( A ) Raji cells stably expressing HIV - 1 YU - 2 ENV ( Raji - Env ) were stained with bNAbs ( 10 - 1074 or 3BNC117 ) or isotype control ( mGO53 ) and analyzed by flow cytometry . The numbers indicate the % of positive cells . One representative experiment is shown .

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(A) Raji cells stably expressing HIV-1 YU-2 ENV (Raji-Env) were stained with bNAbs (10-1074 or 3BNC117) or isotype control (mGO53) and analyzed by flow cytometry. The numbers indicate the % of positive cells. One representative experiment is shown.


( B ) Binding of the 24 indicated antibodies to the surface of Raji - Env cells . Results are expressed as the median fluorescence intensity ( MFI ) of staining . n = 6 independent experiments . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test .

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

(B) Binding of the 24 indicated antibodies to the surface of Raji-Env cells. Results are expressed as the median fluorescence intensity (MFI) of staining. n=6 independent experiments. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( C ) Raji - Env cells were incubated with indicated antibodies and either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 1h , surface levels of C3 were analyzed by flow cytometry . One representative experiment is shown .

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

(C) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 1h, surface levels of C3 were analyzed by flow cytometry. One representative experiment is shown.


( D ) C3 deposition by the 24 indicated antibodies . For each antibody , a normalized MFI is calculated by subtracting the MFI of the " no antibody " condition . ( nd . not done ) . n = 3 donors of serum . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test .

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

(D) C3 deposition by the 24 indicated antibodies. For each antibody, a normalized MFI is calculated by subtracting the MFI of the "no antibody" condition. (nd. not done). n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( E ) Raji - Env cells were incubated with indicated antibodies and either normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , cell death was determined by flow cytometry . The numbers indicate the percentage of dead cells . One representative experiment is shown .

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

(E) Raji-Env cells were incubated with indicated antibodies and either normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, cell death was determined by flow cytometry. The numbers indicate the percentage of dead cells. One representative experiment is shown.


( F ) CDC induction by the 24 indicated antibodies . The % of CDC was calculated as the relative percentage of dead cells compared to the " no antibody " condition . n = 3 donors of serum . Data information : Error bars indicate SEM . Significance was determined by comparing each antibody to mGO53 . Only significant comparisons are depicted ; * P < 0 . 05 , Mann - Whitney test .

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

(F) CDC induction by the 24 indicated antibodies. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum. Data information: Error bars indicate SEM. Significance was determined by comparing each antibody to mGO53. Only significant comparisons are depicted; *P<0.05, Mann-Whitney test.


( G ) Raji - Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs ( 10 - 1074 , 3BNC117 or PGDM1400 ) or isotype control ( mGO53 ) . Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the " no antibody " condition . n = 3 donors of serum .

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

(G) Raji-Env cells were incubated for 24h with NHS and indicated concentrations of WT or E430G mutant bNAbs (10-1074, 3BNC117 or PGDM1400) or isotype control (mGO53). Cell death was measured by flow cytometry and the % of CDC was calculated as the relative % of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( A ) Activated primary CD4 T cells were infected by HIV - 1 ( strain NLAD8 ) and subjected to antibody binding assay . The numbers indicate the % of Gag + cells that are decorated by the indicated antibodies . One representative experiment is shown .

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

(A) Activated primary CD4 T cells were infected by HIV-1 (strain NLAD8) and subjected to antibody binding assay. The numbers indicate the % of Gag+ cells that are decorated by the indicated antibodies. One representative experiment is shown.


( B ) Antibody binding at the surface of HIV - 1 - infected primary CD4 T cells . Results are expressed as median fluorescence ( MFI ) of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 9 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Antibody binding at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as median fluorescence (MFI) of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=9 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( C ) HIV - 1 - infected ( strain NLAD8 ) CD4 T cells were cultured with the indicated antibody and NHS . After 24h , the surface levels of C3 and the frequency of infected cells ( Gag + ) were determined by flow cytometry . One representative experiment is shown .

(C) HIV-1-infected (strain NLAD8) CD4 T cells were cultured with the indicated antibody and NHS. After 24h, the surface levels of C3 and the frequency of infected cells (Gag+) were determined by flow cytometry. One representative experiment is shown.


( D ) C3 surface levels at the surface of HIV - 1 - infected primary CD4 T cells . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 7 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C3 surface levels at the surface of HIV-1-infected primary CD4 T cells. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( E ) HIV - 1 - infected CD4 T cells ( strain NLAD8 ) were cultured with the indicated antibody and normal ( NHS ) or heat - inactivated ( HIHS ) human serum . After 24h , the surface levels of C5b - 9 ( MAC ) on infected ( Gag + ) cells were determined by flow cytometry . One representative experiment is shown .

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

(E) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and normal (NHS) or heat-inactivated (HIHS) human serum. After 24h, the surface levels of C5b-9 (MAC) on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.


( F ) C5b - 9 ( MAC ) surface levels on HIV - 1 - infected cells ( strain NLAD8 ) . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted .

(F) C5b-9 (MAC) surface levels on HIV-1-infected cells (strain NLAD8). Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( G ) HIV - 1 - infected CD4 T cells ( strain NLAD8 ) were cultured with the indicated antibody and NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , the percentage of infected ( Gag + ) cells in each condition was determined by flow cytometry . One representative experiment is shown .

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

(G) HIV-1-infected CD4 T cells (strain NLAD8) were cultured with the indicated antibody and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. One representative experiment is shown.


( H ) Complement - mediated cytotoxicity of HIV - 1 - infected CD4 T cells ( strain NLAD8 ) . For each antibody , the % of CDC was calculated at 24h as the % of disappearance of Gag + cells in NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = 7 donors of CD4 T cells . Data information : Error bars indicate SEM . * P < 0 . 05 , * * P < 0 . 01 , Wilcoxon test . Only significant comparisons are depicted .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Complement-mediated cytotoxicity of HIV-1-infected CD4 T cells (strain NLAD8). For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=7 donors of CD4 T cells. Data information: Error bars indicate SEM. *P<0.05, **P<0.01, Wilcoxon test. Only significant comparisons are depicted.


( A ) Raji - Env cells were subcloned to obtain cell lines expressing increasing amounts of Env . Env surface levels were determined by the binding of 10 - 1074 and measured by flow cytometry . One representative experiment is shown .

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

(A) Raji-Env cells were subcloned to obtain cell lines expressing increasing amounts of Env. Env surface levels were determined by the binding of 10-1074 and measured by flow cytometry. One representative experiment is shown.


( B ) Parental Raji cells and the different Raji - Env clones were cultured with 10 - 1074 or an isotype control ( mGO53 ) and NHS for 24h . The % of CDC was calculated as the relative percentage of dead cells compared to the " no antibody " condition . n = 3 donors of serum .

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

(B) Parental Raji cells and the different Raji-Env clones were cultured with 10-1074 or an isotype control (mGO53) and NHS for 24h. The % of CDC was calculated as the relative percentage of dead cells compared to the "no antibody" condition. n=3 donors of serum.


( C ) Non - infected ( NI ) and HIV - 1 - infected primary CD4 T cells ( strain NLAD8 ) as well as three Raji - Env clones ( D1 , D2 and D4 ) were incubated with 10 - 1074 and antibody surface levels were analyzed by flow cytometry . Results are expressed as median fluorescence ( MFI ) of staining in total cells ( Raji , n = 3 independent experiments ) or in infected ( Gag + ) cells ( CD4 T cells , n = 5 donors ) .

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

(C) Non-infected (NI) and HIV-1-infected primary CD4 T cells (strain NLAD8) as well as three Raji-Env clones (D1, D2 and D4) were incubated with 10-1074 and antibody surface levels were analyzed by flow cytometry. Results are expressed as median fluorescence (MFI) of staining in total cells (Raji, n=3 independent experiments) or in infected (Gag+) cells (CD4 T cells, n=5 donors).


( D ) HIV - or HIV ∆ Vpu - infected CD4 T cells ( strain NLAD8 ) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry ( left panel ) . The staining obtained with mGO53 was similar to the background signal observed on unstained cells . Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected ( Gag + ) cells were determined by flow cytometry ( right panel ) . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were subjected to antibody binding with indicated antibodies and surface levels were determined by flow cytometry (left panel). The staining obtained with mGO53 was similar to the background signal observed on unstained cells. Infected cells were cultured with the indicated antibodies and NHS for 24h and the C3 surface levels on infected (Gag+) cells were determined by flow cytometry (right panel). One representative experiment is shown.


( E ) HIV - or HIV ∆ Vpu - infected CD4 T cells ( strain NLAD8 ) were incubated with NHS and either 10 - 1074 ( left panel ) or 3BNC117 ( right panel ) . Both WT or E430G - mutated antibodies were used . After 24h , C3 surface levels on infected cells were analyzed by flow cytometry . Results are expressed as MFI of staining on infected ( Gag + ) cells . The MFI of C3 staining on Gag - cells was included as a control . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

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

(E) HIV- or HIV∆Vpu-infected CD4 T cells (strain NLAD8) were incubated with NHS and either 10-1074 (left panel) or 3BNC117 (right panel). Both WT or E430G-mutated antibodies were used. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. The MFI of C3 staining on Gag- cells was included as a control. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( A ) Primary CD4 T cells were nucleofected with a control ( CRISPR Control ) or anti - CD59 ( CRISPR CD59 ) guide RNA / Cas9 complex . CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection . Isotype control ( grey ) and CD59 stainings ( blue ) . One representative experiment is shown on the left . Numbers indicate the % of CD59 + cells ( middle ) or the MFI of staining in CD59 - cells ( Right ) . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary CD4 T cells were nucleofected with a control (CRISPR Control) or anti-CD59 (CRISPR CD59) guide RNA/Cas9 complex. CD59 surface levels were analyzed by flow cytometry 7 days after nucleofection. Isotype control (grey) and CD59 stainings (blue). One representative experiment is shown on the left. Numbers indicate the % of CD59+ cells (middle) or the MFI of staining in CD59- cells (Right). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( B ) NLAD8 ∆ Vpu - infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h . Numbers indicate the percentage of infected ( Gag + ) cells . One representative experiment is shown .

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

(B) NLAD8∆Vpu-infected CD59 KO primary CD4 T cells were cultured with indicated antibodies and NHS or HIHS for 24h. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) CD59 - KO ( CRISPR CD59 ) or control ( CRISPR Control ) CD4 T cells infected with HIV WT ( left ) or HIVΔVpu ( right ) ( strain NLAD8 ) were cultured with the indicated antibody , and NHS or HIHS for 24h . The % of CDC was calculated as the % of disappearance of Gag + cells after culture with NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = at least 6 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD59-KO (CRISPR CD59) or control (CRISPR Control) CD4 T cells infected with HIV WT (left) or HIVΔVpu (right) (strain NLAD8) were cultured with the indicated antibody, and NHS or HIHS for 24h. The % of CDC was calculated as the % of disappearance of Gag+ cells after culture with NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n= at least 6 donors of CD4 T cells.


( D ) CD59 surface levels on non - infected ( NI ) or HIV - 1 - infected ( strain NLAD8 ) primary CD4 T cells were analyzed by flow cytometry . Results are expressed as the MFI of staining with an isotype control or the anti - CD59 antibody . n = 4 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CD59 surface levels on non-infected (NI) or HIV-1-infected (strain NLAD8) primary CD4 T cells were analyzed by flow cytometry. Results are expressed as the MFI of staining with an isotype control or the anti-CD59 antibody. n=4 donors of CD4 T cells.


( B ) HIV - 1 - infected primary CD4 T cells were cultured with the indicated antibodies ( E430G ) and with NHS or HIHS . The frequency of infected cells was assessed by flow cytometry in live cells at days 1 , 2 , 3 and 6 . Numbers indicate the percentage of infected ( Gag + ) cells . One representative experiment is shown .

(B) HIV-1-infected primary CD4 T cells were cultured with the indicated antibodies (E430G) and with NHS or HIHS. The frequency of infected cells was assessed by flow cytometry in live cells at days 1, 2, 3 and 6. Numbers indicate the percentage of infected (Gag+) cells. One representative experiment is shown.


( C ) Frequency of live HIV - 1 - infected cells ( NLAD8 WT or ΔVpu strain ) 6 days after incubation with the indicated antibodies , and with NHS or HIHS . Each donor of CD4 T cells is represented by a different symbol . n = 8 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Frequency of live HIV-1-infected cells (NLAD8 WT or ΔVpu strain) 6 days after incubation with the indicated antibodies, and with NHS or HIHS. Each donor of CD4 T cells is represented by a different symbol. n=8 donors of CD4 T cells.


( D ) Elimination of HIV - 1 - infected cells ( strain NLAD8 WT or ΔVpu ) over culture with the indicated antibody ( E430G ) , and NHS or HIHS . The frequency of % infected ( Gag + ) cells is determinined by flow cytometry . The mean relative percentage of infected cells compared to the HIHS condition is depicted . n = 6 - 8 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Elimination of HIV-1-infected cells (strain NLAD8 WT or ΔVpu) over culture with the indicated antibody (E430G), and NHS or HIHS. The frequency of % infected (Gag+) cells is determinined by flow cytometry. The mean relative percentage of infected cells compared to the HIHS condition is depicted. n=6-8 donors of CD4 T cells.


( A ) Primary CD4 T cells infected with HIV - 1 ( Transmitted / Founder strains CH058 and CH077 , and the primary strain vKB18 ) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry . The numbers indicate the % of bNAb + cells among infected ( Gag + ) cells . The percentages of infected cells were 27 % , 30 % and 6 % for CH058 , CH077 and vKB18 , respectively . One representative experiment is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary CD4 T cells infected with HIV-1 (Transmitted/Founder strains CH058 and CH077, and the primary strain vKB18) were incubated with the indicated bNAbs and surface levels were analyzed by flow cytometry. The numbers indicate the % of bNAb+ cells among infected (Gag+) cells. The percentages of infected cells were 27%, 30% and 6% for CH058, CH077 and vKB18, respectively. One representative experiment is shown.


( B ) CH058 - infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS . After 24h , the C3 surface levels on infected ( Gag + ) cells were determined by flow cytometry . One representative experiment is shown .

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

(B) CH058-infected CD4 T cells were cultured with the indicated WT or E430G mutant antibody and NHS. After 24h, the C3 surface levels on infected (Gag+) cells were determined by flow cytometry. One representative experiment is shown.


( C ) CH058 - , CH077 - and vKB18 - infected primary CD4 T cells were incubated with the indicated antibodies and NHS . After 24h , C3 surface levels on infected cells were analyzed by flow cytometry . Results are expressed as MFI of staining on infected ( Gag + ) cells . Each dot represents a donor of CD4 T cells . n = 6 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CH058-, CH077- and vKB18-infected primary CD4 T cells were incubated with the indicated antibodies and NHS. After 24h, C3 surface levels on infected cells were analyzed by flow cytometry. Results are expressed as MFI of staining on infected (Gag+) cells. Each dot represents a donor of CD4 T cells. n=6 donors of CD4 T cells.


( D ) Primary CD4 T cells infected with the indicated HIV - 1 strains ( CH058 , CH077 or vKB18 ) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement - mediated cytotoxicity . After 24h , the percentage of infected ( Gag + ) cells in each condition was determined by flow cytometry . For each antibody , the % of CDC was calculated at 24h as the % of disappearance of Gag + cells in NHS compared to HIHS . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Primary CD4 T cells infected with the indicated HIV-1 strains (CH058, CH077 or vKB18) were cultured with the indicated WT or E430G mutant antibodies and NHS or HIHS to determine complement-mediated cytotoxicity. After 24h, the percentage of infected (Gag+) cells in each condition was determined by flow cytometry. For each antibody, the % of CDC was calculated at 24h as the % of disappearance of Gag+ cells in NHS compared to HIHS. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( A ) HIV - 1 - infected primary CD4 T cells ( strain CH058 ) were incubated with sera of HIV negative ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test .

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

(A) HIV-1-infected primary CD4 T cells (strain CH058) were incubated with sera of HIV negative (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.


( B ) CH058 - infected primary CD4 T cells were cultured for 24h with sera of healthy ( HIV - ; n = 9 ) or ART - treated HIV - 1 - infected ( HIV + ; n = 9 ) individuals ( serum dilution 1 : 100 ) and 50 % NHS from a healthy donor . C3 surface levels on infected ( Gag + ) cells were analyzed by flow cytometry . Results are expressed as the MFI on infected ( Gag + ) cells . Error bars indicate SEM . * * * * P < 0 , 0001 ; Mann - Whitney test .

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

(B) CH058-infected primary CD4 T cells were cultured for 24h with sera of healthy (HIV-; n=9) or ART-treated HIV-1-infected (HIV+; n=9) individuals (serum dilution 1:100) and 50% NHS from a healthy donor. C3 surface levels on infected (Gag+) cells were analyzed by flow cytometry. Results are expressed as the MFI on infected (Gag+) cells. Error bars indicate SEM. ****P<0,0001; Mann-Whitney test.


( C ) CD4 T cells not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with the serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 1000 ) . The levels of IgG binding on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown on the left . On the right , results are expressed as MFI of staining . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells . Error bars indicate SEM . * P < 0 . 05 , Wilcoxon test . Only significant comparisons are depicted .

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(C) CD4 T cells not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with the serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:1000). The levels of IgG binding on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown on the left. On the right, results are expressed as MFI of staining. Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells. Error bars indicate SEM. *P<0.05, Wilcoxon test. Only significant comparisons are depicted.


( D ) Primary CD4 T cells either not infected ( NI ) or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) were incubated with 50 % NHS from a healthy donor and serum of HIV - 1 - infected ART - treated individuals ( sKB104 or sKB105 , dilution 1 : 100 ) . The levels of C3 deposition on infected ( Gag + ) cells were analyzed by flow cytometry . One representative experiment is shown ( left panel ) . Results are expressed as MFI of staining ( right panel ) . Each dot represents a single donor of CD4 T cells . n = 6 donors of CD4 T cells .

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(D) Primary CD4 T cells either not infected (NI) or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058) were incubated with 50% NHS from a healthy donor and serum of HIV-1-infected ART-treated individuals (sKB104 or sKB105, dilution 1:100). The levels of C3 deposition on infected (Gag+) cells were analyzed by flow cytometry. One representative experiment is shown (left panel). Results are expressed as MFI of staining (right panel). Each dot represents a single donor of CD4 T cells. n=6 donors of CD4 T cells.


( E ) Correlation between patients ' sera ( sK104 and sKB105 ) antibody binding and C3 deposition on CD4 T cells either mock - infected or infected with WT , ∆ Nef , ∆ Vpu or ∆ Nef ∆ Vpu HIV - 1 ( strain CH058 ) . Each dot is the mean of 6 donors of CD4 T cells . Correlation was analyzed by Spearman correlation . Correlation coefficient ( r ) and p - value are indicated .

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

(E) Correlation between patients' sera (sK104 and sKB105) antibody binding and C3 deposition on CD4 T cells either mock-infected or infected with WT, ∆Nef, ∆Vpu or ∆Nef∆Vpu HIV-1 (strain CH058). Each dot is the mean of 6 donors of CD4 T cells. Correlation was analyzed by Spearman correlation. Correlation coefficient (r) and p-value are indicated.


( A ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times , and then cell viability was assessed . ( B ) 786 - 0 or 769 - P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT ( 3 * WT indicates 3 - fold expression WT ) and then treated with aa starvation for the indicated time . Cell viability was subsequently assessed . ( Data information : The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t - test . ( * * p < 0 . 01 ; * * * p < 0 . 001 . )

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

(A) 786-0 or 769-P cells stably expressing shNT or shGDH1 were treated with or without aa starvation for the indicated times, and then cell viability was assessed. (B) 786-0 or 769-P cells stably expressing shNT or shGDH1 were reconstituted with or without various amounts of rGDH1 WT (3*WT indicates 3-fold expression WT) and then treated with aa starvation for the indicated time. Cell viability was subsequently assessed. ( Data information: The data are represented as means ± SEM of three independent experiments and all statistical analyses were conducted using unpaired t-test. (**p < 0.01; ***p < 0.001.)


( I ) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed ( left ) . Quantitative analysis of the GDH1 protein levels was performed using unpaired t - test ( right ) . * * * p < 0 . 001 . Scale bars : 50 μm .

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(I) IHC staining of GDH1 in primary KIRC tumors and adjacent normal tissues collected by us was performed (left). Quantitative analysis of the GDH1 protein levels was performed using unpaired t-test (right). ***p < 0.001. Scale bars: 50 μm.


( K ) Overall survival of patients in different tumor stages in the TCGA - KIRC cohort . The R package " survminer " - based maximally selected log - rank statistic was used to determine the best cutoff . The log - rank test was used to determine the p value .

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(K) Overall survival of patients in different tumor stages in the TCGA-KIRC cohort. The R package "survminer"-based maximally selected log-rank statistic was used to determine the best cutoff. The log-rank test was used to determine the p value.


( D ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of GDH1 with or without aa starvation . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O 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) Immunofluorescence (IF) assay was performed to determine the subcellular location of GDH1 with or without aa starvation. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( E ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control .

(E) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of GDH1 with or without aa starvation. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( G ) Immunofluorescence ( IF ) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . GDH1 localization was indicated by GDH1 antibody and MitoTracker ( left ) . Signal intensities and distances were quantified ( right ) . Scale bars : 20 μm .

(G) Immunofluorescence (IF) assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. GDH1 localization was indicated by GDH1 antibody and MitoTracker (left). Signal intensities and distances were quantified (right). Scale bars: 20 μm.


( H ) Cytoplasm - mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted ( dMSP ) GDH1 . The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 ( mitochondrial membrane marker ) and GDH1 . Tubulin was used as loading control .

(H) Cytoplasm-mitochondrial fractionation assay was performed to determine the subcellular location of WT or mitochondrial signal peptide deleted (dMSP) GDH1. The fractionation efficiency and GDH1 location were determined by WB using antibodies against VDAC1 (mitochondrial membrane marker) and GDH1. Tubulin was used as loading control.


( D ) FLAG - tagged GDH1 ( including WT and mutants with different lysine mutations to arginine ) was co - transfected with HA - tagged ubiquitin for 48 hours , and then cells were subjected to aa starvation for 12 hours before collecting cells . WB and qPCR separately tested the levels of GDH1 protein and mRNA , respectively . Data information : The data are represented as the mean ± SEM of three independent experiments . The one - way ANOVA test ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

(D) FLAG-tagged GDH1 (including WT and mutants with different lysine mutations to arginine) was co-transfected with HA-tagged ubiquitin for 48 hours, and then cells were subjected to aa starvation for 12 hours before collecting cells. WB and qPCR separately tested the levels of GDH1 protein and mRNA, respectively. Data information: The data are represented as the mean ± SEM of three independent experiments. The one-way ANOVA test (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


( H ) 786 - 0 or 769 - P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time . Cell viability was determined . Unpaired two - tailed t - test was used . Data information : The data are represented as the mean ± SEM of three independent experiments . The unpaired t - test were used ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

(H) 786-0 or 769-P cells stably expressing shGDH1 were reconstituted with rGDH1 WT or 3KR mutant and then treated with or without aa starvation for the indicated time. Cell viability was determined. Unpaired two-tailed t-test was used. Data information: The data are represented as the mean ± SEM of three independent experiments. The unpaired t-test were used (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


786 - 0 cells were treated with MG132 ( 10 μM ) under aa starvation . Antibodies against GDH1 ( C ) were used to enrich protein complexes in cells with or without GDH1 . IgG was used as a control . WB was performed using the indicated antibodies .

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

786-0 cells were treated with MG132 (10 μM) under aa starvation. Antibodies against GDH1 (C) were used to enrich protein complexes in cells with or without GDH1. IgG was used as a control. WB was performed using the indicated antibodies.


( H ) 786 - 0 or 769 - P cells ( WT or stably expressing shGDH1 ) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times . Cell viability was determined . Unpaired t - test was used with * * * p < 0 . 001 .

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

(H) 786-0 or 769-P cells (WT or stably expressing shGDH1) were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for the indicated times. Cell viability was determined. Unpaired t-test was used with ***p < 0.001.


( I ) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 ( upper panel ) . The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated ( lower panel ) . The cor . test was used to determine the p - value . Scale bars : 50 μm .

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(I) IHCs of KIRC clinical samples were performed using antibodies against GDH1 or RNF213 (upper panel). The correlated IHC signal between GDH1 and RNF213 in KIRC samples was calculated (lower panel). The cor.test was used to determine the p-value. Scale bars: 50 μm.


786 - 0 cells expressing WT or enzymatically dead ( ED ) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours . In the above cells in ( A ) , qPCR was used to measure the mRNA of the indicated RP genes ( C ) .

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

786-0 cells expressing WT or enzymatically dead (ED) GDH1 were transfected with siNC or siRNF213 for 48 hours and then were treated with or without aa starvation for 12 hours. In the above cells in (A), qPCR was used to measure the mRNA of the indicated RP genes (C).


( J ) KIRC clinical samples were collected for αKG measurement , and we assessed overall survival ( left panel ) with log - rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation ( right panel ) . Unpaired t - test was used in the right panel with * * * p < 0 . 001 .

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(J) KIRC clinical samples were collected for αKG measurement, and we assessed overall survival (left panel) with log-rank test according to the dividing groups of high and low intracellular levels of αKG with the best separation (right panel). Unpaired t-test was used in the right panel with ***p < 0.001.


( G ) An in vitro demethylation assay was performed with recombinant KDM4A ( 0 . 05 mM ) or KDM6A ( 0 . 05 mM ) , and the indicated histone H3 lysine methylation peptides under 50 - μM αKG conditions ( no αKG was used as a blank control ) . Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system .

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(G) An in vitro demethylation assay was performed with recombinant KDM4A (0.05 mM) or KDM6A (0.05 mM), and the indicated histone H3 lysine methylation peptides under 50-μM αKG conditions (no αKG was used as a blank control). Various concentrations of 2HG from 0 to 200 μM was used to compete with αKG in the reaction system.


( H ) 786 - 0 cells were supplemented with or without 2HG ( 500 μM ) for 48 hours , and the cells were fixed with 1 % formaldehyde . ChIP assays were performed using antibodies against H3K9me3 or H3K27me3 . qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes .

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

(H) 786-0 cells were supplemented with or without 2HG (500 μM) for 48 hours, and the cells were fixed with 1% formaldehyde. ChIP assays were performed using antibodies against H3K9me3 or H3K27me3. qPCR was used to test the enrichment of the above histone H3 lysine modifications on the promoters of the indicated RP genes.


Xenogeneic inoculation of 786 - 0 cells lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into the left groin of nude mice . The solid tumor formed by the indicated 786 - 0 cells is shown ( B ) .

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Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The solid tumor formed by the indicated 786-0 cells is shown (B).


Xenogeneic inoculation of 786 - 0 cells lacking GDH1 with restored expression of WT , ED or 3KR mutant GDH1 into the left groin of nude mice . The staining of Ki67 ( G , upper panel ) and TUNEL ( G , lower panel ) using tumors from ( B ) are shown and analyzed ( G , right panel ) . We used 6 mice in each group . Scale bars : 50 μm . Data information : All statistical analyses were conducted using unpaired t - test ( n = 6 ) . ( N . S . : not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) .

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

Xenogeneic inoculation of 786-0 cells lacking GDH1 with restored expression of WT, ED or 3KR mutant GDH1 into the left groin of nude mice. The staining of Ki67 (G, upper panel) and TUNEL (G, lower panel) using tumors from (B) are shown and analyzed (G, right panel). We used 6 mice in each group. Scale bars: 50 μm. Data information: All statistical analyses were conducted using unpaired t-test (n = 6). (N.S.: not significant; *p < 0.05; **p < 0.01; ***p < 0.001).


( B ) The confirmative RT - PCRs were resolved in a 2 % agarose gel . Untreated reverse transcriptase RNAs ( - RT ) and genomic DNA served as negative and positive controls , respectively .

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(B) The confirmative RT-PCRs were resolved in a 2% agarose gel. Untreated reverse transcriptase RNAs (-RT) and genomic DNA served as negative and positive controls, respectively.


( D ) Left panel : Absorption spectra of dark - adapted XccBphP after illumination with red , far - red , fluorescent white lamp light , sunlight or sunlight filtered through green leaves ( LF ) . Pr / Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel . Right panel : The XccBphP - C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red , far - red and white light or dark adaptation .

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(D) Left panel: Absorption spectra of dark-adapted XccBphP after illumination with red, far-red, fluorescent white lamp light, sunlight or sunlight filtered through green leaves (LF). Pr/Pfr equilibriums scheme derived from absorption spectra is depicted in the bottom panel. Right panel: The XccBphP-C13S mutant was purified in the presence of BV and its absorption spectra recorded upon illumination with red, far-red and white light or dark adaptation.


( A - B ) A . thaliana plants were inoculated with wild - type , XccbphP , pXccBphP or pC13S bacterial strains cultured prior to inoculation under ( A ) normal laboratory illumination or ( B ) under light or dark conditions . After 1 , 2 or 3 d . p . i . , CFU per plant mg were determined ( n = 3 replicates ) . Data presented here derive from more than 4 independent experiments .

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(A-B) A. thaliana plants were inoculated with wild-type, XccbphP, pXccBphP or pC13S bacterial strains cultured prior to inoculation under (A) normal laboratory illumination or (B) under light or dark conditions. After 1, 2 or 3 d.p.i., CFU per plant mg were determined (n = 3 replicates). Data presented here derive from more than 4 independent experiments.


( C ) Stomatal closure in the presence of bacterial strains or not ( untreated ) after 1 h under light conditions . ( D ) Stomatal opening after 3 h of incubation with bacterial strains or not ( untreated ) in the dark . A control treatment without bacteria and illuminated was included ( Light ) . ( C - D ) Stomatal apertures were recorded ( n = 80 replicates ) . Data are representative of two independent experiments .

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(C) Stomatal closure in the presence of bacterial strains or not (untreated) after 1 h under light conditions. (D) Stomatal opening after 3 h of incubation with bacterial strains or not (untreated) in the dark. A control treatment without bacteria and illuminated was included (Light). (C-D) Stomatal apertures were recorded (n = 80 replicates). Data are representative of two independent experiments.


( E ) A . thalianaleaves were inoculated with wild - type , XccbphP , pXccBphP and pC13S strains , stained for callose deposits and observed by fluorescence microscopy . MgCl2 buffer ( untreated ) and flg22 peptide were used as negative and positive controls , respectively . Top panel : representative pictures of three independent experiments . Scale bar represents 200 µm . Bottom panel : the number of callose deposits per field of view ( 0 . 45 mm2 ) were determined ( n = 8 replicates ) . Data are representative of two independent experiments . ( A - E ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a two - tailed Mann - Whitney test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 )

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

(E) A. thalianaleaves were inoculated with wild-type, XccbphP, pXccBphP and pC13S strains, stained for callose deposits and observed by fluorescence microscopy. MgCl2 buffer (untreated) and flg22 peptide were used as negative and positive controls, respectively. Top panel: representative pictures of three independent experiments. Scale bar represents 200 µm. Bottom panel: the number of callose deposits per field of view (0.45 mm2) were determined (n = 8 replicates). Data are representative of two independent experiments. (A-E) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a two-tailed Mann-Whitney test (*P < 0.05, **P < 0.01, ***P < 0.001)


( A ) Five µL supernatants from wild - type , XccbphP , pXccBphP and pC13 strains bacterial cultures ( OD600 = 1 ) grown under light or dark conditions were plated onto PYM - carboxymethyl cellulose ( CMC ) agar plates and revealed with Congo redstaining ( n = 2 replicates ) . The extracellular β - 1 , 4 - endoglucanase production levels correlated with CMC degradation halo radiuses ( top panel ) . Halo measurements are presented in the bottom panel . Data derive from 10 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(A) Five µL supernatants from wild-type, XccbphP, pXccBphP and pC13 strains bacterial cultures (OD600 = 1) grown under light or dark conditions were plated onto PYM-carboxymethyl cellulose (CMC) agar plates and revealed with Congo redstaining (n = 2 replicates). The extracellular β-1,4-endoglucanase production levels correlated with CMC degradation halo radiuses (top panel). Halo measurements are presented in the bottom panel. Data derive from 10 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).


( B ) The extracellular β - 1 , 4 - endoglucanase activity from bacterial cultures supernatants ( OD600 = 1 ) was determined by a colorimetric assay in solution ( n = 2 replicates ) . Wild - type , XccbphP , pXccBphP and pC13 strains were assayed under normal laboratory conditions . Data derive from 3 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

(B) The extracellular β-1,4-endoglucanase activity from bacterial cultures supernatants (OD600 = 1) was determined by a colorimetric assay in solution (n = 2 replicates). Wild-type, XccbphP, pXccBphP and pC13 strains were assayed under normal laboratory conditions. Data derive from 3 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).


( A ) Five µL from wild - type , XccbphP , pXccBphP and pC13S bacterial cultures ( OD600 = 1 ) were plated onto PYM - glucose agar plates under light or dark conditions . Xanthan production was determined measuring colony diameters ( n = 4 replicates ) for 20 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).


( B ) Wild - type , XccbphP , pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions . Extracellular xanthan was purified by KCl addition and ethanol precipitation , then dried and weighted ( n = 2 replicates ) for 4 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).


( C ) Representative confocal laser - scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions . The scale bar represents 30 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

(C) Representative confocal laser-scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions. The scale bar represents 30 µm.


Northern blotting shows that Gasz depletion ( Gasz KD ) led to the accumulation of flam - piRNA precursors but reduced the level of mature idefix - piRNA arising from the precursors , as in the case of Zuc depletion ( Zuc KD ) . U6 snRNA was detected as a loading control .

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

Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control.


Western blotting showing the protein levels of endogenous Armi , Piwi , SoYb , Yb , Vret , and Gasz in total OSC lysate ( total ) , the mitochondrial fraction ( mito ) , and the cytoplasmic fraction after mitochondrial isolation ( cyto ) . β - Tubulin ( β - Tub ) and HSP60 were detected as markers for the " cyto " and " mito " fractions , respectively .

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

Western blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively.


Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Zuc KD ) Zuc knockdown in OSCs . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi signal ( green ) is strongly overlapped with the mitochondrial signal ( red ) in Zuc - depleted OSCs ( Zuc KD ) . Armi is localized to Yb bodies in control OSCs ( control ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Gasz KD ) Gasz depletion in Zuc - depleted OSCs ( Zuc KD ) . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi mitochondrial signal ( green ) found in Zuc - depleted cells ( Zuc KD / control ) mostly disappeared after additional Gasz depletion in the cells ( Zuc KD / Gasz KD ) , but was found at Yb bodies . Mitochondrial signal is shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Armi and Piwi co - immunoprecipitated with Flag - Gasz ( F - Gasz ) from the mitochondrial fraction ( mito fraction ) of OSCs ( input ) . F - Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation . * : Heavy chains of antibodies used for immunoprecipitation . n . i . : Non - immune IgG used as a negative control .

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

Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control.


Co - immunoprecipitation from whole - cell lysate of OSCs before ( control ) and after ( Zuc KD ) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag - Gasz ( F - Gasz ) complex increased upon Zuc depletion ( Zuc KD ) .

Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD).


Northern blotting shows that the flam - piRNA precursors in the Flag - Gasz complex ( Flag ) accumulated upon Zuc depletion ( Zuc KD ) .

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

Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD).


Upper : The flam - piRNA precursors associated with Piwi disappeared upon Armi depletion ( Armi KD ) in Zuc - lacking OSCs ( Zuc KD ) . Lower : The abundance of immunopurified Piwi is shown by western blotting .

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

Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.


Depletion of endogenous Piwi ( Piwi KD ) in Zuc - lacking OSCs ( Zuc KD ) caused Armi ( green ) to relocate to Yb bodies . Localization of Armi at Yb bodies is shown in B . Mitochondrial ( upper panel ) and Yb ( lower panel ) signals are shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


The Armi complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and Gasz . Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc - lacking OSCs . Co - IP index ( * ) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 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

The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05.


The Flag - Gasz ( F - Gasz ) complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and F - Gasz . F - Gasz was ectopically expressed prior to immunoprecipitation . Co - IP index ( * ) shows that the ratio of Armi amount in the F - Gasz complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 0 . 31 .

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

The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31.


In vitro pull - down assays show that Armi - Flag ( Armi - F ) directly binds with GST - Gasz ∆ C113 but hardly with GST . Armi - F was immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F was detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.


Upper : Northern blotting shows that Flag - Piwi WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with flam - piRNA precursors in Zuc - depleted OSCs . Endogenous Piwi was depleted simultaneously with Zuc ( Zuc KD + Piwi KD ) . Zuc was depleted by RNAi and so residual Zuc may have remained in cells . Therefore , a tiny amount of piRNAs was loaded onto Piwi . F - Piwi WT , PAZmt , and MIDmt were made to be RNAi - resistant by mutagenesis ( see Materials and Methods ) . Lower : The abundance of immunopurified Flag - Piwi ( F - Piwi ) is shown by western blotting .

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

Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting.


Armi localization to Yb bodies observed in Zuc - and Piwi - depleted OSCs ( Zuc KD + Piwi KD ) ( Figs 3A and B ) was restored by ectopic expression of Flag - Piwi WT and PAZmt but not of MIDmt ( see also Fig EV4B ) . Armi , mitochondria , and F - Piwi are shown in green , red , and yellow , respectively . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Western blotting shows that Flag - Piwi ( F - Piwi ) WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with Armi in Zuc / Piwi - depleted OSCs ( Zuc KD + Piwi KD ) .

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

Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).


Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT bound with Piwi and Gasz . The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz . Luciferase - Flag ( Luc - F ) was used as a negative control .

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

Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT bound with Piwi and Gasz. The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz. Luciferase-Flag (Luc-F) was used as a negative control.


Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F N756A mutant ( green ) localized to Yb bodies ( see also Fig EV5D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and N756A mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and N756A mutant were detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.


Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT but not Armi ∆ N34 mutant bound with Gasz in the cells . Both Armi WT and ∆ N34 mutant interacted with Piwi to similar extents . Luciferase - Flag ( Luc - F ) was used as a negative control .

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

Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control.


Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F ∆ N34 mutant ( green ) localized to Yb bodies ( see also Fig EV6D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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

Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and ∆ N34 mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and mutant were detected by western blotting using anti - Flag antibodies .

In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.


Immunoprecipitation and subsequent western blotting shows that Armi - Flag ( Armi - F ) but not Flag - Armi ( F - Armi ) strongly bound with Gasz in Zuc / Armi - depleted OSCs ( Zuc KD + Armi KD ) . Piwi bound with both Armi ( F - Armi and Armi - F ) . Luciferase - Flag ( Luc - F ) was used as a negative control .

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

Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.


( A ) Representative images of immunofluorescence analysis ( with anti - PlxnB2 antibodies ) of the phenotype of COS7 cells transfected with wild - type or mutated PlxnB2 , compared to the collapsed phenotype induced by the cognate ligand Sema4C ( 1 μg / ml ) in cells expressing the WT receptor . ( B ) The stacked bar graph shows the fraction of collapsed PlxnB2 - expressing cells , identified as having a surface minor of 2000 μm2 ( measured by ImageJ software ) , out of 50 - 100 cells counted per each condition , from different fields and n > 3 experiments ) . The statistical analysis was performed comparing WT untreated cells with all other groups , by Fisher ' s Exact Test , which indicated a * * * * * p value < 0 . 00001 for Sema4C - treated and for G842C - PlxnB2 - expressing cells ( confirmed by ANOVA analysis on individual cell values , shown in Appendix Fig . S2 ) . By Fisher ' s test , the only other mutants significantly different from WT were R820H ( p < 0 . 001 ) and R843Q ( p < 0 . 01 ) , although ANOVA analysis on individual cell values shown in Appendix Fig . S2 didn ' t confirm such significance for other PlxnB2 variants .

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

(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.


( A ) The graphs show the analysis of cellular viability in shPlxnB2 - tranduced agnospheres , compared to their respective scramble - transfected controls , after 24 , 48 , 72 , and 96 hours of growth in culture . Values are mean ± SD of n ≥ 3 independent experiments ( 6 technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons ( Graph Pad Prism 8 . 0 . 0 ) : * * p = 0 . 0053 ; * * * * p < 0 . 0001 .

(A) The graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001.


( C ) After transplantation in immunodeficient mice of CUP cells described above , the volume of ADX43 - and ( D ) ADX901 - shPlxnB2 tumors was assessed , in comparison with the respective controls . Plotted values indicate the mean ± SD ( n = 8 each group ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons : * * p = 0 . 0060 ; * * * * p < 0 . 0001 .

(C) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.


( A ) Time course analysis of the cellular viability of AS901 , stably overexpressing either wild - type or G842C - mutated PlxnB2 , or controls transduced with an empty vector . Plotted values are the mean ± SD of n = 3 independent experiments ( six technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs . controls ( CTRL ) : at 24h * * p = 0 . 0097 , 48h * * p = 0 . 0026 , 72h * * p = 0 . 0079 , 96h * p = 0 . 0226 . PlxnB2 - G842C vs . PlxnB2 - WT : at 48h $ p = 0 . 0418 , 72h $ p = 0 . 0318 . ( B ) Tumor growth curves of ADX 901 over - expressing either PlxnB2 - G842C or PlxnB2 - WT , or mock control . Plotted values are the mean ± SD ( n = 8 each groups ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs control ( CTRL ) : at 25 days * * p = 0 . 0029 , 28 days * * p = 0 . 0028 , 32 days * * p = 0 . 0033 . PlxnB2 - G842C vs PlxnB2 - WT : at 25 days $ p = 0 . 0221 , 28 days $ p = 0 . 0234 , 32 days $ $ p = 0 . 0024 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.


( A ) Western blotting showing basal endogenous or overexpressed PlxnB2 , either WT or G842C - mutated isoforms , in MCF7 cells . In this wide view of the hybridized membrane , it is appreciable the presence of full length molecules at 250 kDa , as well as the processed 80 kDa fragments ( reported in ref . 64 ) . Vinculin staining provided protein loading controls .

(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls.


( B ) The graph shows the mean ± SD of relative increase in 24 - hour spontaneous migration across Transwell inserts of MCF7 cells ( analysed above ) over - expressing either wild - type or G842C - mutated PlxnB2 , versus mock - transfected controls ( n = 4 biological replicates ) . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : PlxnB2 - WT vs . mock * p = 0 . 0145 ; PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0277 ; PlxnB2 - G842C vs . mock * * * p = 0 . 0002 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002.


( C ) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( with at least three technical replicates for each ) . The statistical significance was verified by one - way ANOVA with Tukey ' s correction for multiple comparisons : * p = 0 . 0350 ; * * p = 0 . 0029 ; * * * p = 0 . 0002 . Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B.


( D ) Western blotting analysis of endogenous and overexpressed PlxnB2 ( either WT or G842C - mutated isoforms ) in CUP cells AS906 ( both full length molecules at 250 kDa and processed 80 kDa fragments ) . Vinculin staining provided protein loading controls .

O O O O O O O O O O O O O O O O O O O O O O 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) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls.


( E ) The invasive capacity of CUP cells AS906 ( shown in previous panel ) , overexpressing WT or G842 - mutated PlxnB2 ( or mock control ) , was assessed across Matrigel - coated transwell inserts . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( including technical duplicates , in two cases ) . Below the graph , are shown representative fields of the semipermeable membranes ( additional larger size images can be found in appendix Figure S4C ) ; scale bar : 100μm . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : * p = 0 . 0406 ; * * p = 0 . 0045 ; * * * p = 0 . 0003 .

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

(E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.


( A ) Phospho - EGFR analysis by western blotting in control and PlxnB2 - silenced AS43 cells . Images show representative results of n = 3 independent experiments , while the graph at the bottom shows the mean ± SD of normalized band quantification . The statistical significance across replicates was verified by unpaired t - test : * * * * p < 0 . 0001 . ( B ) Phospho - EGFR analysis by western blotting in AS901 and AS906 cells either mock , or overexpressing WT or G842C - mutated PlxnB2 . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by one - way ANOVA followed by Tukey test : AS901 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0434 , PlxnB2 - G842C vs . mock * p = 0 . 0211 ; AS906 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0436 , PlxnB2 - G842C vs . mock * p = 0 . 0481 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481.


( C ) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres , after 6 - hour treatment with the EGFR inhibitor cetuximab ( 50μg / ml ) . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by Sidak ' s multiple comparison test between cetuximab - treated and untreated conditions , per each group : AS43 * p = 0 . 0153 , * * * p = 0 . 0001 , * * * * p < 0 . 0001 ; AS901 * p = 0 . 0201 .

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

(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.


( A ) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab ( 50μg / ml ) . In the plot , each box represents values from 25th to 75th percentiles , the central band indicates the mean , and the whiskers expand from maximum to minimum values of individual cetuximab - treated experimental points ( n ≥ 9 ) , normalized to the average value of untreated cells within each group . The statistical significance across three independent experiments , each comprising ≥ 3 technical replicates , was verified by unpaired t - test multiple comparisons of cetuximab - treated and untreated conditions , per each group : * * * p = 0 . 0002 ; * * * * p < 0 . 0001 .

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

(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.


( B ) The viability of AS43 , in presence ( or absence ) of cetuximab ( 50μg / ml ) , was assessed over the indicated time course , compared to values at T0 . The graph shows the mean ± SD of two independent experiments ( with quadruplicate technical replicates each ) . The statistical significance was verified through multiple comparisons by two - way ANOVA with Bonferroni correction of cetuximab - treated and untreated samples , per each time point : * * * * p < 0 . 0001 .

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

(B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.


( D ) The invasive capacity of AS906 cells ( overexpressing WT or G842C - mutated PlxnB2 , or mock transfected ) was assessed as in Fig . 7E , in the presence or absence of cetuximab 50μg / ml . Images show representative results ( scale bar : 100μm ) , while the graph on the right shows the mean ± SD of n = 3 independent experiments . The statistical significance across replicates was verified by one - way ANOVA with Bonferroni correction : * * p = 0 . 0010 ; * * * p = 0 . 0005 ; * * * * p < 0 . 0001 .

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

(D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.


( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody .

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

(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated THP - 1 shSAMHD1 cells . Mean ± SD , n = 3 .

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

Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.


Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated U87 cells . Mean ± SD , n = 3 .

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Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3.


ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) .

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

ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).


PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . G ) CXCL - 10 protein in cell supernatants from D - F ( ELISA ) .

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

PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).


( H ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ .

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

(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.


( I ) RT products from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP viruses .

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

(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.


( J ) Immunoblot of HIV - 1 R9 BaL virus particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 .

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

(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.