( B ) Flow cytometry quantification of cell live / dead markers Annexin - V and propidium iodide on BV - 2 cells with stable retrovirus expression of Siglec - F constructs . % parent values are shown for early apoptotic ( Annexin - V + ; PI ‑ ) , late apoptotic ( Annexin - V + ; PI ‑ ) , and necrotic ( Annexin - V - ; PI + ) populations . Bars indicate mean ± SD ; n = 8 replicates .

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(B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates.


( C ) Confluency doubling times quantified from BV - 2 cells with dox - inducible expression of Siglec - F constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field images . Bars indicate mean ± SD ; n = 8 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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(C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( D ) Selected gene expression changes ( top ) and enriched gene sets ( bottom ) taken from RNA - seq analysis of BV - 2 cells with stable retroviral expression of Siglec constructs , 2xY - > F , and empty vector controls . Heatmaps share a colormap range of 0 . 25 to 4 for z - score values ( top ) , normalized enrichment scores ( NES , bottom ) .

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(D) Selected gene expression changes (top) and enriched gene sets (bottom) taken from RNA-seq analysis of BV-2 cells with stable retroviral expression of Siglec constructs, 2xY->F, and empty vector controls. Heatmaps share a colormap range of 0.25 to 4 for z-score values (top), normalized enrichment scores (NES, bottom).


( F ) Expression levels of C1qa , IL - 1β , and β - actin by qPCR analysis in BV - 2 cells with inducible Siglec - F expression after 48 hours of treatment with 500 ng / mL doxycycline . Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) Western blot showing pro - IL - 1β and β - tubulin in BV - 2 cells induced to express Siglec - F at 0 and 48 hours .

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(G) Western blot showing pro-IL-1β and β-tubulin in BV-2 cells induced to express Siglec-F at 0 and 48 hours.


( B - C ) Volcano plots showing phosphotyrosine peptides that were ( B ) increased or ( C ) decreased after Siglec - F expression . Plots show Siglec - F compared to 2xY - > F mutant data integrated across all three runs . Protein names are shown for altered peptides . Labels are only shown for peptides with maximum directional change from each protein . Labels colored according to GO terms : magenta = signal transduction , red = endosome , cyan = cell adhesion , yellow = cytoskeleton , green = metabolism .

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(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism.


( D ) Heatmap showing phosphosites in CK - p25 which overlapped in their directional change with BV - 2 Siglec - F - associated sites . Colors indicate fold change relative to 2xY - > F controls .

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(D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls.


Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were increased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) .

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Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) .

Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


( G ) Western blot quantification of myc , SHP - 2 , and SHP - 1 from myc co - IP eluates . Co - IP lysates were prepared from BV - 2 cells stably expressing Siglec - F and optionally treated with 1 μM SHP099 .

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(G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.


( H ) Heatmap showing phosphosites that were perturbed by SHP099 treatment . Color scale is same as ( D ) .

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(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).


( B - C ) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec - F expression for ( B ) Siglec - F and ( C ) Siglec - F 2xY - > F . Siglec - expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates .

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(B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ , Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of 10 , 000 MW Dextran Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) pHrodo Dextran uptake in BV - 2 cells with stable expression of Siglec - F estimated by Incucyte measurements . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( H ) pHrodo dextran uptake in BV - 2 cells stably expressing Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from the same experiment as ( G ) .

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(H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G).


( I ) Confluency doubling times for BV - 2 cells with stable expression of Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from the same experiment as ( G ) . Bars indicate mean ± 95 % CI ; n = 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated 2xY - > F group .

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(I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group.


B . Flow cytometry of cells infected with the lentiCRISPR v2 . 0 library where the expression of GFP indicates latency reactivation . Target genes were enriched through two - round sorting . Continuously cultured C11 latent cells infected with the lentiCRISPRv2 . 0 served as control . Blue dots and the encircled area represent the GFP - positive cells for flow cytometry analysis .

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B. Flow cytometry of cells infected with the lentiCRISPR v2.0 library where the expression of GFP indicates latency reactivation. Target genes were enriched through two-round sorting. Continuously cultured C11 latent cells infected with the lentiCRISPRv2.0 served as control. Blue dots and the encircled area represent the GFP-positive cells for flow cytometry analysis.


C . Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2 . 0 library infection .

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C. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection.


D . Before next generation sequencing , the GFP expression in two - round sorted C11 cells was confirmed by fluorescence microscopy . Scale bar , 100 μm .

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D. Before next generation sequencing, the GFP expression in two-round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm.


E . Fold change ( Log2 ) of the abundance of target genes in sorted C11 cells . Enriched sgRNA genes are highlighted .

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E. Fold change (Log2) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted.


A . Validation of the top candidate genes in the C11 cell line . C11 cells were infected by lentiCRISPR v2 . 0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg / ml puromycin . The percentage of GFP - positive cells was measured by flow cytometry to determine the level of HIV - 1 reactivation .

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A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation.


The effect of candidate genes on HIV latency was further verified in J - Lat 10 . 6 ( B ) models of HIV latency . GFP expression in J - Lat 10 . 6 cells was analyzed by flow

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The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) models of HIV latency. GFP expression in J-Lat 10.6 cells was analyzed by flow cytometry


The effect of candidate genes on HIV latency was further verified in ACH2 ( C ) models of HIV latency . p24 in ACH2 cells was analyzed by ELISA , respectively .

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The effect of candidate genes on HIV latency was further verified in ACH2 (C) models of HIV latency. p24 in ACH2 cells was analyzed by ELISA, respectively.


E . PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1 - sg1 . Mock C11 cells served as control .

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E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control.


A . PEBP1 expression was measured by qPCR ( left panel ) and Western blot ( right panel ) in the latently infected C11 cells and the parental Jurkat cells .

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A. PEBP1 expression was measured by qPCR (left panel) and Western blot (right panel) in the latently infected C11 cells and the parental Jurkat cells.


B . The immunoprecipitation assay was performed in C11 cell lysates with anti - PEBP1 antibody followed by Western blot with anti - Raf1 or anti - IKK antibodies .

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B. The immunoprecipitation assay was performed in C11 cell lysates with anti-PEBP1 antibody followed by Western blot with anti-Raf1 or anti-IKK antibodies.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . The levels of indicated proteins in total protein lysates ( C ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . the levels of NF - κB / p65 protein in nucleus ( D ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. the levels of NF-κB/p65 protein in nucleus (D) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


E . NF - κB / p65 protein recruitment into HIV LTR was analyzed by ChIP - qPCR with specific primers targeting the HIV LTR after normalization to the input .

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E. NF-κB/p65 protein recruitment into HIV LTR was analyzed by ChIP-qPCR with specific primers targeting the HIV LTR after normalization to the input.


F . The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells . 293T cells were co - transfected with PEBP1 - sg1 alone or PEBP1 - sg1 with HIV - 1 LTR - empty plasmids , HIV - 1 wild type LTR - luciferase plasmids , HIV - 1 LTR - Δsp1 - luciferase , HIV - 1 LTR - ΔNF - κB - luciferase , HIV - 1 LTR - ΔAp1 - luciferase or HIV - 1 LTR - ΔYY1 - luciferase . Transcription of HIV - 1 was determined by luciferase reporter assay .

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F. The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells. 293T cells were co-transfected with PEBP1-sg1 alone or PEBP1-sg1 with HIV-1 LTR-empty plasmids, HIV-1 wild type LTR-luciferase plasmids, HIV-1 LTR-Δsp1-luciferase, HIV-1 LTR-ΔNF-κB-luciferase, HIV-1 LTR-ΔAp1-luciferase or HIV-1 LTR-ΔYY1-luciferase. Transcription of HIV-1 was determined by luciferase reporter assay.


A . The expression of PEBP1 was analysed by Western blot in TZM - bl cells infected with mock , Lv - PCDH - empty or Lv - PCDH - PEBP1 plasmids .

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A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids.


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . The transcription of HIV - 1 was evaluated 72 h post - infection by luciferase

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . levels of p24 in the supernatants ( C ) .

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. levels of p24 in the supernatants (C).


D . The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h .

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D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.


E . Induction of PEBP1 by EGCG or DHA in HIV - 1 infected primary CD4 + T cells . Primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . Seventy - two hours post - treatment with 10 µM EGCG or DHA , the expression of PEBP1 was detected by qPCR .

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E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR.


F . The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4 + T cells . The primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B and E , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . The supernatants from HIV - 1 - infected CD4 + T cell were collected 1 , 2 , 3 or 4 days post - infection . Replication of HIV - 1 was analyzed by p24 ELISA .

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F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.


A . EGCG inhibited α - CD3 / CD28 reactivation of latent HIV - 1 . Primary CD4 + T cells were isolated from HIV - 1 positive patients on antiretroviral therapy ( n = 5 , P1 - P5 ) . Primary CD4 + T cells were treated with α - CD3 / CD28 alone or α - CD3 / CD28 and 10 µM EGCG . Cell - associated RNA was extracted 7 days post - treatment . The transcription of HIV - 1 was determined by real - time qPCR .

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A. EGCG inhibited α-CD3/CD28 reactivation of latent HIV-1. Primary CD4+ T cells were isolated from HIV-1 positive patients on antiretroviral therapy (n=5, P1-P5). Primary CD4+ T cells were treated with α-CD3/CD28 alone or α-CD3/CD28 and 10 µM EGCG. Cell-associated RNA was extracted 7 days post-treatment. The transcription of HIV-1 was determined by real-time qPCR.


B . PEBP1 mRNA was induced by EGCG in patient primary CD4 + T cells . Primary CD4 + T cells were treated with 10 μM EGCG . The expression of PEBP1 in the cells was measured by qPCR .

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B. PEBP1 mRNA was induced by EGCG in patient primary CD4+ T cells. Primary CD4+ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.


C . PEBP1 protein was induced by EGCG in patient primary CD4 + T cells , which was determined by Western blot of whole cell lysate from primary CD4 + T cells .

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C. PEBP1 protein was induced by EGCG in patient primary CD4+ T cells, which was determined by Western blot of whole cell lysate from primary CD4+ T cells.


D . EGCG - induced PEBP1 suppressed nuclear entry of NF - κB / p65 . This was measured by Western blot of nuclear protein of primary CD4 + T cells treated with α - CD3 / CD28 or α - CD3 / CD28 plus 10 μM EGCG .

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D. EGCG-induced PEBP1 suppressed nuclear entry of NF-κB/p65. This was measured by Western blot of nuclear protein of primary CD4+ T cells treated with α-CD3/CD28 or α-CD3/CD28 plus 10 μM EGCG.


B . Transcription of HIV - 1 in the primary CD4 + T cells was determined by nanoluc luciferase assays during HIV - 1 infection .

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B. Transcription of HIV-1 in the primary CD4+ T cells was determined by nanoluc luciferase assays during HIV-1 infection.


C . After infection with VSV - G pseudotyped HIV - 1 NL4 . 3 - nanoluc , the mRNA expression of PEBP1 was measured by qPCR .

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C. After infection with VSV-G pseudotyped HIV-1 NL4.3-nanoluc, the mRNA expression of PEBP1 was measured by qPCR.


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The mRNA expression levels of PEBP1 were measured by

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The mRNA expression levels of PEBP1 were measured by qPCR


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The protein expression levels of PEBP1 were measured by Western blot .

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The protein expression levels of PEBP1 were measured by Western blot.


F . PEBP1 knockout enhanced HIV - 1 transcription in the primary CD4 + T cell model of latency . The expression of HIV - 1 was measured by nanoluc after gene knockout where α - CD3 / CD28 stimulation served as a positive control .

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F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control.


( A ) Neural precursors derived from human iPS cells ( iNPCs ) were mock or ZIKV - infected and co - stained for Centrin ( green ) , Sox2 ( blue ) and flavivirus envelope ( red ) . All cells were fixed 16 hours post infection ( hpi ) . ( B ) Mock and ZIKV - infected U87 and H4 cells in S phase co - stained for Centrin ( green ) , flavivirus envelope ( red ) and DNA ( blue ) . ( C ) Quantification of the percentage of mock or ZIKV - infected iNPC , U87 or H4 cells in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 100nm .

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(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi). (B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue). (C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 100nm.


( A ) Mock or ZIKV - infected U87 cells in S phase co - stained for Centrin ( red ) and the centrosomal microcephaly - associated protein , CEP63 ( green ) . ( B ) The fluorescence intensity ± s . d . of CEP63 at the centrosome ( 0 . 5μm around the Centrin signal ) and around the centrosome ( 1μm surrounding the Centrin signal minus the centrosomal CEP63 ) were quantified in mock and ZIKV infected U87 cells . For fluorescence quantifications 8 cells were analyzed per experiment ( n = 3 ) . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

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(A) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green). (B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test). Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( D ) Immunoblot analysis of lysate from mock or ZIKV - infected U87 cells probed for CEP63 and ZIKV NS3 . Actin served as a loading control . Asterisk denotes specific band for CEP63 .

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(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.


( E ) S - phase mock and ZIKV infected control and Cep63gt / gt MEFs co - stained for Centrin ( green ) and flavivirus envelope ( red ) . ( F ) Quantification of the percentage of mock or ZIKV - infected Het and Cep63gt / gt MEFs in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

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(E) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red). (F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( B ) Mock or ZIKV - infected U87 cell lysates were immunoprecipitated for CEP63 or c - Myc , which served as a negative control for this and other immunoprecipitations ( Control ) . Precipitated proteins were immunoblotted for CEP63 and NS3 . Asterisk denotes specific band .

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(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.


( C ) We expressed Myc - tagged Brazilian Zika NS3 in 293T / 17 cells and immunoprecipitated c - Myc . Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63 . The centriole distal end component , CP110 served as a negative control . Asterisk denotes specific band for CEP63 .

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(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.


( G ) HeLa cells transfected with Control or Myc - tagged Brazilian ZIKV NS3 ( Br NS3 ) co - stained for Myc ( red ) and Centrin ( green ) . Data information : Scale bars indicate 5μm for all images .

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(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green). Data information: Scale bars indicate 5μm for all images.


( H ) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . For all quantifications at least 100 cells were counted per experiment ( biological replicates = 3 ) . p < 0 . 005 ( paired Student ' s t test ) for the comparison of NS3 to mock transfected cells was denoted with asterisk .

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(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.


( A ) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1 . Precipitated proteins were immunoblotted for CEP63 and TBK1 .

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(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.


( B ) Mock and ZIKV - infected U87 cells were co - stained for γ - tubulin ( red ) and p - TBK1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green). Data information: Scale bars indicate 5μm for all images.


( C ) Lysate from mock or ZIKV - infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3 , TBK1 , and p - TBK1 . Actin served as a loading control .

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(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.


( D ) RT - ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV - infected H4 cells . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented .

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(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.


( E ) Control and Cep63gt / gt MEFs were co - stained with antibodies to γ - tubulin ( red ) and p - Tbk1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green). Data information: Scale bars indicate 5μm for all images.


( F ) Immunoblot analysis of control and Cep63gt / gt MEFs probed with antibodies to Tbk1 and p - Tbk1 . Actin served as a loading control .

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(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.


( G ) HeLa cell lysate was immunoprecipitated with antibodies to CEP63 , DTX4 or c - Myc ( negative control ) . Precipitated proteins were blotted with antibodies to CEP63 and DTX4 .

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(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.


( H ) Mock and ZIKV - infected U87 cells co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( I ) Total cell lysate from mock and ZIKV - transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4 . Actin served as a loading control .

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(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.


( J ) SC and CEP63 siRNA treated HeLa cells were co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( K ) TBK1 or c - Myc ( negative control ) were immunoprecipitated from Control or Cep63 mutant MEFs . Precipitating proteins were probed with antibodies to TBK1 and K48 - linked ubiquitin .

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(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.


( L ) 16hpi Mock and ZIKV - infected U87 cell lysate was immunoprecipitated for TBK1 or c - Myc . The immunoprecipitations were probed using antibodies to TBK1 and K - 48 - linked ubiquitin .

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(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.


( C ) . The table summarises the number and the sex of the embryos of the indicated genotypes , recovered from timed matings of Rif1 + / - x Rif1 + / - mice , in a C57BL / 6J genetic background . The day of gestation ( E ) is indicated .

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(C). The table summarises the number and the sex of the embryos of the indicated genotypes, recovered from timed matings of Rif1+/- x Rif1+/- mice, in a C57BL/6J genetic background. The day of gestation (E) is indicated.


( C ) . Time course analysis of Xist RNA expression by RT - qPCR during EB differentiation of Rif1 + / + ( Rif1 + / + + OHT ) and Rif1 - / - ( Rif1F / F + OHT ) cells at the indicated timepoints . Rif1 + / + ( solid line ) and Rif1 - / - ( dashed line ) , female ( black ) and male ( grey ) . Data are presented as mean ± standard deviation from three ( female lines ) or two ( male lines ) independent experiments . Statistical significance was determined using 2 - way ANOVA comparing female Rif1 + / + to female Rif1 - / - cell lines ( * * * * p ≤ 0 . 0001 ) . Xist RT - primers Xist ex7 F and R were used . Values are normalised to a geometric mean consisting of the expression of Gapdh , Ubiquitin and β - Actin .

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(C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin.


RIF1 association with the Xist promoter assessed by ChIP - qPCR in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female cell lines , in ESCs ( A ) P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 and 2 are two intergenic regions that serve as negative controls . Peak and cRAD represent two previously identified regions of RIF1 association ( positive control ) . See Appendix Fig . S2C for primer positions within Xist . Mean ± standard deviation from 3 independent experiments ( A ) and 2 independent experiments ( B ) . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association in Rif1 + / + cells on Xist P2 and P1 versus 5 ' . * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 and ns = not significant .

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

RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.


( D ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) and a wild type female mESC line ( grey ) , also harbouring one castaneus and one 129 X chromosome . Allele - specific ChIP - qPCR primers were used , cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2 . Enrichments are presented relative to input DNA . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 , * * * p ≤ 0 . 001 . Below , schematic of the Xist / Tsix alleles in the Fa2L undifferentiated cells . ( E ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) upon differentiation . The analysis was performed as in ( D ) . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 . * p ≤ 0 . 05 . Below , schematic of the Xist / Tsix alleles in the Fa2L differentiated cells . (

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

(D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells. (E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells. (


( F ) . Quantification by ChIP - qPCR of RIF1 association with the indicated regions in the Fa2L cell line , following treatment with DMSO only ( black ) or flavopiridol ( grey ) . Primers as in ( E ) . ( G ) . Same as in ( F ) but for a wild type female mESC line . All enrichments are presented relative to input DNA . Mean ± standard deviation from three ( F ) and two ( G ) independent experiments are presented . Statistical significance was determined using Student ' s two - tailed , paired t test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant ) .

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

(F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E). (G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).


( A ) . Time course analysis of Xist expression by RT - qPCR during EB differentiation of female mESCs following knock down of Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Data are presented as mean ± standard deviation from three independent experiments . Statistical significance was determined using 2 - way ANOVA . Xist primers Xist ex3 F and Xist ex4 R were used . Normalisation was performed using a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . ( * p ≤ 0 . 05 ) . ( B ) . RT - qPCR analysis of Xist expression levels during differentiation of the Fa2L cells , following expression of shRNA against Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Mean ± standard deviation from a minimum of three independent experiments is presented . 2 - way ANOVA was used to determine statistical significance . ns = not significant . (

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

(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. (


( C ) . Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase ( Control , black ) and KAP1 ( Kap1 KD , grey ) , during differentiation . Mean ± standard deviation from four independent experiments are shown . Statistical significance was determined using 2 - way ANOVA . ( * * p ≤ 0 . 01 ) . Values have first been normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha .

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

(C). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.


( D ) . RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase ( control ) or against Kap1 ( Kap1 KD ) . Left : Cells with no ( 0 , red ) , one ( 1 , grey ) and two or more ( 2 , black ) Tsix foci were counted in two independent experiments , shown averaged . Statistical significance was determined by χ2 . A minimum of 110 cells were counted per each time point for each line ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Right : examples of cells with one ( top ) or two ( central and bottom ) Tsix FISH signals . Scale bars : 5μm .

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 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 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 B-MEASURED_VAR O O O O O O O O

(D). RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase (control) or against Kap1 (Kap1 KD). Left: Cells with no (0, red), one (1, grey) and two or more (2, black) Tsix foci were counted in two independent experiments, shown averaged. Statistical significance was determined by χ2. A minimum of 110 cells were counted per each time point for each line (**p ≤ 0.01, ****p ≤ 0.0001). Right: examples of cells with one (top) or two (central and bottom) Tsix FISH signals. Scale bars: 5μm.


( A ) . ChIP - qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs ( Rif1 + / + , same as used in Fig . 3B but without OHT ) and during early differentiation . ZFP629 is a well - characterised KAP1 associated region ( positive control ) . Xite A and C indicate two regions within the Tsix enhancer Xite , Tsix region 1 indicates Tsix major promoter , Tsix region 2 indicates the Dxpas34 region , Tsix region 3 indicates a region slightly downstream of the Dxpas34 region . P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 is an intergenic region . See Appendix Fig . S2C for the positions of the primers within Xist and Tsix . The data are presented as mean ± standard deviation from three ( 2d EB and 1d EB ) and two ( ESCs ) independent experiments . Statistical significance was calculated by Student ' s two - tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB ( * p ≤ 0 . 05 and ns = not significant ) . In the inset , Tsix RNA levels were quantified by RT - qPCR during the differentiation of wild type female ESCs shown in Fig . 4F . The average of two experiments is shown . Tsix values are normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . Error bars indicate standard deviations .

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

(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.


( C ) . KAP1 association with Xist promoter in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female mESC cell lines . Ezr is an additional region known to be associated with KAP1 in mESCs . Enrichments are presented relative to input DNA . Mean ± standard deviation from a minimum of three independent experiments per cell line are displayed . Statistical significance was determined using Student ' s two - tailed , unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1 + / + versus Rif1 - / - cells ( * * p ≤ 0 . 01 ) .

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

(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).


( C ) . HEXIM1 association with the Xist promoter ( P1 and P2 ) in two independent Rif1 + / + and two Rif1 - / - mESC lines , analysed by ChIP - qPCR . Tkbp1 and Myf5 are two control regions . As in the case of KAP1 association , deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2 . Average ± standard deviation of two independent experiments . p values were calculated by two - tailed , unpaired , equal variance t - test . ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O 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). HEXIM1 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and two Rif1-/- mESC lines, analysed by ChIP-qPCR. Tkbp1 and Myf5 are two control regions. As in the case of KAP1 association, deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2. Average ±standard deviation of two independent experiments. p values were calculated by two-tailed, unpaired, equal variance t-test. (**p ≤ 0.01, ***p ≤ 0.001).


( E ) . Upon infection of Fa2L cells with shRNA against Kap1 or control , against Luciferase , HEXIM1 association with P2 was analysed , on both alleles , by ChIP - qPCR . As in the case of KAP1 , HEXIM1 shows preferential association with Xist P2 on the Cast allele ( future Xa , Control ) . The association is lost upon knock down of Kap1 ( Kap1 KD ) . Myf5 serves as a negative and Neat1 as a positive control regions .

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

(E). Upon infection of Fa2L cells with shRNA against Kap1 or control, against Luciferase, HEXIM1 association with P2 was analysed, on both alleles, by ChIP-qPCR. As in the case of KAP1, HEXIM1 shows preferential association with Xist P2 on the Cast allele (future Xa, Control). The association is lost upon knock down of Kap1 (Kap1 KD). Myf5 serves as a negative and Neat1 as a positive control regions.


Pulldowns of pointed end site 1 - 4 mutants using Strep - Hook3 ( D ) or Strep - BICD2 ( E ) . Binding is shown relative to the binding of the wildtype pointed end construct ( dashed lines ) . * shows p < 0 . 05 . For BICD2 , p = 0 . 06 for Site 1 mutant , and p = 0 . 1 for Site 4 mutant . Data presented as mean ± SEM , n = 3 . Statistical significance calculated using ANOVA with Tukey ' s test for multiple comparisons .

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

Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.


A slice through a filtered tomogram showing SR vesicles in contact with a putative T - tubule . Individual RyR1 molecules are indicated by the red circles . The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump . Areas circled in yellow indicate accumulation of density inside the SR . Scale bar : 20 nm . A slice through the structure of RyR1 in contact with putative TT - membrane . A middle slice through the in situ structure of RyR1 at 12 . 6 - Å resolution . The observed additional transmembrane density is indicated by the red arrows . Scale bars in B , C : 10 nm .

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

A slice through a filtered tomogram showing SR vesicles in contact with a putative T-tubule. Individual RyR1 molecules are indicated by the red circles. The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump. Areas circled in yellow indicate accumulation of density inside the SR. Scale bar: 20 nm. A slice through the structure of RyR1 in contact with putative TT-membrane. A middle slice through the in situ structure of RyR1 at 12.6-Å resolution. The observed additional transmembrane density is indicated by the red arrows. Scale bars in B, C: 10 nm.


Slice through the structures of apoRyR1 with the putative TT - membrane ( left ) and the standalone apoRyR1 ( right , with the flipped handendness and reduced resolution for comparison ) 22 Å away from the middle slice ( Y = 92 in the volume coordinates ) . Scale bar : 10 nm .

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

Slice through the structures of apoRyR1 with the putative TT-membrane (left) and the standalone apoRyR1 (right, with the flipped handendness and reduced resolution for comparison) 22 Å away from the middle slice (Y = 92 in the volume coordinates). Scale bar: 10 nm.


The location of the CaM / S100A1 binding site is marked with red circles in slices through maps of the in situ structure ( left ) and the single particle structure ( EMDB : 8393 ) in the same orientation as the in situ structure and filtered to 15 Å ( right ) . Scale bar : 10 nm .

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

The location of the CaM/S100A1 binding site is marked with red circles in slices through maps of the in situ structure (left) and the single particle structure (EMDB: 8393) in the same orientation as the in situ structure and filtered to 15 Å (right). Scale bar: 10 nm.


A slice through the average structure of the apo - state at a position 22 Å away from the middle slice shown in Fig . 1C . The intra - SR extension is boxed on the density in red .

O O O O O O O O O O O O O O O O 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 slice through the average structure of the apo-state at a position 22 Å away from the middle slice shown in Fig. 1C. The intra-SR extension is boxed on the density in red.


Representative SR vesicles with different distributions of inner - SR density : segregated , evenly distributed , empty , and empty with filaments . RyR1 particles are circled in red , areas circled in yellow indicate accumulation of density inside the SR .

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

Representative SR vesicles with different distributions of inner-SR density: segregated, evenly distributed, empty, and empty with filaments. RyR1 particles are circled in red, areas circled in yellow indicate accumulation of density inside the SR.


The fractions of vesicles that fall into each density category for each RyR1 sample ( EDTA : n = 64 ; 1 mM Ca2 + : n = 32 ; 5 mM Ca2 + : n = 21 , technical replicates ) . Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density , orange to segregated density , and grey to empty vesicles .

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

The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles.


Structures of RyR1 determined in presence of increasing Ca2 + concentration ( 1 mM and 5 mM ) , all with intra - SR extensions . Scale bars : 10 nm in ( A ) and ( E ) , 20 nm in ( C ) .

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

Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).


B is domain representation on the grayscale image . Please note the location of two transmembrane densities : yellow - shown in Figure 1C , magenta - shown in Figure 3B .

O O O O O O O O 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 is domain representation on the grayscale image. Please note the location of two transmembrane densities: yellow - shown in Figure 1C, magenta - shown in Figure 3B.


Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15 . 4 , 18 , 18 , and 27 Å , respectively , with the percentages of the total particles in each class shown beneath . Red arrows show domain movements compared to class 1 .

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

Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15.4, 18, 18, and 27 Å, respectively, with the percentages of the total particles in each class shown beneath. Red arrows show domain movements compared to class 1.


The first five modes of correlated movements . Direction of motion is indicated by the arrows . Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode . Scale bars : 10 nm .

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

The first five modes of correlated movements. Direction of motion is indicated by the arrows. Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode. Scale bars: 10 nm.


Structure of ryRyR1 at a resolution of 17 . 5 Å . Two views are through the middle slice ( top ) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below ( bottom panels ) . The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo - structure . This movement is highlighted by red arrows in the right panels . Scale bar : 10 nm .

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

Structure of ryRyR1 at a resolution of 17.5 Å. Two views are through the middle slice (top) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below (bottom panels). The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo-structure. This movement is highlighted by red arrows in the right panels. Scale bar: 10 nm.


( a ) Representative 2D μ - CT scans of RasV12 ; ctrl at day 6 , RasV12 ; scrib - / - larvae at day 6 and day 10 . Muscle ( green ) , fat body ( blue ) and eye - antennal discs / tumor ( red ) are outlined . Scale bar : 1mm . Anterior ( A ) , Right ( R ) .

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

(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).


( a ) Quantification of tumor growth of RasV12 ; ctrl day 6 ( n = 15 ) RasV12 ; scrib - / - tumors , day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 4 ) , day 9 ( n = 5 ) and day 10 ( n = 5 ) .

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

(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).


( b ) Quantifications of muscle volume , of larvae carrying tumors of RasV12 ; ctrl ( n = 15 ) , and RasV12 ; scrib - / - at day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 5 ) , day 9 ( n = 5 ) and dat 10 ( n = 5 ) .

O 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

(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).


( c ) Quantification of width of Dorsal Oblique 3 ( DO3 ) muscle in larvae carrying tumors of RasV12 ; ctrl at day 6 ( n = 29 ) and RasV12 ; scrib - / - at days 6 ( n = 29 ) and day 10 ( n = 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).


( d ) Quantification of adipose tissue volume in larvae carrying tumors of RasV12 ; ctrl at day 6 and RasV12 ; scrib - / - at days 6 and day 10 .

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

(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.


( e ) Representative confocal images of adipose tissue in RasV12 ; ctrl and RasV12 ; scrib - / - tumors bearing animals at indicated ages . Lipid droplets are highlighted with Lipid Tox staining . Scale bar :

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

(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm


( f ) Quantification of dry weight of RasV12 ; ctrl ( n = 9 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) and day 10 ( n = 18 ) tumor bearing larvae excluding the tumor weight .

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

(f) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.


( g ) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12 ; ctrl ( n = 14 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) , day 8 ( n = 16 ) and day 10 ( n = 14 ) , each colored line represents a single larva .

O O 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

(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.


( h ) Coomassie feeding assay to asses food intake of RasV12 ; scrib - / - over time , three repeated measurements of an average food intake of 20 larvae .

(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.


( a - e ) Cartoon ( top ) illustrates the genotypes of the larvae , eye - antennal disc ( EAD , circle : tumor cells in green and microenvironment cell in black ) , systemic cells are illustrated as a square ( wild type cells in light brown and atg13 mutant cells in light orange ) at indicated ages . Cartoon ( left ) illustrates the structure of cephalic complex attached to mouth hook . Representative images of Larva ( image of larva using the backlight of microscope ) , cephalic complex ( green highlights the GFP - labelled tumor clones ) , muscle ( Phalloidin in green stains actin and Hoechst in blue stains nucleus ) and adipose tissue ( Lipid Tox in red stains for lipid droplets , Hoechst stains nucleus ) from top to bottom . ( a ) RasV12 ; ctrl tumor bearing animal at day 6 . ( b ) RasV12 ; scrib - / - tumor bearing animal at day 6 . ( c ) RasV12 ; scrib - / - tumor bearing animal at day 8 . ( d ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - at day 8 . ( e ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - animal complemented with transgenic atg13 , rescuing the tumor growth , at day 8 .

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

(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.


( f ) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 ( shown in yellow dashed line in 2a ) , of animals bearing tumors at day 6 RasV12 ; ctrl ( n = 45 ) , RasV12 ; scrib - / - ( n = 30 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 30 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 29 ) and at day 10 RasV12 ; scrib - / - ( n = 40 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 25 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 28 ) .

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

(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).


( g ) Quantification of tumor volumes day 6 RasV12 ; ctrl ( n = 7 ) , RasV12 ; scrib - / - ( n = 11 ) and day 8 RasV12 ; scrib - / - ( n = 13 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 9 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 9 ) .

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

(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).


( h ) Quantification of Ventral Longitudinal muscle 4 ( VL4 ) ( shown in yellow dashed line in 2a ) of third segment of larvae carrying at day 6 RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - ( n = 14 ) , and at day8 RasV12 ; scrib - / - ( n = 11 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 12 ) and ey3 . 5 - atg13 ; RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - ( n = 14 ) .

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

(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).


( i ) Quantification of dry weight excluding tumor of RasV12 ; scrib - - , atg13 - / - / / atg13 - / - at day 6 ( n = 8 ) and day 10 ( n = 7 ) .

(i) Quantification of dry weight excluding tumor of RasV12; scrib- -, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).


( j ) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - at day 6 ( n = 15 ) and at day 8 ( n = 15 ) , each colored line represents a single larva .

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

(j) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.


( a ) Changes in groups ( carbohydrates , amino acids and fatty acids ) of storage metabolites measured in circulating haemolymph with progressing wasting , shown as log2 changes measured by LC - MS and calculated per larvae relative to RasV12 ; ctrl at day 6 .

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

(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.


( b ) Volcano plot showing autophagy - dependent changes to amounts of circulating metabolites at day 6 . X - axis shows the log2 of fold change of RasV12 ; atg13 - / - scrib - / - / / atg13 - / - vs . RasV12 ; scrib - / - , y - axis shows - log10 p - value , calculated by t - test . Metabolite names are shown for metabolites with log2 ( FC ) > ± 1 and / or - log10 ( P ) < 2 . Green points indicate log2 ( FC ) > ± 1 , blue indicate - log10 ( P ) < 2 and red indicates for above both thresholds .

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

(b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.


( c ) Autophagy - driven wasting releases metabolites into circulation of 113 reliably detected metabolites , those showing significant differences in any of the three comparisons are shown . Color indicates the log2 ( fold change ) difference and the numbers show the p - value of the comparison . The statistical test to define significance was FDR - adjusted t - test p - value < 0 .

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

(c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05


( a ) The amount of glycogen in the whole larvae measured by biochemical assay ( n = 4 ) , normalized to RasV12 ; ctrl at day 6 , and per number of larvae .

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

(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.


( b - e ) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12 ; ctrl , RasV12 ; scrib - / - and RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - showing glycogen levels ( white ) at day 6 and day 8 .

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

(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.


( g ) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor . A similar , but independent experiment is reported in Holland P . , et al . , 2021 BMC Biol .

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

(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.


Immunohistochemistry of NSMCE2 on mouse testis . The inset illustrates the accumulation of NSMCE2 on the XY body . Scale bar , 50 μm .

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

Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.


Immunofluorescence ( IF ) of NSMCE2 and SCP3 on isolated mouse spermatocytes . Threads of NSMCE2 can be detected on regions of weak SCP3 signal , indicative of asynapsis ( arrowheads ) . Scale bar , 2 . 5 μm .

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

Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.


Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes .

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

Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.


Representative image of NSMCE2 and BRCA1 foci in control and MMS ‐ treated MEFs . Scale bar , 2 . 5 μm .

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

Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.


Quantification of NSMCE2 and BRCA1 foci in wild ‐ type MEFs exposed to IR ( 10 Gy ; 1 ‐ h recovery ) , MMS ( 1 mM for 1 h ; 24 ‐ h recovery ) , and PARP inhibitor ( 1 μM for 1 h ; 24 ‐ h recovery ) . Data indicate mean values and are representative of three independent experiments . Error bars indicate SD .

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

Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.


Representative image of IR ‐ ( 10 Gy ; 1 ‐ h recovery ) and MMS ‐ ( 1 mM for 1 ‐ h ; 24 ‐ h recovery ) induced NSMCE2 and γH2AX foci in MEFs . Scale bar , 2 . 5 μm .

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

Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.


Brightfield microscopy of 2 . 5 dpc embryos obtained from crosses of Nsmce2GT / + mice . The right panel is of the same 2 embryos shown on the left panel , 36 h after being cultured in vitro . The arrow indicates embryos that showed asymmetric cell sizes when isolated , which degenerated upon culture . These embryos were identified as Nsmce2GT / GT through genotyping .

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

Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.


Examples of the three genotypes found at Mendelian frequencies ( see Appendix Table S1 ) on 2 . 5 dpc embryos .

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Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.


Examples of the type of nuclei found on Nsmce2GT / GT and Nsmce2 + ( + / + or + / GT ) 2 . 5 dpc embryos , 12 h after in vitro culture . Mutant embryos were identified due to the absence of NSMCE2 signal ( green ) . DAPI was used to stain DNA ( blue ) . White arrows indicate examples of cells with condensed chromosomes or irregular shapes , which are hardly seen in wild type embryos .

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

Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.


Immunoprecipitation of SMC5 ( and IgG , as a control ) from a 48 ‐ h culture of B lymphocytes of wt and Nsmce2SD / SD animals . NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB . The image of the Ponceau staining is shown as a loading control .

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Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.


IF of NSMCE2 foci in MMS ‐ treated Nsmce2 + / + and Nsmce2SD / SD MEFs . Scale bar , 2 . 5 μm .

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IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.


Representative picture of 6 ‐ month ‐ old Nsmce2 + / + and Nsmce2SD / SD littermates .

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Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.


Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2SD / SD mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test .

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Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.


A Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2 + / GT mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test . * * * P 0 . 001 .

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A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001.


B Tumor incidence found on Nsmce2 + / + and Nsmce2 + / GT mice at the time of death ( i . e . moribund mice ) .

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B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).