A and B Influenza virus specific activation of the murine FcγRI , FcγRII , FcγRIII and FcγRIV by antibodies in pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 mice per group ) . MDCKII cells were infected with Cal / 7 / 9 ( A ) or PR8 ( B ) before mouse serum and FcγR - expressing reporter cells were added . Influenza specific Fc - FcγR interaction induces the production of IL - 2 in the FcγR - expressing reporter cell , which was measured by anti - IL2 ELISA ( n = 3 , technical duplicates ) . Mean ± SE .

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

A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE.


C HA - specific activation of FcγR by AAV - HA , AAV - cHA or WIV pooled pre - challenge sera ( n = 18 mice per group ) . MDCKII cells were transfected with wildtype HA ( pAAV - HA ) or a stalk - only HA ( pAAV - mHL1 ) before mouse serum and FcγR - I - expressing reporter cells were added ( n = 2 , technical duplicates ) . Mean ± SE .

O B-GENEPROD O O O O B-GENEPROD O B-ORGANISM O B-GENEPROD O B-ORGANISM O O O B-ORGANISM O O O O B-TISSUE O O O O B-ORGANISM O O O O B-CELL_LINE O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.


A to F Mice were vaccinated three times with AAV - vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus ( Cal / 7 / 9 ) ( A , B ; n = 5 ) or two different lethal doses of the heterologous PR8 strain ( C , D ; n = 6 and E , F ; n = 7 ) . Survival was monitored over a 14 - day period and is depicted as Kaplan - Meier plot ( A , C , E ) . Weight loss was determined during the 14 - day period , which is shown in relation to the weight at day 0 ( B , D , F ) . Mean ± SE . Statistical significance between negative control group ( AAV - GFP ) or WIV group ( E ) and each vaccine group was determined using Log - Rank ( Mantel - Cox ) test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

A to F Mice were vaccinated three times with AAV-vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus (Cal/7/9) (A, B; n = 5) or two different lethal doses of the heterologous PR8 strain (C, D; n = 6 and E, F; n = 7). Survival was monitored over a 14-day period and is depicted as Kaplan-Meier plot (A, C, E). Weight loss was determined during the 14-day period, which is shown in relation to the weight at day 0 (B, D, F). Mean ± SE. Statistical significance between negative control group (AAV-GFP) or WIV group (E) and each vaccine group was determined using Log-Rank (Mantel-Cox) test (*p<0.05, **p<0.01).


B Cal / 7 / 9 - specific serum antibody titers in individual sera at indicated time points . Black lines : individual animals , red lines : mean titer . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) . The lower table indicates Cal / 7 / 9 - specific MN50 and HAI mean titers . Assays were performed in technical duplicates .

O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-TISSUE O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O

B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates.


C to E Clinical score ( C ) , body temperature ( D ) , and relative weight ( E ) during challenge period . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ± SD

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

C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD


C to E Clinical score ( C ) , body temperature ( D ) , and relative weight ( E ) during challenge period . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ±

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


F Virus titers in nasal turbinates ( NT ) , trachea or lung homogenates . Statistical significance between AAV - GFP and vaccine groups was determined using Kruskal - Wallis test with Dunn ' s multiple comparison testing ( * p < 0 . 05 ) . Lines indicate the GMT . Assays were performed in technical duplicates .

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

F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates.


G and H IHC staining of submucosal glands ( SMG ) ( G ) or bronchi ( H ) with an anti - H1N1 polyclonal antibody ( bar : 100 µm ; red : influenza antigen ( neufuchsin ) ; blue : nuclei ( haematoxylin ) ) .

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

G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)).


I and J H & E stained histological sections of bronchi ( I ) and lung parenchyma with alveolar epithelial cells ( AEC , J ) . Arrowheads : areas of suppurative bronchitis / bronchiolitis ; arrows : interstitial pneumonia , necrosis of AEC ( bar : ( I ) 100 µm ; ( J ) 50 µm ) .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O O B-TISSUE I-TISSUE O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O O O O O O B-DISEASE O B-DISEASE O O O B-DISEASE I-DISEASE O B-DISEASE O B-CELL_TYPE O O O O O O O O O O O O O O O O

I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).


( B ) The concentration - effect relationships of PARP1 inhibition by thioparib , talazoparib , and olaparib , as assayed by histone - based ELISA . Data are obtained from three biological replicates and depicted as mean ± SEM . ( C ) Concentration - dependent increase in PARP1 - DNA binding by three PARP inhibitors using DSB FA assays . FA was measured 60 min after adding NAD + , and the ΔFA values represent the changes in PARP1 - DNA binding . Data from four biological replicates are presented as mean ± SEM . (

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

(B) The concentration-effect relationships of PARP1 inhibition by thioparib, talazoparib, and olaparib, as assayed by histone-based ELISA. Data are obtained from three biological replicates and depicted as mean ± SEM. (C) Concentration-dependent increase in PARP1-DNA binding by three PARP inhibitors using DSB FA assays. FA was measured 60 min after adding NAD+, and the ΔFA values represent the changes in PARP1-DNA binding. Data from four biological replicates are presented as mean ± SEM. (


( F ) and ( G ) Effects of thioparib on BRCA1 - deficient MDA - MB - 436 ( F ) and BRCA2 - deficient Capan - 1 ( G ) xenografts . Mice bearing MDA - MB - 436 tumors were dosed orally with 10 mg / kg thioparib , 100 mg / kg olaparib , or vehicle ( Veh ) once daily for 3 weeks ; and mice bearing Capan - 1 tumors were dosed orally with 5 or 25 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( Veh ) once daily for 3 weeks ( n = 6 ) . Data are shown as mean ± SEM . Statistical analysis was performed by two - way ANOVA . * * * P < 0 . 0001 . Representative images of xenograft tumors are shown , and mice tails represent complete regression of tumors .

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

(F) and (G) Effects of thioparib on BRCA1-deficient MDA-MB-436 (F) and BRCA2-deficient Capan-1 (G) xenografts. Mice bearing MDA-MB-436 tumors were dosed orally with 10 mg/kg thioparib, 100 mg/kg olaparib, or vehicle (Veh) once daily for 3 weeks; and mice bearing Capan-1 tumors were dosed orally with 5 or 25 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (Veh) once daily for 3 weeks (n = 6). Data are shown as mean ± SEM. Statistical analysis was performed by two-way ANOVA. ***P < 0.0001. Representative images of xenograft tumors are shown, and mice tails represent complete regression of tumors.


( C ) The IC50 values of thioparib in six acquired PARPi - resistant cell lines . Numbers 1 - 6 represent the PARPi - resistant Capan - 1 / OP , Capan - 1 / TP , MDA - MB - 436 / OP , MDA - MB - 436 / TP , U251 / OP , and U251 / TP cells , respectively . Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay . The IC50 values are expressed as the mean ± SD from three separate experiments .

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

(C) The IC50 values of thioparib in six acquired PARPi-resistant cell lines. Numbers 1-6 represent the PARPi-resistant Capan-1/OP, Capan-1/TP, MDA-MB-436/OP, MDA-MB-436/TP, U251/OP, and U251/TP cells, respectively. Cells were treated with the indicated PARP inhibitors for 7 days and then subjected to SRB assay. The IC50 values are expressed as the mean ± SD from three separate experiments.


( E ) Efficacy of thioparib in the PDX model BR - 05 - 0028 ( BRCA1 - deficient ) . Mice bearing BR - 05 - 0028 tumors derived from a patient with breast cancer ( n = 6 ) were treated with 10 or 30 mg / kg thioparib or 100 mg / kg olaparib for 6 weeks . Data are depicted as mean ± SEM . * * * P < 0 . 0001 .

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

(E) Efficacy of thioparib in the PDX model BR-05-0028 (BRCA1-deficient). Mice bearing BR-05-0028 tumors derived from a patient with breast cancer (n = 6) were treated with 10 or 30 mg/kg thioparib or 100 mg/kg olaparib for 6 weeks. Data are depicted as mean ± SEM. ***P < 0.0001.


( B ) Concentration - dependent effects of thioparib on the viability of malignant hematologic cells . Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay . Data from three independent experiments were presented as mean ± SEM .

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

(B) Concentration-dependent effects of thioparib on the viability of malignant hematologic cells. Cells were treated with thioparib or other PARP inhibitors for 72 h and then subjected to CCK8 assay. Data from three independent experiments were presented as mean ± SEM.


( D ) Kaplan - Meier survival curve of M - NSG mice transplanted with JeKo - 1 cells treated with 10 mg / kg thioparib , 0 . 3 mg / kg talazoparib , or vehicle ( n = 10 ) . Statistical analysis was performed by log - rank ( Mantel - Cox ) test . * * * P < 0 . 0001 , ns : P = 0 . 3526 .

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

(D) Kaplan-Meier survival curve of M-NSG mice transplanted with JeKo-1 cells treated with 10 mg/kg thioparib, 0.3 mg/kg talazoparib, or vehicle (n = 10). Statistical analysis was performed by log-rank (Mantel-Cox) test. ***P < 0.0001, ns: P = 0.3526.


( B ) Western blotting of γH2AX and phosphor - RPA32 in cells exposed to thioparib , talazoparib , or olaparib for 12 h .

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

(B) Western blotting of γH2AX and phosphor-RPA32 in cells exposed to thioparib, talazoparib, or olaparib for 12 h.


( E ) and ( F ) . Thioparib caused changes in apoptosis - related proteins in JeKo - 1 or THP - 1 cells , as determined by western blotting . Cells were exposed to thioparib , talazoparib , olaparib , or Cpd 391 for 24 h and then subjected to western blotting .

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

(E) and (F). Thioparib caused changes in apoptosis-related proteins in JeKo-1 or THP-1 cells, as determined by western blotting. Cells were exposed to thioparib, talazoparib, olaparib, or Cpd 391 for 24 h and then subjected to western blotting.


( G ) and ( H ) . Depletion of PARP1 in HT - 29 and Capan - 1 / TP cells resulted in a decrease in DNA damage and thioparib resistance . The upper panel shows the changes in γH2AX and phospho - RPA32 in HT - 29 PARP1 knockout ( KO ) clones ( G ) or Capan - 1 / TP PARP1 KO clones ( H ) and their parental cells after exposure to thioparib for 24 h . The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells . Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments . Data are presented as the mean ± SD . ThP : Thioparib , OP : Olaparib , TP : Talazoparib , 391 : Cpd 391 .

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

(G) and (H). Depletion of PARP1 in HT-29 and Capan-1/TP cells resulted in a decrease in DNA damage and thioparib resistance. The upper panel shows the changes in γH2AX and phospho-RPA32 in HT-29 PARP1 knockout (KO) clones (G) or Capan-1/TP PARP1 KO clones (H) and their parental cells after exposure to thioparib for 24 h. The lower panel shows the IC50 values of thioparib in these PARP1 KO clones and parental cells. Cells were treated with thioparib for 5 or 7 d and the IC50 values were determined by SRB assay from three separate experiments. Data are presented as the mean ± SD. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391.


( A ) HR repair assays in U2OS - DR - GFP cells treated with increasing concentrations of thioparib . Cells were incubated with the indicated drugs at the time of I - SceI introduction for 48 h . GFP - positive cells were analyzed after I - SceI transfection for 72 h by flow cytometry . The ATR inhibitor VE - 821 was used as a positive control . Three independent experiments were performed and data are expressed as mean ± SD . Statistical analysis was performed by one - way ANOVA , and all groups were compared with control . * * * P < 0 . 0001 , * P = 0 . 0205 , 0 . 0101 ( from left to right ) .

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

(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).


( C ) Representative images of RAD51 foci in U2OS - DR - GFP cells with or without radiation . Graphs ( right panel ) show quantification of cells with ≥ 5 RAD51 foci in vehicle - and drug - treated cells . Scale bar : 5 μm . Data are obtained from three biological replicates and presented as mean ± SD . Statistical analysis was performed by one - way ANOVA . Left to right : P < 0 . 0001 , = 0 . 0033 , < 0 . 0001 , = 0 . 0006 , = 0 . 0031 , = 0 . 0015 .

O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.


( H ) HR repair assay . U2OS - DR - GFP ( WT ) and PARP1 KO cells ( KO1 and KO2 ) were treated with the indicated drugs for 48 h and collected after 72 h of I - SceI transfection as in ( A ) . VE - 821 served as a positive control . PARP1 knockout efficiency was evaluated by western blotting . Data from three biological replicates are presented as mean ± SD . Statistical analysis was performed by one - way ANOVA , and each group was compared with the control . * * * P < 0 . 0001 , * P = 0 . 0143 , 0 . 0483 , ns : P = 0 . 3061 , 0 . 8269 , 0 . 9997 , 0 . 3906 ( from left to right ) .

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

(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).


( C ) Effects of thioparib on CXCL9 , CXCL10 , and IL15 mRNA in JeKo - 1 cells . Data from three independent experiments are shown as the mean ± SD . Statistical analysis was performed by two - way ANOVA . Left panel : * P = 0 . 0367 , * * P = 0 . 0011 ; median panel : * * * P = 0 . 0002 , < 0 . 0001 , < 0 . 0001 ; right panel : * * * P = 0 . 0005 , 0 . 0009 , 0 . 0001 ( from left to right ) .

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(C) Effects of thioparib on CXCL9, CXCL10, and IL15 mRNA in JeKo-1 cells. Data from three independent experiments are shown as the mean ± SD. Statistical analysis was performed by two-way ANOVA. Left panel: *P = 0.0367, **P = 0.0011; median panel: ***P = 0.0002, < 0.0001, < 0.0001; right panel: ***P = 0.0005, 0.0009, 0.0001 (from left to right).


( D ) The protein levels of p - STAT1 , p - TBK1 , γH2AX , and phospho - RPA32 in HT - 29 parent , PARP1 − / − ( # KO1 ) , and PARP7 − / − cell lines after 24 or 48 h thioparib treatment .

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(D) The protein levels of p-STAT1, p-TBK1, γH2AX, and phospho-RPA32 in HT-29 parent, PARP1−/− (#KO1), and PARP7−/− cell lines after 24 or 48 h thioparib treatment.


( F ) Western blot analysis of p - STAT1 , p - TBK1 , and γH2AX in HT - 29 parent , STING − / − , and TBK1 − / − cells after 24 or 48 h thioparib treatment .

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

(F) Western blot analysis of p-STAT1, p-TBK1, and γH2AX in HT-29 parent, STING−/−, and TBK1−/−cells after 24 or 48 h thioparib treatment.


( H ) Immunohistochemistry staining of CD4 , CD8 , and CD45 expression in MC38 tumor tissue sections . Quantification of cells positive of CD4 , CD8 , CD45 were shown . Scale bar : 25 μm . Data from ten technical replicates from each of the two mice are presented as mean ± SD . Statistical analysis was performed by unpaired t - test . * * * P < 0 . 0001 , ns : P = 0 . 1958 .

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

(H) Immunohistochemistry staining of CD4, CD8, and CD45 expression in MC38 tumor tissue sections. Quantification of cells positive of CD4, CD8, CD45 were shown. Scale bar: 25 μm. Data from ten technical replicates from each of the two mice are presented as mean ± SD. Statistical analysis was performed by unpaired t-test. ***P < 0.0001, ns: P = 0.1958.


Western blot analysis of soluble and insoluble fractions ( Red - and yellow rectangles , highlights CTF - 35 and CTF - 25 respectively in the insoluble fraction ) .

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Western blot analysis of soluble and insoluble fractions (Red- and yellow rectangles, highlights CTF-35 and CTF-25 respectively in the insoluble fraction).


Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP - 43 ( pTDP - 43 ) . Scale bar : 20µm . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . I represents its corresponding profile intensity plot of the selected ( square ) region . Scale bar : 10µm , J quantification pooled control ( C0 = 10 , C1 = 10 and C2 = 10 ) n = 30 and pooled mutant TDP - 43 ( P1 = 10 , P2 = 10 and P3 = 10 ) n = 30 cells from each differentiation . Unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of phosphorylated TDP-43 (pTDP-43). Scale bar: 20µm. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). I represents its corresponding profile intensity plot of the selected (square) region. Scale bar: 10µm, J quantification pooled control (C0=10, C1=10 and C2=10) n=30 and pooled mutant TDP-43 (P1=10, P2=10 and P3=10) n=30 cells from each differentiation. Unpaired Mann-Whitney-test.


Western blots showing nucleo - cytoplasmic fractionation of WT - mCherry and MUT - mCherry iPSC - derived motor neurons with F ( left panel ) quantification of total mCherry and total untagged TDP - 43 on α - tubulin and ( right panel ) relative cytoplasmic mCherry and untagged TDP - 43 on total mCherry and total untagged TDP - 43

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Western blots showing nucleo-cytoplasmic fractionation of WT-mCherry and MUT-mCherry iPSC-derived motor neurons with F (left panel) quantification of total mCherry and total untagged TDP-43 on α-tubulin and (right panel) relative cytoplasmic mCherry and untagged TDP-43 on total mCherry and total untagged TDP-43 respectively


Representative kymographs from control and mutant TDP - 43 iPSC - derived motor neurons . The x - axis represent distance in µm while on the y - axis time in seconds is revealed . Stationary mitochondria are visible as straight vertical lines , while moving mitochondria are depicted as skewed lines . B Quantification of percentage of moving mitochondria .

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Representative kymographs from control and mutant TDP-43 iPSC-derived motor neurons. The x-axis represent distance in µm while on the y-axis time in seconds is revealed. Stationary mitochondria are visible as straight vertical lines, while moving mitochondria are depicted as skewed lines. B Quantification of percentage of moving mitochondria.


Immunofluorescent analysis showing fluorescence intensity of pTDP - 43 in motor neurons . Scale bar : 10µm and its perspective intensity plot in F . G Quantification of E , each dot represents one analysed cell , unpaired Mann - Whitney - test .

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Immunofluorescent analysis showing fluorescence intensity of pTDP-43 in motor neurons. Scale bar: 10µm and its perspective intensity plot in F. G Quantification of E, each dot represents one analysed cell, unpaired Mann-Whitney-test.


axonal transport analysis . Quantification of percentage of moving mitochondria ( J ) , number of moving mitochondria ( K ) and number of stationary mitochondria ( L ) normalized to neurite length 100µm .

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

axonal transport analysis. Quantification of percentage of moving mitochondria (J), number of moving mitochondria (K) and number of stationary mitochondria (L) normalized to neurite length 100µm.


Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot , representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment .

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Total cell lysates were fractionated into soluble and insoluble fractions and analysed by Western blot, representative Western blots of soluble and insoluble fraction without C and with D HDAC6 inhibitor treatment.


Immunofluorescent analysis of TDP - 43 in control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor after 12 h treatment . Presented immunofluorescent images of control ( C2 ) and mutant TDP - 43 ( P2 ) . Scale bar : 25µm . B Profile intensity plot of control , mutant TDP - 43 and mutant TDP - 43 + HDAC6 inhibitor . C Quantification of nucleo - cytoplasmic ratio ( N / C ) fluorescent intensity of TDP - 43 in motor neurons , each dot represents one analyzed cell , unpaired Mann - Whitney - test .

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Immunofluorescent analysis of TDP-43 in control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor after 12 h treatment. Presented immunofluorescent images of control (C2) and mutant TDP-43 (P2). Scale bar: 25µm. B Profile intensity plot of control, mutant TDP-43 and mutant TDP-43+HDAC6 inhibitor. C Quantification of nucleo-cytoplasmic ratio (N/C) fluorescent intensity of TDP-43 in motor neurons, each dot represents one analyzed cell, unpaired Mann-Whitney-test.


Quantification of relative nuclear mCherry TDP - 43 on total fraction , ratio paired - t - test .

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

Quantification of relative nuclear mCherry TDP-43 on total fraction, ratio paired-t-test.


Quantification of percentage of moving mitochondria ,

Quantification of percentage of moving mitochondria,


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . A - F . Clinical parameters determined in the study include age ( A ) , gender ( B ) , parasitemia ( C ) , hemoglobin ( g / dL blood ) ( D ) , hematocrit ( E ) , and platelet count ( F ) . Boxes represent the 25th to 75th percentiles , whiskers show the range ( minimum to maximum ) , and lines represent the median . Significance was determined by the Kruskal - Wallis test with Dunn ' s multiple comparisons ( A , D - F ) , the Chi - square test ( B ) , and the Mann - Whitney test ( C ) using 30 ( symptomatic ) , 40 ( asymptomatic ) and 31 ( healthy immune controls ) biological replicates . * * * * p < 0 . 0001 .

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

P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. A-F. Clinical parameters determined in the study include age (A), gender (B), parasitemia (C), hemoglobin (g/dL blood) (D), hematocrit (E), and platelet count (F). Boxes represent the 25th to 75th percentiles, whiskers show the range (minimum to maximum), and lines represent the median. Significance was determined by the Kruskal-Wallis test with Dunn's multiple comparisons (A, D-F), the Chi-square test (B), and the Mann-Whitney test (C) using 30 (symptomatic), 40 (asymptomatic) and 31 (healthy immune controls) biological replicates. ****p<0.0001.


P . falciparum symptomatic ( n = 30 ) and asymptomatic ( n = 40 ) infected individuals , as well as light - microscopy and PCR parasite - negative healthy immune controls ( n = 31 ) were recruited for the study . G . Antibody titers specific for P . falciparum parasite lysate . Bars represent the mean ± SD of using 29 ( symptomatic ) , 35 ( asymptomatic ) and 26 ( healthy immune controls ) biological replicates . The dotted line depicts the average antibody background levels of malaria - naive healthy Melbourne controls . H . Mean antibody titers as determined by ELISA to the P . falciparum recombinant proteins EBA - 175 , EBA - 140 , PfRh2 , PfRh4 , PfRh5 , and PfRipr .

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

P. falciparum symptomatic (n=30) and asymptomatic (n=40) infected individuals, as well as light-microscopy and PCR parasite-negative healthy immune controls (n=31) were recruited for the study. G. Antibody titers specific for P. falciparum parasite lysate. Bars represent the mean ± SD of using 29 (symptomatic), 35 (asymptomatic) and 26 (healthy immune controls) biological replicates. The dotted line depicts the average antibody background levels of malaria-naive healthy Melbourne controls. H. Mean antibody titers as determined by ELISA to the P. falciparum recombinant proteins EBA-175, EBA-140, PfRh2, PfRh4, PfRh5, and PfRipr.


PBMCs from P . falciparum symptomatic ( n = 16 ) and asymptomatic ( n = 24 ) infected individuals as well as healthy immune controls ( n = 24 ) were stained with a panel of metal - labelled antibodies and analysed by CyTOF . tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub - populations within gated : A . TH1 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 + ) B . TH2 - like memory CD4 + T cells ( CD19 - CD3 + CD4 + CD45RA - CCR6 - CXCR3 - ) C . Circulating memory TFH cells ( CD19 - CD3 + CD4 + CD45RA - CXCR5 + ) D . Classical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 + ) E . Atypical MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 - CD21 - ) F . Activated MBCs ( CD3 - CD19 + CD20 + CD10 - CD27 + CD21 - ) The tSNE plots in the top panel display cell density and represent the pooled data for each group , while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot . Heatmaps show the median marker expression for each FlowSOM cluster .

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

PBMCs from P. falciparum symptomatic (n=16) and asymptomatic (n=24) infected individuals as well as healthy immune controls (n=24) were stained with a panel of metal-labelled antibodies and analysed by CyTOF. tSNE analysis was performed and FlowSOM clustering was used to identify individual cell sub-populations within gated: A. TH1-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3+) B. TH2-like memory CD4+ T cells (CD19-CD3+CD4+CD45RA-CCR6-CXCR3-) C. Circulating memory TFH cells (CD19-CD3+CD4+CD45RA-CXCR5+) D. Classical MBCs (CD3-CD19+CD20+CD10-CD27+CD21+) E. Atypical MBCs (CD3-CD19+CD20+CD10-CD27-CD21-) F. Activated MBCs (CD3-CD19+CD20+CD10-CD27+CD21-) The tSNE plots in the top panel display cell density and represent the pooled data for each group, while the lower panel shows a projection of the FlowSOM clusters on a tSNE plot. Heatmaps show the median marker expression for each FlowSOM cluster.


Unsupervised identification of differentially abundant high - dimensional cell populations between clinical groups was performed using CITRUS ( FDR < 5 % ) . between P . falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls . A - E . tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers , relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3 + CCR6 - memory CD4 + T cells ( A ) , CXCR3 - CCR6 - memory CD4 + T cells ( B ) , circulating memory TFH cells ( C ) , classical MBCs ( D ) , and atypical MBCs ( E ) . The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays . The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay , whiskers showing the range ( minimum to maximum ) , with lines representing the median of 16 ( symptomatic ) , 24 ( asymptomatic ) and 24 ( healthy immune controls ) biological replicates , while the pink histograms illustrate marker expression in identified differentially abundant populations , relative to background expression , shown in lilac .

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Unsupervised identification of differentially abundant high-dimensional cell populations between clinical groups was performed using CITRUS (FDR<5%). between P. falciparum symptomatic infected individuals and asymptomatic participants or healthy immune controls. A-E. tSNE plots of clinical groups and viSNE plots depicting expression of selected surface markers, relative abundance and cellular phenotype of differentially abundant clusters identified among CXCR3+CCR6- memory CD4+ T cells (A), CXCR3-CCR6- memory CD4+ T cells (B), circulating memory TFH cells (C), classical MBCs (D), and atypical MBCs (E). The tSNE plots in the top or each panel display cell density and represent pooled data for each group as calculated in the clustering analysis while the viSNE plots on each top panel depict relevant marker expression on tSNE overlays. The lower panels from left to right show differentially abundant populations identified in purple on a tSNE overlay, whiskers showing the range (minimum to maximum), with lines representing the median of 16 (symptomatic), 24 (asymptomatic) and 24 (healthy immune controls) biological replicates, while the pink histograms illustrate marker expression in identified differentially abundant populations, relative to background expression, shown in lilac.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . A . Estimated proportions of PBMC sub - populations determined from cell - type deconvolution from study participant transcriptional profiles .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. A. Estimated proportions of PBMC sub-populations determined from cell-type deconvolution from study participant transcriptional profiles.


PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) infected individuals as well as healthy immune controls ( n = 6 ) were selected for RNA - seq analysis . D . Blood transcriptional module ( BTM ) analysis showing significant BTMs differentially enriched for pairwise comparisons ( FDR < 15 % ) .

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PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=5) infected individuals as well as healthy immune controls (n=6) were selected for RNA-seq analysis. D. Blood transcriptional module (BTM) analysis showing significant BTMs differentially enriched for pairwise comparisons (FDR<15%).


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . A . Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes ( DEGs ) between symptomatic P . falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance . B . Mean - difference plot displaying genes differentially expressed between symptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in symptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. A. Unsupervised hierarchical clustering heat map of the 922 differentially expressed genes (DEGs) between symptomatic P. falciparum malaria and healthy immune controls using the complete method and Euclidian measure of distance. B. Mean-difference plot displaying genes differentially expressed between symptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in symptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . F . Upstream regulator analysis of the 922 DEGs between symptomatic P . falciparum malaria and healthy immune controls . The red lines represent a significant activation z - score of ± 2 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. F. Upstream regulator analysis of the 922 DEGs between symptomatic P. falciparum malaria and healthy immune controls. The red lines represent a significant activation z-score of ±2.


Gene expression profiles of PBMCs from P . falciparum symptomatic and healthy immune controls were compared . Hierarchical clustering heatmap ( G ) displaying DEGs in symptomatic P . falciparum malaria and healthy immune controls predicted to be controlled by IFN - γ and IL - 13 upstream regulators .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and healthy immune controls were compared. Hierarchical clustering heatmap (G) displaying DEGs in symptomatic P. falciparum malaria and healthy immune controls predicted to be controlled by IFN-γ and IL-13 upstream regulators.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . C . Heat map of the 418 DEGs in P . falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls . E . Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2 - fold - change and - Log10 ( P - value ) differentially expressed between P . falciparum symptomatic and asymptomatic infected individuals . Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. C. Heat map of the 418 DEGs in P. falciparum symptomatic and asymptomatic infected participants as well as in healthy immune controls. E. Volcano plot displaying selected genes clusters 1 and 2 from heatmap in C scaled by Log2-fold-change and -Log10(P-value) differentially expressed between P. falciparum symptomatic and asymptomatic infected individuals. Genes in red are overrepresented in symptomatic malaria and genes in blue are overrepresented in asymptomatic malaria.


Gene expression profiles of PBMCs from P . falciparum symptomatic and asymptomatic infected participants were compared . F . Mean RPKMs + SEM of selected genes in P . falciparum malaria symptomatic ( n = 6 ) and asymptomatic ( n = 5 ) individuals . Dotted red lines depict transcriptional levels of healthy immune controls , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of PBMCs from P. falciparum symptomatic and asymptomatic infected participants were compared. F. Mean RPKMs + SEM of selected genes in P. falciparum malaria symptomatic (n=6) and asymptomatic (n=5) individuals. Dotted red lines depict transcriptional levels of healthy immune controls, Mann-Whitney test of biological replicates,*p<0.05.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . A . Hierarchical clustering heat map of the 171 differentially expressed genes ( DEGs ) in P . falciparum asymptomatic and healthy immune control participants . C . Mean - difference plot displaying DEGs between asyptomatic P . falciparum malaria and healthy immune controls . Each gene is plotted as a single point determined by log - fold - change and average transcript abundance . Red genes are overrepresented , and blue genes are underrepresented in asymptomatic malaria .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. A. Hierarchical clustering heat map of the 171 differentially expressed genes (DEGs) in P. falciparum asymptomatic and healthy immune control participants. C. Mean-difference plot displaying DEGs between asyptomatic P. falciparum malaria and healthy immune controls. Each gene is plotted as a single point determined by log-fold-change and average transcript abundance. Red genes are overrepresented, and blue genes are underrepresented in asymptomatic malaria.


Gene expression profiles of bulk PBMCs from P . falciparum - infected asymptomatic individuals and healthy immune controls were compared . E . Mean RPKMs + SEM of selected genes in P . falciparum malaria asymptomatic individuals ( n = 5 ) and healthy immune controls ( n = 6 ) . Dotted red lines depict transcriptional levels of P . falciparum - infected symptomatic individuals , Mann - Whitney test of biological replicates , * p < 0 . 05 .

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Gene expression profiles of bulk PBMCs from P. falciparum-infected asymptomatic individuals and healthy immune controls were compared. E. Mean RPKMs + SEM of selected genes in P. falciparum malaria asymptomatic individuals (n=5) and healthy immune controls (n=6). Dotted red lines depict transcriptional levels of P. falciparum-infected symptomatic individuals, Mann-Whitney test of biological replicates, *p<0.05.


A . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM + activated MBCs . B . Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T - bet + CD4 + T cells .

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A. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with IgM+ activated MBCs. B. Volcano plot showing genes upregulated in symptomatic individuals that were significantly correlated with T-bet+ CD4+ T cells.


B - G . PBMCs from P . falciparum symptomatic ( n = 6 ) and asymptomatic ( n = 6 ) infected individuals as well as healthy immune controls ( n = 6 ) were stained with a panel of antibodies and analyzed by flow cytometry . tSNE plots ( B ) display cell density of the pooled data for each group , ( C ) shows a projection of the FlowSOM clusters on a tSNE plot , and ( D ) is a heatmap depicting the celluar phenotype of clusters of CTLA - 4 + CD3 + CD19 - cells . Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups ( E ) , Mann - Whitney test , * p < 0 . 05 . The panel on the left shows the average CTLA - 4 mean fluorescent intensity ( MFI ) or 6 biological replicates in cluster 4 + SEM , Mann - Whitney test , * * p < 0 . 01 , while representative histograms of CTLA - 4 expression in cluster 4 are shown on the right ( F ) . Marker intensity within clusters 1 and 6 ( G ) .

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B-G. PBMCs from P. falciparum symptomatic (n=6) and asymptomatic (n=6) infected individuals as well as healthy immune controls (n=6) were stained with a panel of antibodies and analyzed by flow cytometry. tSNE plots (B) display cell density of the pooled data for each group, (C) shows a projection of the FlowSOM clusters on a tSNE plot, and (D) is a heatmap depicting the celluar phenotype of clusters of CTLA-4+CD3+CD19- cells. Bar plots representing mean relative abundance of biological replicates + SEM of relevant clusters among clinical groups (E), Mann-Whitney test, *p<0.05. The panel on the left shows the average CTLA-4 mean fluorescent intensity (MFI) or 6 biological replicates in cluster 4 + SEM, Mann-Whitney test, **p<0.01, while representative histograms of CTLA-4 expression in cluster 4 are shown on the right (F). Marker intensity within clusters 1 and 6 (G).


Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA , B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice ( heterozygous ; 3 ( n = 14 ) , 6 ( n = 12 ) , 8 ( n = 10 ) , 12 ( n = 11 ) , 19 ( n = 9 ) , and 25 ( n = 6 ) months of age ) . CSF Aβ40 ( F ( 1 , 56 ) = 22 . 351 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 56 ) = 38 . 597 , P < 0 . 001 ) followed a significant quadratic trend .

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Human Aβ exhibits a biphasic profile in CSF of APP transgenic miceA, B Aβ40 and Aβ42 concentrations in CSF of male APP23 mice (heterozygous; 3 (n = 14), 6 (n = 12), 8 (n = 10), 12 (n = 11), 19 (n = 9), and 25 (n = 6) months of age). CSF Aβ40 (F(1, 56) = 22.351, P < 0.001) as well as CSFAβ42 (F(1, 56) = 38.597, P < 0.001) followed a significant quadratic trend.


C Aβ42 / 40 ratio in CSF of APP23 mice showed a delayed decrease with age ( F ( 1 , 56 ) = 53 . 894 , P < 0 . 001 ) .

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C Aβ42/40 ratio in CSF of APP23 mice showed a delayed decrease with age (F(1, 56) = 53.894, P < 0.001).


D , E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice ( homozygous ; 2 ( n = 13 ) , 3 - 4 ( n = 16 ) , 7 - 8 ( n = 15 ) , 18 - 19 ( n = 14 ) , 24 ( n = 16 ) , and 30 ( n = 18 ) months of age ) . CSF Aβ40 followed a significant quadratic trend ( F ( 1 , 86 ) = 6 . 678 , P = 0 . 011 ) and CSF Aβ42 best fitted a cubic trend ( F ( 1 , 86 ) = 30 . 599 , P < 0 . 001 ) .

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D, E Aβ40 and Aβ42 concentrations in CSF of male and female APP24 mice (homozygous; 2 (n = 13), 3-4 (n = 16), 7-8 (n = 15), 18-19 (n = 14), 24 (n = 16), and 30 (n = 18) months of age). CSF Aβ40 followed a significant quadratic trend (F(1, 86) = 6.678, P = 0.011) and CSF Aβ42 best fitted a cubic trend (F(1, 86) = 30.599, P < 0.001).


F Aβ42 / 40 ratio in CSF of APP24 mice showed a delayed decrease with age ( F ( 1 , 86 ) = 64 . 936 , P < 0 . 001 ) .

F Aβ42/40 ratio in CSF of APP24 mice showed a delayed decrease with age (F(1, 86) = 64.936, P < 0.001).


G , H Aβ40 and Aβ42 in the CSF of female APP51 mice ( heterozygous ; 3 ( n = 6 ) , 15 ( n = 8 ) , and 24 ( n = 8 ) months of age ; 22 mice in total ) . CSF Aβ40 ( F ( 1 , 19 ) = 37 . 349 , P < 0 . 001 ) as well as CSFAβ42 ( F ( 1 , 19 ) = 107 . 670 , P < 0 . 001 ) followed a significant quadratic trend .

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G, H Aβ40 and Aβ42 in the CSF of female APP51 mice (heterozygous; 3 (n = 6), 15 (n = 8), and 24 (n = 8) months of age; 22 mice in total). CSF Aβ40 (F(1, 19) = 37.349, P < 0.001) as well as CSFAβ42 (F(1, 19) = 107.670, P < 0.001) followed a significant quadratic trend.


I Aβ42 / 40 ratio in CSF of APP51 mice showed a delayed decrease with age ( F ( 1 , 19 ) = 26 . 367 , P < 0 . 001 ) .

I Aβ42/40 ratio in CSF of APP51 mice showed a delayed decrease with age (F(1, 19) = 26.367, P < 0.001).


HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1 . CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group .

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HumanAβ in CSF and brain of APP transgenic miceA APP23CSFAβ40 and Aβ42 in the same animals as shown in Fig1. CSFAβ42 and Aβ40 are expressed as percentages of levels measured in the youngest age group.


B , C Aβ40 and Aβ42 ( pmol / g wet brain ) in the FA - soluble brain extract from the same APP23mice showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 56 ) = 221 . 114 , P < 0 . 001 and F ( 1 , 56 ) = 370 . 947 , P < 0 . 001 , respectively ) .

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B, C Aβ40 and Aβ42 (pmol/g wet brain) in the FA-soluble brain extract from the same APP23mice showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 56) = 221.114, P < 0.001 and F(1, 56) = 370.947, P < 0.001, respectively).


D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group .

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D APP24 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentage of the youngest age group.


E , F Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP24mice also showed a robust increase with age ; ANOVA revealed a significant cubic trend ( F ( 1 , 86 ) = 202 . 173 , P < 0 . 001 and F ( 1 , 86 ) = 139 . 941 , P < 0 . 001 , respectively ) .

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E, F Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP24mice also showed a robust increase with age; ANOVA revealed a significant cubic trend (F(1, 86) = 202.173, P < 0.001 and F(1, 86) = 139.941, P < 0.001, respectively).


G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group .

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G APP51 CSF Aβ40 and Aβ42 in the same animals shown in Fig1 as percentages of levels in the youngest age group.


H , I Aβ40 and Aβ42 ( pmol / g wet brain ) in the brain from the same APP51 mice showed a robust increase with age ; ANOVA revealed a significant quadratic trend ( F ( 1 , 19 ) = 12 . 960 , P = 0 . 002 and F ( 1 , 19 ) = 19 . 366 , P < 0 . 001 , respectively ) .

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H, I Aβ40 and Aβ42 (pmol/g wet brain) in the brain from the same APP51 mice showed a robust increase with age; ANOVA revealed a significant quadratic trend (F(1, 19) = 12.960, P = 0.002 and F(1, 19) = 19.366, P < 0.001, respectively).


Aβplaque pathology in the brains of APP23miceAβimmunostaining ( CN3 antibody , dark blue ) in 25 - μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6 - to 8 - month - old APP23mice . At 12 months and thereafter , there is a progressive increase in plaque number and size and a progressive involvement of different brain regions . Insets highlight the plaque characteristics at the different ages . Scale bar , 100 μm . The mean number of Aβ plaques per section per hemibrain increased with age in 3 - to 12 - month - old mice . Only four mice were analyzed in the 3 - month - old age group , as APP23mice do not develop plaques at this age ( Sturchler - Pierrat et al , 1997 ) . The 6 - , 8 - , and 12 - month - old age groups included 12 , 10 , and 11 mice , respectively ( these are the same mice that were used for CSF and brainAβ measurements ) . Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age . Data are represented as group means ± SEM .

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Aβplaque pathology in the brains of APP23miceAβimmunostaining (CN3 antibody, dark blue) in 25-μm sagittal brain sections shows only sparse Aβ deposits primarily in the frontal cortex of 6- to 8-month-old APP23mice. At 12 months and thereafter, there is a progressive increase in plaque number and size and a progressive involvement of different brain regions. Insets highlight the plaque characteristics at the different ages. Scale bar, 100 μm.The mean number of Aβ plaques per section per hemibrain increased with age in 3- to 12-month-old mice. Only four mice were analyzed in the 3-month-old age group, as APP23mice do not develop plaques at this age (Sturchler-Pierrat et al, 1997). The 6-, 8-, and 12-month-old age groups included 12, 10, and 11 mice, respectively (these are the same mice that were used for CSF and brainAβ measurements). Note that the Aβ plaques became too numerous and often could no longer be individually distinguished in the age groups > 12 months of age. Data are represented as group means ± SEM.


Brain sAPPβ shows an age - related increase in APP23 , APP24 , and APP51 micesAPPβ was measured in Triton X - 100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group . Swedish sAPPβ showed an age - dependent increase in APP23 mice following a linear trend ( F ( 1 , 83 ) = 52 . 914 , P < 0 . 001 ) ; APP23 from two independent batches were included in this analysis ( see Materials and Methods and Supplementary Fig S2 for details ) . Swedish sAPPβ showed an age - dependent increase in APP24mice following a linear trend ( F ( 1 , 84 ) = 11 . 264 , P = 0 . 001 ) . Human wild - type sAPPβ showed an age - dependent increase in APP51 following a quadratic trend ( F ( 1 , 18 ) = 68 . 980 , P < 0 . 001 ) . Data information : Post hoc Dunnett ' s test group comparisons were always conducted between the youngest group and all other groups . All data are represented as group means ± SEM ; * P < 0 . 05 ; * * P < 0 . 01 ; and * * * P < 0 . 001 . For absolute values , see Supplementary Fig S2 .

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Brain sAPPβ shows an age-related increase in APP23, APP24, and APP51 micesAPPβ was measured in Triton X-100 brain extracts from largely the same mice as analyzed in Figs1 and2 and is expressed as percentages of levels measured in the youngest age group.Swedish sAPPβ showed an age-dependent increase in APP23 mice following a linear trend (F(1, 83) = 52.914, P < 0.001); APP23 from two independent batches were included in this analysis (see Materials and Methods and Supplementary Fig S2 for details).Swedish sAPPβ showed an age-dependent increase in APP24mice following a linear trend (F(1, 84) = 11.264, P = 0.001).Human wild-type sAPPβ showed an age-dependent increase in APP51 following a quadratic trend (F(1, 18) = 68.980, P < 0.001).Data information: Post hoc Dunnett's test group comparisons were always conducted between the youngest group and all other groups. All data are represented as group means ± SEM; *P < 0.05; **P < 0.01; and ***P < 0.001. For absolute values, see Supplementary Fig S2.


( B ) Quantification of embryos that expressing the ZGA reporter ( MERVL : : tdTomato ) after injection with a siRNA - repressor or mRNA - inducer . N , the total number of embryos analyzed for each condition . Error bars , mean ± S . D . ; n = 3 biological replicates per group .

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(B) Quantification of embryos that expressing the ZGA reporter (MERVL::tdTomato) after injection with a siRNA-repressor or mRNA-inducer. N, the total number of embryos analyzed for each condition. Error bars, mean ± S.D.; n = 3 biological replicates per group.


( C ) Representative fluorescence image of SCNT embryos derived by different methods . The SCNT embryos produced by transfer of MERVL : : tdTomato cumulus cells ( B6D2F1 background ) into WT enucleated oocytes . n = 3 biological replicates per group . Scale bar , 100 μm .

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(C) Representative fluorescence image of SCNT embryos derived by different methods. The SCNT embryos produced by transfer of MERVL::tdTomato cumulus cells (B6D2F1 background) into WT enucleated oocytes. n = 3 biological replicates per group. Scale bar, 100 μm.


( D ) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA , as indicated . Error bars , mean ± S . D . ; n = 3 biological replicates per group ; N , total number of embryos analyzed for each condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with control group , by two - tailed Student ' s t - test .

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(D) Stacked bar plots show the fraction of embryos at blastocyst stages after injection with different mRNA, as indicated. Error bars, mean ± S.D.; n = 3 biological replicates per group; N, total number of embryos analyzed for each condition; **P < 0.01, ***P < 0.001 as compared with control group, by two-tailed Student's
 t-test.


( E ) Representative live - cell images of dynamic ZGA - reporter expression during SCNT embryo development . The time after initial observation is shown at the bottom of the image . n = 3 biological replicates per group . Scale bar , 50 μm .

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(E) Representative live-cell images of dynamic ZGA-reporter expression during SCNT embryo development. The time after initial observation is shown at the bottom of the image. n = 3 biological replicates per group. Scale bar, 50 μm.


( G ) Preimplantation development of SCNT embryos . The percentage of embryos reaching each indicated stage is shown . The D - SCNT embryos produced by transfer of dox - Dux cumulus cell ( B6D2F1 background ) into WT enucleated oocytes ( B6D2F1 background ) . Error bars , mean ± S . D . ; n = 3 biological replicates per group . Scale bar , 100 μm .

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(G) Preimplantation development of SCNT embryos. The percentage of embryos reaching each indicated stage is shown. The D-SCNT embryos produced by transfer of dox-Dux cumulus cell (B6D2F1 background) into WT enucleated oocytes (B6D2F1 background). Error bars, mean ± S.D.; n = 3 biological replicates per group. Scale bar, 100 μm.


( J ) Image of full - term cloned mice derived by canonical SCNT and D - SCNT method ( left ) . The birth rate of SCNT embryos derived by different methods is indicated ( right ) . N , the total number of cloned pups obtained from each condition .

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(J) Image of full-term cloned mice derived by canonical SCNT and D-SCNT method (left). The birth rate of SCNT embryos derived by different methods is indicated (right). N, the total number of cloned pups obtained from each condition.


( B ) Durations of dox addition at different time points during chemical induction ( upper ) and assessment of AP + colonies ( bottom ) . Error bars , mean ± S . D . ; n = 3 biological replicates per condition ; * * P < 0 . 01 , * * * P < 0 . 001 as compared with condition - 1 , by two - tailed Student ' s t - test . n . s . , not significant .

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(B) Durations of dox addition at different time points during chemical induction (upper) and assessment of AP+ colonies (bottom). Error bars, mean ± S.D.; n = 3 biological replicates per condition; **P < 0.01, ***P < 0.001 as compared with condition-1, by two-tailed Student's
 t-test. n.s., not significant.


( C ) Morphological changes at distinct time points during D - CiPS induction . Scale bar , 100 μm .

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(C) Morphological changes at distinct time points during D-CiPS induction. Scale bar, 100 μm.


( D ) Representative images of the number of AP + positive colonies . n = 5 biological replicates per condition .

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(D) Representative images of the number of AP+ positive colonies. n = 5 biological replicates per condition.


( E ) RT - qPCR analysis of select ZGA - related genes in dox - untreated D - CiPSCs and canonical CiPSCs at passage 1 . The value in CiPSCs was set as 1 , and data shown are mean expression values relative to GAPDH . Error bars , mean ± S . E . M . ; n = 3 biological replicates per group ; * * * P < 0 . 001 by two - tailed Student ' s t - test .

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(E) RT-qPCR analysis of select ZGA-related genes in dox-untreated D-CiPSCs and canonical CiPSCs at passage 1. The value in CiPSCs was set as 1, and data shown are mean expression values relative to GAPDH. Error bars, mean ± S.E.M.; n = 3 biological replicates per group; ***P < 0.001 by two-tailed Student's t-test.


( F ) Representative immunostaining images of D - CiPSCs expressing pluripotency markers . n = 5 biological replicates . Scale bar , 50 μm .

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(F) Representative immunostaining images of D-CiPSCs expressing pluripotency markers. n = 5 biological replicates. Scale bar, 50 μm.


( G ) Histology by H & E staining of teratoma tissues derived by D - CiPS cells . n = 3 biological replicates . Scale bar , 200 μm .

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(G) Histology by H&E staining of teratoma tissues derived by D-CiPS cells. n = 3 biological replicates. Scale bar, 200 μm.


( I ) Karyotype analysis of D - CiPSCs and canonical CiPSCs . The majority of D - CiPSCs and CiPSCs cells possessed a normal karyotype . Scale bar , 10 μm .

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(I) Karyotype analysis of D-CiPSCs and canonical CiPSCs. The majority of D-CiPSCs and CiPSCs cells possessed a normal karyotype. Scale bar, 10 μm.


( J ) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D - CiPSCs or canonical CiPSCs . Filled and empty squares represent methylated and unmethylated CpGs , respectively .

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(J) Bisulfite sequencing analysis of demethylation of Oct4 and Nanog promoters in D-CiPSCs or canonical CiPSCs. Filled and empty squares represent methylated and unmethylated CpGs, respectively.


Ookinete formation in vitro . After 12 h of culture , the ookinetes were Giemsa - stained and analyzed for ookinete morphology . The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes .

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Ookinete formation in vitro. After 12 h of culture, the ookinetes were Giemsa-stained and analyzed for ookinete morphology. The ookinete conversion rate was calculated as the number of mature ookinetes per 100 female gametocytes.


Oocyst counts in the mosquitoes at day 7 post blood - feeding . x / y on the top is the number of mosquitoes containing oocyst / the number of mosquitoes dissected ; the percentage number is the mosquito infection prevalence .

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Oocyst counts in the mosquitoes at day 7 post blood-feeding. x/y on the top is the number of mosquitoes containing oocyst/the number of mosquitoes dissected; the percentage number is the mosquito infection prevalence.


Co - staining of P28 and GAP45 ( IMC protein ) in female gametes ( P28 + only ) and zygotes ( P28 + / GAP45 + ) of the 17XNL , Δap2 - o3 and Δcdpk4 strains . Percentages of female gametes and zygotes were indicated respectively . n is the number of cells counted . Scale bars = 5 μm . Note : the signal of P28 and GAP45 is reduced similarly in all Δap2 - o3 female gametes and zygotes compared to that of 17XNL .

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Co-staining of P28 and GAP45 (IMC protein) in female gametes (P28+ only) and zygotes (P28+/GAP45+) of the 17XNL, Δap2-o3 and Δcdpk4 strains. Percentages of female gametes and zygotes were indicated respectively. n is the number of cells counted. Scale bars = 5 μm. Note: the signal of P28 and GAP45 is reduced similarly in all Δap2-o3 female gametes and zygotes compared to that of 17XNL.


Day 7 midgut oocyst counts from mosquitoes infected with parasites , including 17XNL , Δap2 - o3 , Δmap2 , or Δnek4 strain alone as well as mixtures of Δmap2 / Δnek4 , Δap2 - o3 / Δmap2 , and Δap2 - o3 / Δnek4 . Δnek4 and Δmap2 are female and male gamete - defect parasites , respectively . n is the number of mosquitoes dissected , Mann - Whitney test applied , two experiments repeated .

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Day 7 midgut oocyst counts from mosquitoes infected with parasites, including 17XNL, Δap2-o3, Δmap2, or Δnek4 strain alone as well as mixtures of Δmap2/Δnek4, Δap2-o3/Δmap2, and Δap2-o3/Δnek4. Δnek4 and Δmap2 are female and male gamete-defect parasites, respectively. n is the number of mosquitoes dissected, Mann-Whitney test applied, two experiments repeated.


Transcript expression heatmap by RNA - seq of 26 genes involving DNA replication and DNA repair between DFsc7 ( WT ) and DFsc7 ; Δap2 - o3 ( KO ) strains .

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Transcript expression heatmap by RNA-seq of 26 genes involving DNA replication and DNA repair between DFsc7 (WT) and DFsc7;Δap2-o3 (KO) strains.


Representative fluorescence microscopy images of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 ( ccp2 : : mCherry ; dpod2 : : gfp ) and DTS1 ; Δap2 - o3 strains . mCherry is specifically expressed in female gametocytes . Flow cytometry detection of mCherry and DPOD2 : : GFP expression in female and male gametocytes of the DTS1 and DTS1 ; Δap2 - o3 strains . The female and male gametocyte populations are circled by solid and dashed line respectively . E , F Similar analysis ( as in C , D ) in DTS2 ( ccp2 : : mCherry ; dpod1 : : gfp ) and DTS2 ; Δap2 - o3 strains . G , H Similar analysis ( as in C , D ) in DTS3 ( ccp2 : : mCherry ; rpa1 : : gfp ) and DTS3 ; Δap2 - o3 strains . I , J Similar analysis ( as in C , D ) in DTS4 ( ccp2 : : mCherry ; mcm7 : : gfp ) and DTS4 ; Δap2 - o3 strains .

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Representative fluorescence microscopy images of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 (ccp2::mCherry;dpod2::gfp) and DTS1;Δap2-o3 strains. mCherry is specifically expressed in female gametocytes. Flow cytometry detection of mCherry and DPOD2::GFP expression in female and male gametocytes of the DTS1 and DTS1;Δap2-o3 strains. The female and male gametocyte populations are circled by solid and dashed line respectively. E,F Similar analysis (as in C, D) in DTS2 (ccp2::mCherry;dpod1::gfp) and DTS2;Δap2-o3 strains. G,H Similar analysis (as in C, D) in DTS3 (ccp2::mCherry;rpa1::gfp) and DTS3;Δap2-o3 strains. I,J Similar analysis (as in C, D) in DTS4 (ccp2::mCherry;mcm7::gfp) and DTS4;Δap2-o3 strains.


EMSA using the recombinant GST - fused AP2 domain of AP2 - O3 and a synthesized DNA probe containing three repeats of predicted motif . GST was used as a negative control . An arrowhead indicates the shifted band .

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EMSA using the recombinant GST-fused AP2 domain of AP2-O3 and a synthesized DNA probe containing three repeats of predicted motif. GST was used as a negative control. An arrowhead indicates the shifted band.


ChIP - qPCR validation of the binding between AP2 - O3 and the upstream region of four male genes The regions detected via ChIP - qPCR are indicated by pink line ( in the peak ) and purple line ( out of the peak ) . mean ± SEM from three independent experiments .

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ChIP-qPCR validation of the binding between AP2-O3 and the upstream region of four male genes The regions detected via ChIP-qPCR are indicated by pink line (in the peak) and purple line (out of the peak). mean±SEM from three independent experiments.


qRT - PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID .

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qRT-PCR detecting mRNA transcript level of ten selected highly expressed genes The numbers indicate the gene ID.


Western blot of GAP45 protein expression in non - activated ( NAG ) and activated gametocyte ( AG ) of 17XNL and Δap2 - o3 strains . BiP as loading control .

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Western blot of GAP45 protein expression in non-activated (NAG) and activated gametocyte (AG) of 17XNL and Δap2-o3 strains. BiP as loading control.


IFA of AP2 - O3 protein expression in activated gametocytes of Pccp2 strains .

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IFA of AP2-O3 protein expression in activated gametocytes of Pccp2 strains.


( a ) Brains of the ' rescued ' mice ( Becn1 − / − ; Becn1EGFP / + ) and of control Becn1 + / + littermates were lysed and subjected to immune isolation using an anti - GFP antibody . Proteins bound to anti - GFP antibody were isolated , detected and identified by SDS - PAGE , Coomassie brilliant blue staining and mass spectrometry . Seven bands pulled down by anti - GFP antibody specifically from ' rescued ' mice lysate are numbered ( this is a copy of image from our previous report with modification ) .

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(a) Brains of the 'rescued' mice (Becn1−/−; Becn1EGFP/+) and of control Becn1+/+ littermates were lysed and subjected to immune isolation using an anti-GFP antibody. Proteins bound to anti-GFP antibody were isolated, detected and identified by SDS-PAGE, Coomassie brilliant blue staining and mass spectrometry. Seven bands pulled down by anti-GFP antibody specifically from 'rescued' mice lysate are numbered (this is a copy of image from our previous report with modification).


( b ) Mouse brain lysates were subjected to co - immunoprecipitation ( co - IP ) followed by WB analysis using the indicated antibodies .

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(b) Mouse brain lysates were subjected to co-immunoprecipitation (co-IP) followed by WB analysis using the indicated antibodies.


( c ) Mouse brain lysates were subjected to co - IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies .

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(c) Mouse brain lysates were subjected to co-IP by NRBF2 antibody and followed by WB analysis using the indicated antibodies.


( d ) Gel filtration analysis . The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10 / 30 column and each fraction was immunoblotted with the indicated antibodies . Relative amounts of each fraction determined by Odyssey software are plotted .

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(d) Gel filtration analysis. The supernatant fraction of mouse brain lysates was subjected to a Superdex 200 HR 10/30 column and each fraction was immunoblotted with the indicated antibodies. Relative amounts of each fraction determined by Odyssey software are plotted.


( e ) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2 . NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h , respectively , and then were lysed and subjected to IP using Beclin 1 antibody . The immunoprecipitants were immunoblotted using the indicated antibodies .

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(e) The deficiency of Atg14L impairs the protein interaction between Beclin 1 and NRBF2. NIH3T3 cells were transfected with Atg14L or NRBF2 RNAi for 72 h, respectively, and then were lysed and subjected to IP using Beclin 1 antibody. The immunoprecipitants were immunoblotted using the indicated antibodies.


( f ) Direct binding between Atg14L and NRBF2 . Flag - tagged Atg14L purified from HEK293T cells overexpressing Flag - Atg14L was incubated with GST or NRBF2 - GST purified from Escherichia coli . The GST pull - down assay was performed to examine the direct binding between Flag - Atg14L and NRBF2 - GST .

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(f) Direct binding between Atg14L and NRBF2. Flag-tagged Atg14L purified from HEK293T cells overexpressing Flag-Atg14L was incubated with GST or NRBF2-GST purified from Escherichia coli. The GST pull-down assay was performed to examine the direct binding between Flag-Atg14L and NRBF2-GST.


( b ) NRBF2 KO MEFs were transfected with CFP , NRBF2 - CFP , dMIT - CFP or dCCD - CFP for 48 h . Atg14L - linked Vps34 was pulled down by an anti - Atg14L antibody and subjected to KA . IP products and inputs were immunoblotted using the antibodies indicated . ( c ) Quantification of IPed Vps34 kinase activity ( PI ( 3 ) P ) , Vps34 and Vps15 protein versus IPed Atg14L protein . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , multiple t - test are followed by Bonferroni correction , n = 4 ( otherwise indicated , n means number of independent experiments ; NS , not significant ) ) .

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(b) NRBF2 KO MEFs were transfected with CFP, NRBF2-CFP, dMIT-CFP or dCCD-CFP for 48 h. Atg14L-linked Vps34 was pulled down by an anti-Atg14L antibody and subjected to KA. IP products and inputs were immunoblotted using the antibodies indicated. (c) Quantification of IPed Vps34 kinase activity (PI(3)P), Vps34 and Vps15 protein versus IPed Atg14L protein. (Data are shown as mean±s.e.m., P values are indicated on the figures, multiple t-test are followed by Bonferroni correction, n=4 (otherwise indicated, n means number of independent experiments; NS, not significant)).


( d ) Overexpression of NRBF2 enhances Atg14L - linked Vps34kinase activity . HEK293T cells were co - transfected with Myc - Vps34 - Vps15 - His and Flag - Atg14L in the presence of CFP or NRBF2 - CFP . Atg14L - linked Vps34 is pulled down by an anti - Flag antibody and subjected to KA ( upper panel ) . IP products and inputs were immunoblotted using the antibodies indicated ( upper panel ) . ( e ) Quantification of Vps34kinase activity ( lower panel ) shows the significant difference between two groups . ( Data are shown as mean ± s . e . m . , P value is indicated on the figures , one sample t - test versus hypothetical mean 1 , n = 5 ) .

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(d) Overexpression of NRBF2 enhances Atg14L-linked Vps34kinase activity. HEK293T cells were co-transfected with Myc-Vps34-Vps15-His and Flag-Atg14L in the presence of CFP or NRBF2-CFP. Atg14L-linked Vps34 is pulled down by an anti-Flag antibody and subjected to KA (upper panel). IP products and inputs were immunoblotted using the antibodies indicated (upper panel). (e) Quantification of Vps34kinase activity (lower panel) shows the significant difference between two groups. (Data are shown as mean±s.e.m., P value is indicated on the figures, one sample t-test versus hypothetical mean 1, n=5).


( a ) Starvation - induced long - lived protein degradation is impaired after NRBF2 knockdown . Compared with control ( con ) siRNA , NRBF2 siRNA decreases long - lived protein degradation ( proteolysis rate ) in NIH3T3 cells under starvation ( ST ) condition . This difference is diminished when the starved cells are co - treated with wortmannin ( WM , 100 nM ) . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one - way analysis of variance with Dunnett as post hoc test , n = 4 ; NS , not significant ) .

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

(a) Starvation-induced long-lived protein degradation is impaired after NRBF2 knockdown. Compared with control (con) siRNA, NRBF2 siRNA decreases long-lived protein degradation (proteolysis rate) in NIH3T3 cells under starvation (ST) condition. This difference is diminished when the starved cells are co-treated with wortmannin (WM, 100 nM). (Data are shown as mean±s.e.m., P values are indicated on the figures, one-way analysis of variance with Dunnett as post hoc test, n=4; NS, not significant).


( b ) Rapamycin ( Rap ) - induced autophagy is impaired in the NRBF2 KO cells . The MEFs were treated with ( dimethyl sulfoxide ) DMSO or 50 μg ml − 1 of Rap for 4 h then the cell lysates were immunoprobed with anti - LC3 and anti - p62 antibodies . ( c ) LC3 - II and p62 levels were quantified and normalized with β - actin . Quantification results show that Rap - induced LC3 lipidation ( upper ) and p62 degradation ( lower ) were markedly impaired in the NRBF2 KO MEFs . ( Data are shown as mean ± s . e . m . , P values are indicated on the figures , one sample t - test ( WT con versus KO con ) or unpaired t - test ( WT Rap versus KO Rap ) ) . p62 blots quantification , n = 6 ; LC3 blots quantification , n = 3 ) .

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

(b) Rapamycin (Rap)-induced autophagy is impaired in the NRBF2 KO cells. The MEFs were treated with (dimethyl sulfoxide) DMSO or 50 μg ml−1 of Rap for 4 h then the cell lysates were immunoprobed with anti-LC3 and anti-p62 antibodies. (c) LC3-II and p62 levels were quantified and normalized with β-actin. Quantification results show that Rap-induced LC3 lipidation (upper) and p62 degradation (lower) were markedly impaired in the NRBF2 KO MEFs. (Data are shown as mean±s.e.m., P values are indicated on the figures, one sample t-test (WT con versus KO con) or unpaired t-test (WT Rap versus KO Rap)). p62 blots quantification, n=6; LC3 blots quantification, n=3).