were determined by LC-MS, and ROS levels were determined by DHE staining assay.C MARS was knocked down by small hairpin RNAs in NE4C cells. MARS knockdown efficiencies were confirmed by western blot. HTL and superoxide levels were quantified (n=4) relative to vector-transfected NE4C cells. Cells were cultured in 20 μM Hcy-containing media.D Relative superoxide levels were determined by DHE staining assay and compared (n=4) between NE4C cells with and without MARS knockdown by shRNA. Cells were cultured in 20 μM Hcy-containing media. MARS2 knockdown efficiencies were confirmed by western blot.E NE4C cells with and without MARS knockdown were treated with either Hcy (20 μM) or Met (75 μM), and cellular superoxide levels were detected 6 h after the start of the respective treatment and quantified relative to the untreated NE4C cells (n=4).F The cellular ROS (superoxide) levels were determined (n=4) by DHE staining of NE4C cells treated with various HTL concentrations for 4 h. The ROS levels were normalized to those of untreated NE4C cells.G Superoxide levels in response to Hcy treatment (20 μM, 4 h) in MARS-expressing and control cells were determined relative to the untreated NE4C cells.H NE4C cells were cultured in DMEM supplemented with different HTL levels as indicated. N-Hcy levels in HTL-treated and control cells were detected by western blot.I Protein N-Hcy levels in response to MARS knockdown (by shRNA) and over-expression were detected by western blot.J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and the N-Hcy (J) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J, K Untreated and MARS/MARS2 knockdown NE4C cells were treated with Hcy (20 μM) or HTL (10 μM), and superoxide (K) levels (n=4) in cells were detected 6 h after the start of the respective treatment and quantified relative to untreated NE4C cells. Error bars indicate SEM. Data were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 4C SOD activities were down - regulated by in vitro N - homocysteinylation . Flag - tagged SOD1 , SOD1K23W , K122W , K128W ( 3KW ) mutant , SOD2 , and SOD2K44W , K51W , K98W , K106W , K178W ( 5KW ) mutant were expressed in HEK293T cells , affinity purified , and incubated with or without 10 μM HTL in solution ( in vitro ) . After 12 h , the reaction mixture was desalted and concentrated with filter tubes . N - Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2 , respectively ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Upon switching N - Hcy substrate lysine ( K ) sites to nonmodifiable tryptophan ( W ) , the N - Hcy unmodifiable mutants displayed lower specific activity than the wide - type ( WT ) SOD . SOD1 ( WT , K23W , K122W , K128W , and 3KW ) and SOD2 ( WT , K44W , K51W , K98W , K106W , K178W , and 5KW ) were expressed in HEK293T cells . The catalytic activity of affinity - purified SOD proteins was determined and normalized to protein levels . Wild - type SOD1 and SOD2 activities were set at 100 % ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E , F Hcy and HTL treatment in NE4C cells increased N - homocysteinylation of SODs and decreased their activities . Flag - tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells . The cells were transferred to serum - free DMEM with or without 20 μM Hcy ( E ) or 10 μM HTL ( F ) 6 h before harvesting . After affinity purification , the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . G N - Hcy SOD levels were down - regulated in MARS / MARS2 knockdown cells . Flag - tagged SOD1 and SOD2 were each over - expressed in untreated and MARS / MARS2 knockdown NE4C cells . After affinity purification , the N - Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4C SOD activities were down-regulated by in vitro N-homocysteinylation. Flag-tagged SOD1, SOD1K23W,K122W,K128W (3KW) mutant, SOD2, and SOD2K44W, K51W,K98W,K106W,K178W (5KW) mutant were expressed in HEK293T cells, affinity purified, and incubated with or without 10 μM HTL in solution (in vitro). After 12 h, the reaction mixture was desalted and concentrated with filter tubes. N-Hcy levels and SOD activities were determined and normalized to untreated WT SOD1 and SOD2, respectively (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Upon switching N-Hcy substrate lysine (K) sites to nonmodifiable tryptophan (W), the N-Hcy unmodifiable mutants displayed lower specific activity than the wide-type (WT) SOD. SOD1 (WT, K23W, K122W, K128W, and 3KW) and SOD2 (WT, K44W, K51W, K98W, K106W, K178W, and 5KW) were expressed in HEK293T cells. The catalytic activity of affinity-purified SOD proteins was determined and normalized to protein levels. Wild-type SOD1 and SOD2 activities were set at 100% (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E, F Hcy and HTL treatment in NE4C cells increased N-homocysteinylation of SODs and decreased their activities. Flag-tagged SOD1 and SOD2 were each ectopically expressed in NE4C cells. The cells were transferred to serum-free DMEM with or without 20 μM Hcy (E) or 10 μM HTL (F) 6 h before harvesting. After affinity purification, the specific activities of SOD1 and SOD2 were determined and normalized to SOD1 and SOD2 activity in untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.G N-Hcy SOD levels were down-regulated in MARS/MARS2 knockdown cells. Flag-tagged SOD1 and SOD2 were each over-expressed in untreated and MARS/MARS2 knockdown NE4C cells. After affinity purification, the N-Hcy levels and relative specific activities of purified SOD1 and SOD2 were determined and quantified relative to the NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 5A Levels of β - catenin in response to Hcy treatment in NE4C cells were detected by western blot ( n = 3 ) . Representative western blots are shown , and band intensities were quantified relative to the untreated group . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . B Levels of β - catenin in response to HTL treatment in NE4C cells were detected . Band intensities were normalized to that of untreated cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . C TCF activities were measured with a Topflash / Fopflash luciferase reporter system ( n = 4 ) . TCF activities responding to either 20 μM Hcy - or 10 μM HTL - treated were measured by quantifying Topflash / Fopflash relative to the untreated groups . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Levels of β - catenin in untreated and MARS knockdown NE4C cells were detected ( n = 3 ) with the presence or absence of 20 μM Hcy in the culture media . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were co - transfected with NRX - Flag and DVL1 - Myc plasmids and treated with different concentrations of Hcy ( E ) The relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were co - transfected with NRX - Flag and DVL1 - Myc plasmids and treated with different concentrations of HTL ( F ) . The relative amount of DVL1 that coimmunoprecipitated ( co - IPed ) with NRX - Flag was determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . G NRX - Flag and DVL1 - Myc were co - transfected in untreated or MARS knockdown NE4C cells . The relative DVL1 levels that co - IPed with NRX - Flag were determined and quantified ( n = 3 ) for treated and control cells cultured with or without 20 μM Hcy . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . Flag - tagged SOD1 and SOD2 were co - over - expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL . ( H ) The levels of β - catenin in cells with and without SOD1 / 2 expression were determined ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . Flag - tagged SOD1 and SOD2 were co - over - expressed in NE4C cells cultured in DMEM and supplemented with HTL . ( I ) Relative levels of DVL1 that co - IPed with NRX - Flag were quantified in SOD - expressing and control cells cultured with or without 10 μM HTL ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Levels of β-catenin in response to Hcy treatment in NE4C cells were detected by western blot (n=3). Representative western blots are shown, and band intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.B Levels of β-catenin in response to HTL treatment in NE4C cells were detected. Band intensities were normalized to that of untreated cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.C TCF activities were measured with a Topflash/Fopflash luciferase reporter system (n=4). TCF activities responding to either 20 μM Hcy- or 10 μM HTL-treated were measured by quantifying Topflash/Fopflash relative to the untreated groups. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Levels of β-catenin in untreated and MARS knockdown NE4C cells were detected (n=3) with the presence or absence of 20 μM Hcy in the culture media. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of Hcy (E) The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were co-transfected with NRX-Flag and DVL1-Myc plasmids and treated with different concentrations of HTL (F). The relative amount of DVL1 that coimmunoprecipitated (co-IPed) with NRX-Flag was determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.G NRX-Flag and DVL1-Myc were co-transfected in untreated or MARS knockdown NE4C cells. The relative DVL1 levels that co-IPed with NRX-Flag were determined and quantified (n=3) for treated and control cells cultured with or without 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with Hcy or HTL. (H) The levels of β-catenin in cells with and without SOD1/2 expression were determined (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.Flag-tagged SOD1 and SOD2 were co-over-expressed in NE4C cells cultured in DMEM and supplemented with HTL. (I) Relative levels of DVL1 that co-IPed with NRX-Flag were quantified in SOD-expressing and control cells cultured with or without 10 μM HTL (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 6B MARS - or MARS2 - catalysed HTL production from Hcy ( 20 μM ) was carried out in vitro in the presence of different AHT concentrations . The amount of HTL produced in each reaction was determined by thin layer chromatography . C MARS and MARS2 activities were determined ( n = 4 ) in the presence of the indicated AHT concentrations in the reactions . AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . D Relative HTL levels were determined ( n = 4 ) for NE4C cells treated with various AHT concentrations for 4 h . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E AHT ( 71 or 110 μg / kg per day ) was injected into C57BL / 6 mice for 6 days . Plasma HTL levels were detected by LC - MS . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels ; N - homocysteinylation levels were detected by western blot . NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels ; Band intensities were quantified relative to the untreated group using Image J ( H ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . I Cellular superoxide levels were determined ( n = 4 ) for NE4C cells treated with various AHT concentrations for 4 h . Superoxide levels were normalized to those of untreated NE4C cells . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT - treated NE4C cells ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . K Levels of β - catenin in untreated , Hcy - , or HTL - treated NE4C cells were detected ( n = 3 ) with the presence or absence of 70 μM AHT in the culture media . Mean β - catenin levels in untreated NE4C cells were set at 100 % . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B MARS- or MARS2-catalysed HTL production from Hcy (20 μM) was carried out in vitro in the presence of different AHT concentrations. The amount of HTL produced in each reaction was determined by thin layer chromatography.C MARS and MARS2 activities were determined (n=4) in the presence of the indicated AHT concentrations in the reactions. AHT IC50 values were determined for MARS and MARS2 with 20 μM Hcy. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.D Relative HTL levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E AHT (71 or 110 μg/kg per day) was injected into C57BL/6 mice for 6 days. Plasma HTL levels were detected by LC-MS. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.F Hcy levels were compared in NE4C or H9C2 cells without and with 100 μM AHT treatments. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; N-homocysteinylation levels were detected by western blot.NE4C cells were cultured in DMEM with or without 20 μM Hcy and supplemented with the indicated AHT levels; Band intensities were quantified relative to the untreated group using Image J (H). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.I Cellular superoxide levels were determined (n=4) for NE4C cells treated with various AHT concentrations for 4 h. Superoxide levels were normalized to those of untreated NE4C cells. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J Apoptotic rates were determined in the absence or presence of 20 μM Hcy in the culture media in control and 70 μM AHT-treated NE4C cells (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.K Levels of β-catenin in untreated, Hcy-, or HTL-treated NE4C cells were detected (n=3) with the presence or absence of 70 μM AHT in the culture media. Mean β-catenin levels in untreated NE4C cells were set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 7A , B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS ( green ) and N - Hcy ( red ) . Scale bar : 150 μm . The relative ( to untreated ) fluorescence intensities were quantified ( n = 3 ) and are presented in ( B ) . C , D Double immunofluorescent staining ( C ) for MARS ( green ) and N - Hcy ( red ) in E10 heart sections . Scale bar : 150 μm . The relative ( to untreated ) fluorescence intensities were quantified ( n = 3 ) and are presented in ( D ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . E Goss morphology of E10 . 5 ATRA - treated and untreated mice embryos . The ATRA - treated embryo exhibits an open neural tube ( indicated by white arrows ) compared to littermate controls . Scale bar : 500 μm . F Examples of normally developed and maldeveloped mice hearts : in the normal heart ( i ) , the ink travelled from the right ventricle ( RV ) into the pulmonary trunk ( PT ) ; however , in ATRA - treated group hearts , ink travelled into the aorta ( Ao ) ( ii , transposition of great arteries ( TGA ) ) or both the PT and Ao ( iii , double outlet right ventricle ( DORV ) ) . Scale bar : 1000 μm . G Cardiac malformations were analysed by hematoxylin and eosin ( H & E ) staining of heart sections from E14 . 5 mice . The cardiac defects observed in ATRA treated mice are as follows : Ventricular septal defect ( VSD ) and single ventricle defects ( SVD ) . Heart compartments are marked : RA : right atria , RV : right ventricle , LA : left atria , LV : left ventricle . Scale bar : 1000 μmH Total protein N - Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses ( i ) . Western blot was used to detect N - Hcy levels ( ii ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . I Neural tube tissues at E10 were homogenized from litters of untreated , ATRA - treated , and ATRA + AHT co - treated pregnant mice . Neural tube homogenates of each group were mixed from 6 embryos . Endogenous SOD1 and SOD2 were affinity purified from the homogenates . N - Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined . The SOD activities of the untreated group were set at 100 % ; error bar indicates assay replicates ( n = 4 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . J Superoxide ( a ) , apoptosis ( b ) , and Wnt signalling ( c , d ) were over - activated in ATRA - treated mice neural tubes and restored by AHT or NAC treatment . ( a ) Whole mount DHE staining of E10 . 5 mice embryos . Scale bar : 500 μm . Neural tube defect ( white arrows ) and heart ( green arrow ) regions show especially higher fluorescence intensities . ( b ) TUNEL ( green ) staining detected apoptotic cell death in E8 . 5 neural tube ( E ) sections . MARS ( red ) . Scale bar : 300 μm . ( c , d ) IHC staining for β - catenin ( green ) in E8 . 5 ( c ) and E10 ( d ) mice neural tube sections . Scale bar : 300 μm ( c ) , 150 μm ( d ) . DAPI ( blue ) . K Average ( n = 3 ) DHE fluorescence intensities were quantified relative to the untreated group . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . L Quantification of TUNEL - positive cells Three sections from three embryos were pooled for analysis . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . M Quantification of β - catenin fluorescence intensities in experiment J ( b ) and J ( c ) . The average ( n = 3 ) β - catenin level in the untreated group was set at 100 % . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons . N Hcy - treated eggs exhibited NTDs phenotypes at embryonic stage 8 ( E8 ) . A normal embryo ( a ) and neural tube defect phenotypes ( b , c ) are shown . Exencephaly ( white arrows ) and spina bifida ( blue arrows ) are indicated . Scale bar : 500 μm . O Total protein N - Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy + AHT - treated groups were detected by western blot . P Whole mount DHE staining of 72 h chicken embryos from untreated , Hcy - , Hcy + AHT - , and Hcy + NAC - treated groups . Scale bar : 2000 μm . Q Quantification of DHE fluorescence intensities in ( P ) ( n = 3 ) . Data are presented as the mean ± SEM and were compared using an unpaired Student ' s t test . nsnot significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 . One - way ANOVA with Dunnett ' s correction was used for multiple comparisons .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B Microscope images of E10 neural tube sections with the indicated treatments were stained with antibodies against MARS (green) and N-Hcy (red). Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (B). C, D Double immunofluorescent staining (C) for MARS (green) and N-Hcy (red) in E10 heart sections. Scale bar: 150 μm. The relative (to untreated) fluorescence intensities were quantified (n=3) and are presented in (D).Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.E Goss morphology of E10.5 ATRA-treated and untreated mice embryos. The ATRA-treated embryo exhibits an open neural tube (indicated by white arrows) compared to littermate controls. Scale bar: 500 μm.F Examples of normally developed and maldeveloped mice hearts: in the normal heart (i), the ink travelled from the right ventricle (RV) into the pulmonary trunk (PT); however, in ATRA-treated group hearts, ink travelled into the aorta (Ao) (ii, transposition of great arteries (TGA)) or both the PT and Ao (iii, double outlet right ventricle (DORV)). Scale bar: 1000 μm.G Cardiac malformations were analysed by hematoxylin and eosin (H&E) staining of heart sections from E14.5 mice. The cardiac defects observed in ATRA treated mice are as follows: Ventricular septal defect (VSD) and single ventricle defects (SVD). Heart compartments are marked: RA: right atria, RV: right ventricle, LA: left atria, LV: left ventricle. Scale bar: 1000 μmH Total protein N-Hcy levels in maternal mice liver homogenates were determined and quantified in correlation to the corresponding prevalence of NTDs in affected foetuses (i). Western blot was used to detect N-Hcy levels (ii). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.I Neural tube tissues at E10 were homogenized from litters of untreated, ATRA-treated, and ATRA+AHT co-treated pregnant mice. Neural tube homogenates of each group were mixed from 6 embryos. Endogenous SOD1 and SOD2 were affinity purified from the homogenates. N-Hcy levels and the specific activities of each purified SOD1 and SOD2 were determined. The SOD activities of the untreated group were set at 100%; error bar indicates assay replicates (n=4). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.J Superoxide (a), apoptosis (b), and Wnt signalling (c, d) were over-activated in ATRA-treated mice neural tubes and restored by AHT or NAC treatment. (a) Whole mount DHE staining of E10.5 mice embryos. Scale bar: 500 μm. Neural tube defect (white arrows) and heart (green arrow) regions show especially higher fluorescence intensities. (b) TUNEL (green) staining detected apoptotic cell death in E8.5 neural tube (E) sections. MARS (red). Scale bar: 300 μm. (c, d) IHC staining for β-catenin (green) in E8.5 (c) and E10 (d) mice neural tube sections. Scale bar: 300 μm (c), 150 μm (d). DAPI (blue).K Average (n=3) DHE fluorescence intensities were quantified relative to the untreated group. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.L Quantification of TUNEL-positive cells Three sections from three embryos were pooled for analysis. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.M Quantification of β-catenin fluorescence intensities in experiment J (b) and J (c). The average (n=3) β-catenin level in the untreated group was set at 100%. Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.N Hcy-treated eggs exhibited NTDs phenotypes at embryonic stage 8 (E8). A normal embryo (a) and neural tube defect phenotypes (b, c) are shown. Exencephaly (white arrows) and spina bifida (blue arrows) are indicated. Scale bar: 500 μm.O Total protein N-Hcy levels of 72 h chicken embryo homogenates from Hcy and Hcy+AHT-treated groups were detected by western blot.P Whole mount DHE staining of 72 h chicken embryos from untreated, Hcy-, Hcy+AHT-, and Hcy+NAC-treated groups. Scale bar:2000 μm. Q Quantification of DHE fluorescence intensities in (P) (n=3). Data are presented as the mean ± SEM and were compared using an unpaired Student's t test. nsnot significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. One-way ANOVA with Dunnett's correction was used for multiple comparisons.


Figure 1 Time - lapse microscopy of sporozoites , 10 min after intradermal inoculation into mice with CD31 - labeled vascular endothelia . Maximum intensity projection over 240 sec shows trajectories of moving sporozoites ( green ) and blood vessels ( magenta ) . The light green structures in the first two panels are autofluorescent hair follicles . Scale bar , 50 μm .

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

Figure 1 Time-lapse microscopy of sporozoites, 10 min after intradermal inoculation into mice with CD31-labeled vascular endothelia. Maximum intensity projection over 240 sec shows trajectories of moving sporozoites (green) and blood vessels (magenta). The light green structures in the first two panels are autofluorescent hair follicles. Scale bar, 50 μm.


Figure 2 ( A ) P . berghei sporozoite trajectories obtained with manual ( left ) and automated ( right ) tracking methods . ( B ) Image processing and automated tracking method . Shown are images illustrating each step of automated tracking : The raw data image shows the first frame of time - lapse microscopy of P . berghei sporozoites ( red ) after intradermal inoculation , followed by the same image after background subtraction and thresholding using Fiji software . Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software . Scale bars , 50 μm . ( C ) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median speed . No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found ( Kruskal - Wallis test ) . ( D ) Displacement of sporozoites 5 to 120 min after intradermal inoculation , obtained with manual and automated tracking methods . To normalize between tracks of different duration , displacement is displayed as final displacement per 30 seconds interval . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal line indicates the median . The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes ( Kruskal - Wallis test , * * * * p < 0 . 0001 ) . For panels C & D , a varying number of videos were processed for each time point : C : 5 min ( 3 videos / 37 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 112 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 95 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 63 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 77 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 44 manual tracks / 151 automated tracks ) . D : 5 min ( 3 videos / 61 manual tracks / 203 automated tracks ) , 10 min ( 6 videos / 74 manual tracks / 292 automated tracks ) , 20 min ( 4 videos / 47 manual tracks / 184 automated tracks ) , 30 min ( 6 videos / 53 manual tracks / 235 automated tracks ) , 60 min ( 7 videos / 65 manual tracks / 162 automated tracks ) , and 120 min ( 6 videos / 48 manual tracks / 151 automated tracks ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(A) P. berghei sporozoite trajectories obtained with manual (left) and automated (right) tracking methods. (B) Image processing and automated tracking method. Shown are images illustrating each step of automated tracking: The raw data image shows the first frame of time-lapse microscopy of P. berghei sporozoites (red) after intradermal inoculation, followed by the same image after background subtraction and thresholding using Fiji software. Spot detection was run using ICY software and the final image shows the overlay with tracks generated using the spot tracking plugin of ICY software. Scale bars, 50 μm. (C) Apparent speed of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median speed. No statistically significant differences between the corresponding timepoints of manual and automated tracking methods were found (Kruskal-Wallis test). (D) Displacement of sporozoites 5 to 120 min after intradermal inoculation, obtained with manual and automated tracking methods. To normalize between tracks of different duration, displacement is displayed as final displacement per 30 seconds interval. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal line indicates the median. The only statistically significant difference in sporozoite displacement between the manual and automated tracking methods was at 120 minutes (Kruskal-Wallis test, ****p<0.0001). For panels C&D, a varying number of videos were processed for each time point: C: 5 min (3 videos/37 manual tracks/203 automated tracks), 10 min (6 videos/112 manual tracks/292 automated tracks), 20 min (4 videos/95 manual tracks/184 automated tracks), 30 min (6 videos/63 manual tracks/235 automated tracks), 60 min (7 videos/77 manual tracks/162 automated tracks), and 120 min (6 videos/44 manual tracks/151 automated tracks). D: 5 min (3 videos/61 manual tracks/203 automated tracks), 10 min (6 videos/74 manual tracks/292 automated tracks), 20 min (4 videos/47 manual tracks/184 automated tracks), 30 min (6 videos/53 manual tracks/235 automated tracks), 60 min (7 videos/65 manual tracks/162 automated tracks), and 120 min (6 videos/48 manual tracks/151 automated tracks).


Figure 3 ( A - C ) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P . berghei ( A ) , P . yoelii ( B ) and P . falciparum ( C ) at 5 , 10 , 30 and 60 min after intradermal inoculation . For each timepoint , the tracks are representative of the average displacement of the entire sporozoite population . Track numbers used for each timepoint were : 5 min , 40 tracks ; 10 min , 32 tracks ; 30 min , 25 tracks ; 60 min , 21 tracks . ( D - F ) Displacement of P . berghei ( D ) , P . yoelii ( E ) and P . falciparum ( F ) sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show median displacement at each time point . Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point . Within a given species , the difference in sporozoite displacement between 5 min and 120 min is statistically significant ( Kruskal - Wallis test : P . berghei p < 0 . 0001 , P . yoelii p < 0 . 0001 , P . falciparum p < 0 . 01 ) . Comparisons between species showed no statistically significant difference in displacements for any given time point , with the exception of the 10 min time point in which there was a significant difference between P . berghei and P . falciparum ( Kruskal - Wallis test , * p < 0 . 05 ) . For panels ( D - F ) , a varying number of videos were processed for each time point after inoculation : ( D ) Same dataset as used for Figure 2D : 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) and 120 min ( 6 videos / 151 tracks ) . ( E ) 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . ( F ) 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

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

Figure 3  (A-C) Tracks generated through automated tracking were plotted to a common origin to visualize parasite dispersal P. berghei (A), P. yoelii (B) and P. falciparum (C) at 5, 10, 30 and 60 min after intradermal inoculation. For each timepoint, the tracks are representative of the average displacement of the entire sporozoite population. Track numbers used for each timepoint were: 5 min, 40 tracks; 10 min, 32 tracks; 30 min, 25 tracks; 60 min, 21 tracks. (D-F) Displacement of P. berghei (D), P. yoelii (E) and P. falciparum (F) sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show median displacement at each time point. Percentage values in boxes indicate the fraction of tracks displacing over 75 micrometers at each time point. Within a given species, the difference in sporozoite displacement between 5 min and 120 min is statistically significant (Kruskal-Wallis test: P. berghei p<0.0001, P. yoelii p<0.0001, P. falciparum p<0.01). Comparisons between species showed no statistically significant difference in displacements for any given time point, with the exception of the 10 min time point in which there was a significant difference between P. berghei and P. falciparum (Kruskal-Wallis test, *p<0.05). For panels (D-F), a varying number of videos were processed for each time point after inoculation: (D) Same dataset as used for Figure 2D: 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks) and 120 min (6 videos/151 tracks). (E) 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). (F) 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


Figure 4 ( A ) Speed of P . berghei , P . yoelii and P . falciparum sporozoites 5 - 120 min after inoculation in box and whisker plots with whiskers displaying the 10 - 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point ( Kruskal - Wallis test , * * * * p < 0 . 0001 for both comparisons ) and the 120 min time point ( Kruskal - Wallis test , * * p < 0 . 006 for both comparisons ) . ( B ) Linear regression of apparent speed shows a more rapid decrease for P . berghei ( slope = - 0 . 005 ) than for P . yoelii and P . falciparum ( slopes = - 0 . 002 ) . Symbols indicate mean values + / - standard deviation . A varying number of videos were processed for each time point after inoculation , using the same data set used for Figure 3 : P . berghei ; 5 min ( 3 videos / 203 tracks ) , 10 min ( 6 videos / 292 tracks ) , 20 min ( 4 videos / 184 tracks ) , 30 min ( 6 videos / 235 tracks ) , 60 min ( 7 videos / 162 tracks ) , and 120 min ( 6 videos / 151 tracks ) . P . yoelii ; 5 min ( 7 videos / 714 tracks ) , 10 min ( 16 videos / 1514 tracks ) , 20 min ( 4 videos / 415 tracks ) , 30 min ( 4 videos / 319 tracks ) , 60 min ( 4 videos / 255 tracks ) , and 120 min ( 4 videos / 180 tracks ) . P . falciparum ; 5 min ( 6 videos / 389 tracks ) , 10 min ( 6 videos / 464 tracks ) , 20 min ( 3 videos / 184 tracks ) , 30 min ( 2 videos / 195 tracks ) , 60 min ( 2 videos / 239 tracks ) , and 120 min ( 2 videos / 103 tracks ) .

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

Figure 4 (A) Speed of P. berghei, P. yoelii and P. falciparum sporozoites 5-120 min after inoculation in box and whisker plots with whiskers displaying the 10-90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. Comparisons among the species showed statistically significant difference in sporozoite speeds at the 20 min time point (Kruskal-Wallis test, ****p<0.0001 for both comparisons) and the 120 min time point (Kruskal-Wallis test, **p<0.006 for both comparisons). (B) Linear regression of apparent speed shows a more rapid decrease for P. berghei (slope = -0.005) than for P. yoelii and P. falciparum (slopes = -0.002). Symbols indicate mean values +/- standard deviation. A varying number of videos were processed for each time point after inoculation, using the same data set used for Figure 3: P. berghei; 5 min (3 videos/203 tracks), 10 min (6 videos/292 tracks), 20 min (4 videos/184 tracks), 30 min (6 videos/235 tracks), 60 min (7 videos/162 tracks), and 120 min (6 videos/151 tracks). P. yoelii; 5 min (7 videos/714 tracks), 10 min (16 videos/1514 tracks), 20 min (4 videos/415 tracks), 30 min (4 videos/319 tracks), 60 min (4 videos/255 tracks), and 120 min (4 videos/180 tracks). P. falciparum; 5 min (6 videos/389 tracks), 10 min (6 videos/464 tracks), 20 min (3 videos/184 tracks), 30 min (2 videos/195 tracks), 60 min (2 videos/239 tracks), and 120 min (2 videos/103 tracks).


Figure 5 ( A ) The number of motile and non - motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation , for P . berghei ( left panel ) , P . yoelii ( center panel ) , and P . falciparum ( right panel ) . ( B ) Fold - change in the number of motile sporozoites at 60 and 120 min post - inoculation compared to the number of motile sporozoites at 5 min . The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites . ( P . berghei : n = 2 , P . yoelii n = 4 , P . falciparum n = 2 ) .

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

Figure 5  (A) The number of motile and non-motile sporozoites moving in the dermis of mice was manually counted in time courses spanning 120 minutes and is displayed as a percentage of total sporozoites observed 5 min after inoculation, for P. berghei (left panel), P. yoelii (center panel), and P. falciparum (right panel). (B) Fold-change in the number of motile sporozoites at 60 and 120 min post-inoculation compared to the number of motile sporozoites at 5 min. The data shown only includes complete imaging sessions over 120 min after intradermal injection of sporozoites. (P. berghei: n=2, P. yoelii n=4, P. falciparum n=2).


Figure 6 ( A ) Maximum intensity projection shows trajectories of moving P . berghei , P . yoelii and P . falciparum sporozoites ( green ) , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bars , 25 μm . See movies EV4 - EV6 . ( B ) Lymphatic and blood vessel invasion of P . berghei , P . yoelii and P . falciparum sporozoites moving in mouse dermis . Combined invasion events observed in 4 min videos recorded at 5 , 10 and 20 min after intradermal injection of sporozoites ( left panel ) and 60 and 120 min after intradermal injection ( right panel ) . n = number of videos scored for each Plasmodium species . To view a blood vessel entry event by P . yoelii or P . falciparum , see movies EV7 and EV8 . There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P . falciparum sporozoites compared to P . berghei and P . yoelii sporozoites at 60 to 120 min after inoculation ( Kruskal - Wallis test , p < 0 . 05 ) .

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

Figure 6 (A) Maximum intensity projection shows trajectories of moving P. berghei, P. yoelii and P. falciparum sporozoites (green), engaging with CD31-labeled vascular endothelia (magenta). Scale bars, 25 μm. See movies EV4 - EV6. (B) Lymphatic and blood vessel invasion of P. berghei, P. yoelii and P. falciparum sporozoites moving in mouse dermis. Combined invasion events observed in 4 min videos recorded at 5, 10 and 20 min after intradermal injection of sporozoites (left panel) and 60 and 120 min after intradermal injection (right panel). n = number of videos scored for each Plasmodium species. To view a blood vessel entry event by P. yoelii or P. falciparum, see movies EV7 and EV8. There were no statistically significant differences in lymphatic or blood vessel entry among the three Plasmodium species at 5 to 20 min after sporozoite inoculation whereas there was a statistically significant increase in lymphatic entry by P. falciparum sporozoites compared to P. berghei and P. yoelii sporozoites at 60 to 120 min after inoculation (Kruskal-Wallis test, p<0.05).


Figure 7 ( A ) Maximum intensity projection of video acquired 10 min after inoculation of P . falciparum sporozoites visualizes trajectories of parasites , engaging with CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 50 μm . See movie EV9 . ( B ) Displacement of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median at each time point . Percentage values indicate the fraction of tracks displacing over 75 microns . ( C ) Apparent speed of sporozoites 5 to 60 min after inoculation . Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median speed at each time point . For panels B - C , data shown originates from the analysis of 3 - 4 videos per time point and a varying number of tracks were processed for each time point after inoculation : 5 min ( 157 tracks ) , 10 min ( 568 tracks ) , 20 min ( 286 tracks ) , 30 min ( 171 tracks ) , 60 min ( 101 tracks ) . No motile sporozoites were observed 120 min after inoculation . ( D ) Proportion of motile and non - motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation . The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites . ( E ) Sporozoite track straightness , the ratio of displacement to track length of P . falciparum sporozoites moving in mouse dermis , human skin ex vivo , and human skin graft , at 5 - 10 min post intradermal injection . Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values . Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin ( * * * * p = 0 . 0001 ) and human skin ex vivo ( * * * * p < 0 . 0001 ) using Tukey ' s multiple comparisons test . A varying number of tracks were processed for each environment : mouse skin : 17 videos , 822 tracks ; human skin ex vivo : 12 videos , 761 tracks ; human skin graft : 9 videos , 718 tracks . ( F ) P . falciparum sporozoites engage with and enter blood vessels in human skin graft . Maximum intensity projection shows trajectories of moving P . falciparum sporozoites ( green ) , engaging with human CD31 - labeled vascular endothelia ( magenta ) . Scale bar , 25 μm . See movie EV10 . Graph shows lymphatic and blood vessel entry events of P . falciparum sporozoites in human skin graft . Combined invasion events observed at 5 - 20 min after intradermal injection in n number of videos . To view a blood vessel invasion event P . falciparum in human skin graft ,

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

Figure 7 (A) Maximum intensity projection of video acquired 10 min after inoculation of P. falciparum sporozoites visualizes trajectories of parasites, engaging with CD31-labeled vascular endothelia (magenta). Scale bar, 50 μm. See movie EV9. (B) Displacement of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median at each time point. Percentage values indicate the fraction of tracks displacing over 75 microns. (C) Apparent speed of sporozoites 5 to 60 min after inoculation. Data is displayed in box and whisker plots with whiskers showing 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median speed at each time point. For panels B-C, data shown originates from the analysis of 3-4 videos per time point and a varying number of tracks were processed for each time point after inoculation: 5 min (157 tracks), 10 min (568 tracks), 20 min (286 tracks), 30 min (171 tracks), 60 min (101 tracks). No motile sporozoites were observed 120 min after inoculation. (D) Proportion of motile and non-motile sporozoites was manually counted and is displayed as percentage of sporozoites observed 5 min after inoculation. The data shown was obtained from one complete imaging session over 120 min after intradermal injection of sporozoites.  (E) Sporozoite track straightness, the ratio of displacement to track length of P. falciparum sporozoites moving in mouse dermis, human skin ex vivo, and human skin graft, at 5-10 min post intradermal injection. Data is displayed in box and whisker plots with horizontal line showing the median and the whiskers extending to the minimum and maximum values. Comparisons showed statistically significant differences in track straightness between the human skin graft and both mouse skin (****p=0.0001) and human skin ex vivo (****p<0.0001) using Tukey's multiple comparisons test. A varying number of tracks were processed for each environment: mouse skin: 17 videos, 822 tracks; human skin ex vivo: 12 videos, 761 tracks; human skin graft: 9 videos, 718 tracks.  (F) P. falciparum sporozoites engage with and enter blood vessels in human skin graft. Maximum intensity projection shows trajectories of moving P. falciparum sporozoites (green), engaging with human CD31-labeled vascular endothelia (magenta). Scale bar, 25 μm. See movie EV10. Graph shows lymphatic and blood vessel entry events of P. falciparum sporozoites in human skin graft. Combined invasion events observed at 5-20 min after intradermal injection in n number of videos. To view a blood vessel invasion event P. falciparum in human skin graft,


Figure 8 ( A ) Activity of mAb 2A10 on sporozoite speed and displacement : Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually . Horizontal lines show the median . For the displacement data , percentage values indicate the fraction of tracks displacing over 75 microns . Statistical analysis : Mann - Whitney , p = 0 . 0001 ( speed data ) and p = 0 . 0098 ( displacement data ) . Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions , which generated a total number of 217 ( naïve ) and 136 ( 2A10 ) sporozoite tracks . ( B ) Activity of mAb 2A10 on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P . falciparum sporozoite inoculation . 5 videos for each condition , from 5 independent imaging sessions were scored for motility . 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry . Statistical analysis : Percent motile , Mann - Whitney , p = 0 . 0317 ; Blood vessel entry , Fisher ' s exact test , p = 0 . 0163 ( C ) Activity of human mAbs 10 and CIS43 , specific for P . falciparum CSP , and VRC01 , an isotype control human mAb specific for HIV - 1 , on the proportion of motile sporozoites ( left panel ) and blood vessel entry ( right panel ) . Four - minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted . Data are pooled from 7 biological replicates , naïve and VRC01 ( n = 2 ) , naïve and mAb 10 ( n = 2 ) , VRC01 and CIS43 ( n = 3 ) . No comparisons were statistically significant ( Mann - Whitney for percent motile and Fisher ' s exact test for blood vessel entry ) .

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

Figure 8 (A) Activity of mAb 2A10 on sporozoite speed and displacement: Data are displayed in box and whisker plots with whiskers displaying the 10 to 90 percentiles and values below and above the whiskers shown individually. Horizontal lines show the median. For the displacement data, percentage values indicate the fraction of tracks displacing over 75 microns. Statistical analysis: Mann-Whitney, p=0.0001 (speed data) and p=0.0098 (displacement data). Data shown originates from the analysis of 5 videos obtained at 5 separate imaging sessions, which generated a total number of 217 (naïve) and 136 (2A10) sporozoite tracks.  (B) Activity of mAb 2A10 on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with mAb 2A10 and naïve controls were manually counted at 10 min post P. falciparum sporozoite inoculation. 5 videos for each condition, from 5 independent imaging sessions were scored for motility. 4 videos per condition from 4 independent imaging sessions were analyzed for blood vessel entry. Statistical analysis: Percent motile, Mann-Whitney, p=0.0317; Blood vessel entry, Fisher's exact test, p=0.0163   (C) Activity of human mAbs 10 and CIS43, specific for P. falciparum CSP, and VRC01, an isotype control human mAb specific for HIV-1, on the proportion of motile sporozoites (left panel) and blood vessel entry (right panel). Four-minute videos of mice inoculated with the indicated mAbs were taken 10 min after sporozoite inoculation and manually counted. Data are pooled from 7 biological replicates, naïve and VRC01 (n=2), naïve and mAb 10 (n=2), VRC01 and CIS43 (n=3). No comparisons were statistically significant (Mann-Whitney for percent motile and Fisher's exact test for blood vessel entry).


Figure 1B . Diluting concentrations of 4D7 to bound full - length 30 amino acid peptide compared to scrambled , C - terminal and N - terminal 11 amino acid peptides as shown in A ( n = 3 biological replicates ) . F . Proliferation curves of factor - independent TF - 1 TpoR CALRdel61 cells cultured with 2 , 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . G Proliferation curves of factor - independent TF - 1 TpoR CALRdel52 cells cultured with 10 or 20 µg / mL 4D7 or 20 µg / mL of control IgG antibody ( n = 3 biological replicates ) . Data information : bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate , with a one - way ANOVA with Bonferroni correction used to determine statistical significance .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. Diluting concentrations of 4D7 to bound full-length 30 amino acid peptide compared to scrambled, C-terminal and N-terminal 11 amino acid peptides as shown in A (n=3 biological replicates).F. Proliferation curves of factor-independent TF-1 TpoR CALRdel61 cells cultured with 2, 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). G Proliferation curves of factor-independent TF-1 TpoR CALRdel52 cells cultured with 10 or 20 µg/mL 4D7 or 20 µg/mL of control IgG antibody (n=3 biological replicates). Data information: bars represent standard error of the mean summarizing 3 biological replicates performed in triplicate, with a one-way ANOVA with Bonferroni correction used to determine statistical significance.


Figure 2B . Similar experiment using TPO - independent TF - 1 TpoR CALRdel61 cells . C . Similar experiment using TPO - independent TF - 1 TpoR CALRdel52 cells at 8 hours . D . Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours . Additionally , cells were treated with 280 nM of ruxolitinib as a positive control . F . Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF - 1 TpoR cells . An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg / mL 4D7 in TF - 1 TpoR CALRdel61 and TF - 1 TpoR CALRdel52 cells . G . Western blot of TpoR immunoprecipitation under non - reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers ( red arrowheads ) present only in TF - 1 TpoR CALRdel61 disrupted by 8 - hour treatment with 20 µg / mL 4D7 but not PBS or 20 µg / mL IgG . CALR monomers & dimers are detectable by polyclonal anti - wild type CALR or anti - mutant CALR monoclonal antibodies . Red arrowheads , detected mutant CALR protein ; brown arrowheads , detected wildtype CALR protein , asterisk , non - specific bands .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B. Similar experiment using TPO-independent TF-1 TpoR CALRdel61 cells. C. Similar experiment using TPO-independent TF-1 TpoR CALRdel52 cells at 8 hours. D. Similar experiment using PBMNCs from CALRdel52 PMF primary cells at 8 hours. Additionally, cells were treated with 280 nM of ruxolitinib as a positive control.F. Western blot showing caspase 3 cleavage occurring within 24 hours of TPO withdrawal in TPO dependent TF-1 TpoR cells. An increase in cleaved caspase 3 is observed after 48 hours of treatment with 20 µg/mL 4D7 in TF-1 TpoR CALRdel61 and TF-1 TpoR CALRdel52 cells.G. Western blot of TpoR immunoprecipitation under non-reducing conditions showing associated CALR 50 kDa monomers and 100 kDa dimers (red arrowheads) present only in TF-1 TpoR CALRdel61 disrupted by 8-hour treatment with 20 µg/mL 4D7 but not PBS or 20 µg/mL IgG. CALR monomers & dimers are detectable by polyclonal anti-wild type CALR or anti-mutant CALR monoclonal antibodies. Red arrowheads, detected mutant CALR protein; brown arrowheads, detected wildtype CALR protein, asterisk, non-specific bands.


Figure 3B . Graph showing the decreased fold change of CD41 + CD61 + megakaryocytes cultured in 4D7 compared to IgG . FACS - sorted CD34 + from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF , IL - 6 and IL - 9 . Black columns show JAK2V617F mutation positive samples ( n = 4 technical replicates of samples from different patients ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant . C . Number of CD41 + megakaryocytes derived from isolated CD34 + progenitors from myelofibrosis patients . Number of CD41 / CD61 + cells counted on day 12 using trypan blue exclusion ( n = 1 technical replicate ) . D . Summary of fold change reduction of CD41 / CD61 + megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type , normalised to IgG ( n = 11 samples from different patients , with 4 technical replicates per sample ) . Data information : Bars represent standard error of means Unpaired students t - test used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant . E Number of CD41 + megakaryocyte colonies from patient samples after 4D7 treatment . CD34 + from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen - based matrix in presence of 20 µg / mL 4D7 or IgG control ( n = 3 biological replicates ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant . F . Representative micrographs showing CD41 + colonies in pink and CD41 - colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification . Scale bar indicates 100µm . G . Summary of fold change reduction of CD41 + megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG ( n = 4 biological replicates ) . Data information : Bars represent standard error of means Unpaired students t - test used to determine statistical significance * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant . H . Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample ( n = 3 biological replicates ) . Data information : Unpaired students t - test used to determine statistical significance Bars represent standard deviations * , P = 0 . 05 - 0 . 01 , * * , P = 0 . 01 - 0 . 001 , * * * , P = 0 . 001 - 0 . 0001 , * * * * , P < 0 . 0001 , n . s , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. Graph showing the decreased fold change of CD41+CD61+ megakaryocytes cultured in 4D7 compared to IgG. FACS-sorted CD34+ from myelofibrosis patients with CALR or JAK2 mutation were cultured over 12 days without TPO in the presence of SCF, IL-6 and IL-9. Black columns show JAK2V617F mutation positive samples (n= 4 technical replicates of samples from different patients). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.C. Number of CD41+ megakaryocytes derived from isolated CD34+ progenitors from myelofibrosis patients. Number of CD41/CD61+ cells counted on day 12 using trypan blue exclusion (n=1 technical replicate). D. Summary of fold change reduction of CD41/CD61+ megakaryocytes by 4D7 in all tested CALR mutated patient samples compared to CALR wild type, normalised to IgG (n=11 samples from different patients, with 4 technical replicates per sample). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.E Number of CD41+ megakaryocyte colonies from patient samples after 4D7 treatment. CD34+ from patients with myelofibrosis with CALRdel52 or CALRins5 were plated on collagen-based matrix in presence of 20 µg/mL 4D7 or IgG control (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.F. Representative micrographs showing CD41+ colonies in pink and CD41- colonies in blue after treatment with 4D7 or IgG at 100 X or 40X magnification. Scale bar indicates 100µm. G. Summary of fold change reduction of CD41+ megakaryocytes in CALR mutated samples cultured MegaCult treated with 4D7 compared to IgG (n= 4 biological replicates). Data information: Bars represent standard error of means Unpaired students t-test used to determine statistical significance *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.H. Number of megakaryocyte colony forming units grouped according to colony cell number after 4D7 treatment in a mutant CALRins5 sample (n=3 biological replicates). Data information: Unpaired students t-test used to determine statistical significance Bars represent standard deviations *, P = 0.05 - 0.01, **, P = 0.01 - 0.001, ***, P = 0.001 - 0.0001, ****, P <0.0001, n.s, not significant.


Figure 4B . Effect of 4D7 on TPO - dependent megakaryocyte differentiation of healthy CD34 + cells cultured with 20 µg / mL 4D7 or IgG control ( n = 2 biological replicates , with 2 technical replicates ) . Data information : Error bars indicate standard error of mean . C . Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG . CFU - GM , colony forming unit - granulocyte macrophage ; BFU - E , blast forming unit - erythroid ; CFU - GEMM , colony forming unit granulocyte , erythroid , monocyte , megakaryocyte ( n = 3 biological replicates ) . Data information : Error bars indicate standard error of mean . F . Western blot showing signalling in ruxolitinib - resistant TF - 1 TpoR CALRdel61 compared to ruxolitinib sensitive TF - 1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho - STAT5 , total STAT5 , phospho - ERK , total ERK and actin as indicated . Ruxolitinib - sensitive cells were non - viable after 16 hours of treatment . G . Comparison of cell growth after DMSO , 20 µg / mL 4D7 , IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib - resistant TF - 1 TpoR CALRdel61 . Cells counted using Trypan Blue exclusion ( n = 3 biological replicates with 3 technical replicates ) . H . Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO , 20 µg / mL 4D7 , IgG or 100nM ruxolitinib performed in ruxolitinib - resistant TF - 1 TpoR CALRdel61 cells ( n = 3 biological replicates with 3 technical replicates ) . Data information : Error bars on represent standard deviation . All P values are unpaired Student ' s unpaired t - test .

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Figure 4B. Effect of 4D7 on TPO-dependent megakaryocyte differentiation of healthy CD34+ cells cultured with 20 µg/mL 4D7 or IgG control (n= 2 biological replicates, with 2 technical replicates). Data information: Error bars indicate standard error of mean.C. Total numbers of hematopoietic colonies plated in MethoCult from healthy cord blood after treatment with 4D7 or IgG. CFU-GM, colony forming unit- granulocyte macrophage; BFU-E, blast forming unit-erythroid; CFU-GEMM, colony forming unit granulocyte, erythroid, monocyte, megakaryocyte (n=3 biological replicates). Data information: Error bars indicate standard error of mean.F. Western blot showing signalling in ruxolitinib-resistant TF-1 TpoR CALRdel61 compared to ruxolitinib sensitive TF-1 TpoR CALRdel61 cells after treatment with 100nM ruxolitinib for 16 hours or 30 minutes and blotted for phospho-STAT5, total STAT5, phospho-ERK, total ERK and actin as indicated. Ruxolitinib-sensitive cells were non-viable after 16 hours of treatment.G. Comparison of cell growth after DMSO, 20 µg/mL 4D7, IgG or 100 nM ruxolitinib treatment over 4 days of ruxolitinib-resistant TF-1 TpoR CALRdel61. Cells counted using Trypan Blue exclusion (n=3 biological replicates with 3 technical replicates). H. Number of colonies observed from cells plated in MethoCult following 72 hours of treatment with DMSO, 20 µg/mL 4D7, IgG or 100nM ruxolitinib performed in ruxolitinib-resistant TF-1 TpoR CALRdel61 cells (n=3 biological replicates with 3 technical replicates). Data information: Error bars on represent standard deviation. All P values are unpaired Student's unpaired t-test.