Figure 7HEK - 293 cells were transfected with a plasmid expressing schistosomal - miR - 10 at the indicated concentration together with either psiCHECK - II vector , psiCHECK - WT - MAP3K7 - 3 ' UTR or psiCHECK - mut - MAP3K7 . After 24h , the cells lysates were subjected to luciferase assay . The results are presented as the ratio of renilla / luciferase expression that was normalized relative to cells transfected only with psiCHECK - II . Values are expressed as the mean + / - SD of at least 3 independent experiments . A representative experiment in which protein extracted from Jurkat - T cells stably over expressing stably control vector or miR - 10 expressing vector were subjected to WB analysis with anti - MAP3K7 or anti - GAPDH antibodies . The graph presents densitometry analysis of 3 WBs analyses performed as in ( C ) . The mean + / - SD was calculated from 4 independent experiments . Statistics were performed using t - test ( * p < 0 . 05 ) . Jurkat - T cells stably expressing either control vector or a vector containing schistosomal - miR - 10 were co - transfected with a control Luciferase plasmid or NF - κB luciferase reporter vector . In all transfections , a plasmid expressing Renilla vector as an internal control was added . 48h post - transfection , cells were harvested and were analyzed by luciferase reporter assay . Values were normalized to the Renilla activity . Data are represented as mean + / - SEM from 5 independent experiments . Statistics were performed using t - test , * * p < 0 . 01 . Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal - exposed Th cells F ) or to Jurkat ( H ) in comparison to the control unexposed cells . ( G , I ) The graphs present densitometry analysis of 3 WB analyses performed as in ( F and H ) , respectively . The mean + / - SD was calculated from 3 independent experiments . Statistics were performed using paired one - tailed t - test ( * p < 0 . 05 ) .

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

Figure 7HEK-293 cells were transfected with a plasmid expressing schistosomal-miR-10 at the indicated concentration together with either psiCHECK-II vector, psiCHECK-WT-MAP3K7-3'UTR or psiCHECK-mut-MAP3K7. After 24h, the cells lysates were subjected to luciferase assay. The results are presented as the ratio of renilla/luciferase expression that was normalized relative to cells transfected only with psiCHECK-II. Values are expressed as the mean+/-SD of at least 3 independent experiments.A representative experiment in which protein extracted from Jurkat-T cells stably over expressing stably control vector or miR-10 expressing vector were subjected to WB analysis with anti-MAP3K7 or anti-GAPDH antibodies. The graph presents densitometry analysis of 3 WBs analyses performed as in (C). The mean +/- SD was calculated from 4 independent experiments. Statistics were performed using t-test (*p<0.05).Jurkat-T cells stably expressing either control vector or a vector containing schistosomal-miR-10 were co-transfected with a control Luciferase plasmid or NF-κB luciferase reporter vector. In all transfections, a plasmid expressing Renilla vector as an internal control was added. 48h post-transfection, cells were harvested and were analyzed by luciferase reporter assay. Values were normalized to the Renilla activity. Data are represented as mean +/-SEM from 5 independent experiments. Statistics were performed using t-test, **p<0.01.Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal-exposed Th cells F) or to Jurkat (H) in comparison to the control unexposed cells. (G, I) The graphs present densitometry analysis of 3 WB analyses performed as in (F and H), respectively. The mean +/- SD was calculated from 3 independent experiments. Statistics were performed using paired one-tailed t-test (*p<0.05).


Figure 1A sWGS performed on sorted CD3 + / Vβ + malignant T - cells isolated from the blood of P1 , shown as a representative example . Copy number profiles and ploidy estimates were calculated using ichorCNA . Blue represents balanced genomic regions , green indicates loss / µdeletion , and red indicates gain / amplification of regions on chromosome 17 . Darker color shades indicate higher log - 2 ratios . B Heatmap of extracted CNA [ log2 ratios ] of STAT genes on chr17 ( i . e . , STAT3 , STAT5A and STAT5B ) , and other relevant tumor suppressors ( i . e , STAT1 , SOCS1 and TP53 ) and oncogenes ( MYC ) . Red indicates gain / amplification , blue indicates loss / deletion . The balance threshold was set to + / - 0 . 1 . P33 presented with two malignant clones in the blood and both were sequenced ( P33 _ 1 ( Clone 1 ) , P33 _ 2 ( Clone 2 ) ) . * Tetraploid malignant clone .

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

Figure 1A sWGS performed on sorted CD3+/Vβ+ malignant T-cells isolated from the blood of P1, shown as a representative example. Copy number profiles and ploidy estimates were calculated using ichorCNA. Blue represents balanced genomic regions, green indicates loss/µdeletion, and red indicates gain/amplification of regions on chromosome 17. Darker color shades indicate higher log-2 ratios.B Heatmap of extracted CNA [log2 ratios] of STAT genes on chr17 (i.e., STAT3, STAT5A and STAT5B), and other relevant tumor suppressors (i.e, STAT1, SOCS1 and TP53) and oncogenes (MYC). Red indicates gain/amplification, blue indicates loss/deletion. The balance threshold was set to +/- 0.1. P33 presented with two malignant clones in the blood and both were sequenced (P33_1 (Clone 1), P33_2 (Clone 2)). *Tetraploid malignant clone.


Figure 2A - C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3 + cells detected in patients for ( A ) STAT3 , ( B ) STAT5A and ( C ) STAT5B . D - F RT - qPCR expression data of ( C ) STAT3 , ( D ) STAT5A , and ( E ) STAT5B genes from whole PBMCs isolated from patient blood ( n = 5 ) or healthy controls ( n = 9 ) . GAPDH expression was used for normalization . Statistical significance was calculated using two - tailed unpaired t - test . P - value : < 0 . 01 ( * * ) , < 0 . 0001 ( * * * * ) . One experiment was performed in technical triplicates . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range . G Immunoblotting for total STAT3 / 5 protein isolated from the respective whole PBMCs from patient blood ( n = 3 ) , or from healthy controls ( n = 3 ) . HSC70 was used as loading control . One experiment was performed . H Representative image of H & E , CD3 , pY - STAT5 single staining and CD3 / pY - STAT5 double staining on skin biopsy tissue from P3 ( n = 1 ) , which demonstrates infiltration of CD3 + pY - STAT5 + ( 13 . 45 % ) cells into the dermal and subcutaneous layer of the skin . Arrows indicate pY - STAT5 + single and CD3 + pY - STAT5 + cells . I - M Quantification of IHC staining from skin - biopsy sections of L - CTCL patients ( n = 6 ) and healthy individuals ( n = 5 ) As the number of STAT5A / B positive cells was negligible ( less than 0 . 03 / per mm2 ) in healthy skin biopsy sections , healthy control samples were considered negative for pY - STAT5 , and levels were set to 0 . Statistical significance was calculated using an unpaired Welch ' s t - test . P - value : < 0 . 05 ( * ) . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range .

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

Figure 2A-C Spearman correlation analysis using CNA log2 ratios and the percentage of clonal CD3+ cells detected in patients for (A) STAT3, (B) STAT5A and (C) STAT5B.D-F RT-qPCR expression data of (C) STAT3, (D) STAT5A, and (E) STAT5B genes from whole PBMCs isolated from patient blood (n = 5) or healthy controls (n = 9). GAPDH expression was used for normalization. Statistical significance was calculated using two-tailed unpaired t-test. P-value: <0.01 (**), <0.0001 (****). One experiment was performed in technical triplicates. The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.G Immunoblotting for total STAT3/5 protein isolated from the respective whole PBMCs from patient blood (n = 3), or from healthy controls (n = 3). HSC70 was used as loading control. One experiment was performed.H Representative image of H&E, CD3, pY-STAT5 single staining and CD3/pY-STAT5 double staining on skin biopsy tissue from P3 (n = 1), which demonstrates infiltration of CD3+ pY-STAT5+ (13.45%) cells into the dermal and subcutaneous layer of the skin. Arrows indicate pY-STAT5+ single and CD3+ pY-STAT5+ cells.I-M Quantification of IHC staining from skin-biopsy sections of L-CTCL patients (n = 6) and healthy individuals (n = 5) As the number of STAT5A/B positive cells was negligible (less than 0.03/per mm2) in healthy skin biopsy sections, healthy control samples were considered negative for pY-STAT5, and levels were set to 0. Statistical significance was calculated using an unpaired Welch's t-test. P-value: <0.05 (*).The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


Figure 3A - C Graphic depicts the aberration patterns on ( A ) chromosome 17 ( STAT3 / 5 ) , ( B ) chromosome 2 ( STAT1 ) and ( C ) chromosome 16 ( SOCS1 ) in the CTCL cell lines used in the study . Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs . reference DNA and were generated by aCGH . Immunoblotting for total protein STAT1 / 3 / 5 and activation levels : phospo - Tyr ( 701 ) - STAT1 / phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5 , and SOCS1 protein levels . The cytokine - dependent cell line , SeAx , was collected 30 min after 5 ng / ml IL - 2 and IL - 4 cytokine addition . HSC70 was used as loading control . One representative out of three independent experiments is shown .

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

Figure 3A-C Graphic depicts the aberration patterns on (A) chromosome 17 (STAT3/5), (B) chromosome 2 (STAT1) and (C) chromosome 16 (SOCS1) in the CTCL cell lines used in the study. Aberration patterns are represented as the log2 ratio of the fluorescence intensity of the tumor DNA vs. reference DNA and were generated by aCGH. Immunoblotting for total protein STAT1/3/5 and activation levels: phospo-Tyr (701)-STAT1/phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5, and SOCS1 protein levels. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. One representative out of three independent experiments is shown.


Figure 4A Heatmap of IC50 values calculated from drug response analysis using CellTiter - Glo viability assays upon 48h drug treatment . One representative , out of three independent experiments performed in triplicates , is shown . B , Immunoblot showing total protein STAT3 / 5 and activation levels : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( B ) JPX - 0750 The cytokine - dependent cell line , SeAx , was collected 30 min after 5 ng / ml IL - 2 and IL - 4 cytokine addition . HSC70 was used as loading control . The normalized levels of phospho - and total STAT3 / 5 quantified by densitometry , are shown below the respective blots . One representative , out of three independent experiments is shown . C Immunoblot showing total protein STAT3 / 5 and activation levels : phospho - Tyr ( 705 ) - STAT3 / phospho - Tyr ( 694 / 699 ) - STAT5A / B after 24h treatment with ( C ) IQDMA . The cytokine - dependent cell line , SeAx , was collected 30 min after 5 ng / ml IL - 2 and IL - 4 cytokine addition . HSC70 was used as loading control . The normalized levels of phospho - and total STAT3 / 5 quantified by densitometry , are shown below the respective blots . One representative , out of three independent experiments is shown . Real - Time apoptosis / necrosis assay in the ( D , SeAx and ( F , Myla cell lines treated with 2 . 5µM JPX - 0750 RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative fluorescence units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed . Real - Time apoptosis / necrosis assay in the E ) SeAx and G ) Myla cell lines treated with 2 . 5µM IQDMA . RLU , Apoptosis [ RLU ] = relative luminescence ; Necrosis [ RFU ] = relative fluorescence units . Error bars represent mean + / - SD . One experiment in technical triplicates was performed . H - I The effect of ( H ) JPX - 0750 and ( I ) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts ( JF ) and keratinocytes ( HaCat ) at a low dose range ( 0 . 7 - 3µM ) . One experiment , in triplicates , was performed with JF and HaCat cells , and three experiments , in triplicates with the CTCL cells . Error bars represent mean + / - SD . Statistical significance was calculated by two - way ANOVA with Tukey ' s multiple comparisons test . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) , < 0 . 0001 ( * * * * ) .

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

Figure 4A Heatmap of IC50 values calculated from drug response analysis using CellTiter-Glo viability assays upon 48h drug treatment. One representative, out of three independent experiments performed in triplicates, is shown.B, Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (B) JPX-0750 The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.C Immunoblot showing total protein STAT3/5 and activation levels: phospho-Tyr (705)-STAT3/phospho-Tyr (694/699)-STAT5A/B after 24h treatment with (C) IQDMA. The cytokine-dependent cell line, SeAx, was collected 30 min after 5 ng/ml IL-2 and IL-4 cytokine addition. HSC70 was used as loading control. The normalized levels of phospho- and total STAT3/5 quantified by densitometry, are shown below the respective blots. One representative, out of three independent experiments is shown.Real-Time apoptosis/necrosis assay in the (D, SeAx and (F, Myla cell lines treated with 2.5µM JPX-0750 RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.Real-Time apoptosis/necrosis assay in the E) SeAx and G) Myla cell lines treated with 2.5µM IQDMA. RLU, Apoptosis [RLU] = relative luminescence; Necrosis [RFU] = relative fluorescence units. Error bars represent mean +/-SD. One experiment in technical triplicates was performed.H-I The effect of (H) JPX-0750 and (I) IQDMA on the viability of CTCL cells in comparison to viability of primary juvenile fibroblasts (JF) and keratinocytes (HaCat) at a low dose range (0.7-3µM). One experiment, in triplicates, was performed with JF and HaCat cells, and three experiments, in triplicates with the CTCL cells. Error bars represent mean +/-SD. Statistical significance was calculated by two-way ANOVA with Tukey's multiple comparisons test. P-value: <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****).


Figure 5B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY - STAT5 and total STAT5 . The cells were collected 30 min after 1 ng / ml IL - 2 cytokine addition . α - Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions , respectively . The normalized levels of phospho - and total STAT5 in the nucleus and cytoplasm , quantified by densitometry , are shown below the respective blots . One representative out of two independent experiments is shown . D - F Scatter plot depicting the profile of SeAx cells treated with ( D ) 1µM IQDMA , ( E ) 2 . 5µM IQDMA and ( F ) 10µM IQDMA . Protein abundances were determined using TMT - based quantification mass spectrometry Significant changes were assessed by a modified t - test as implemented in the limma package , with the negative log10 p values on the x - axis , and log2 fold change shown on the y - axis . G Heatmap of IC50 values calculated from drug response analysis using CellTiter - Blue or CellTiter - Glo viability assays upon 48h drug treatment . One representative out of three independent experiments performed in triplicates is shown . Grey represents the presence of STAT3 / 5 gain .

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

Figure 5B Subcellular fractions of SeAx cells treated with IQDMA for 24h and immunoblotted for pY-STAT5 and total STAT5. The cells were collected 30 min after 1 ng/ml IL-2 cytokine addition. α-Tubulin and Histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively. The normalized levels of phospho- and total STAT5 in the nucleus and cytoplasm, quantified by densitometry, are shown below the respective blots. One representative out of two independent experiments is shown.D-F Scatter plot depicting the profile of SeAx cells treated with (D) 1µM IQDMA, (E) 2.5µM IQDMA and (F) 10µM IQDMA. Protein abundances were determined using TMT-based quantification mass spectrometry Significant changes were assessed by a modified t-test as implemented in the limma package, with the negative log10 p values on the x-axis, and log2 fold change shown on the y-axis.G Heatmap of IC50 values calculated from drug response analysis using CellTiter-Blue or CellTiter-Glo viability assays upon 48h drug treatment. One representative out of three independent experiments performed in triplicates is shown. Grey represents the presence of STAT3/5 gain.


Figure 6A Inhibitor treatment was carried out with increased dose escalation for 24h . SeAx cells were collected 2h after 7 , 5 ng / ml IL - 2 and IL - 4 cytokine addition . STAT3 / 5 target gene products MYC , PIM1 , MCL - 1 , and AKT1 / 2 / 3 , as well as proteins regulating the cell cycle , such as CYCLIN D2 and D3 , and CDK6 were probed by Western blotting . HSC70 served as loading control . The normalized protein levels , quantified by densitometry , are shown below the respective blots . One representative out of three independent experiments is shown . Synergy analysis of the indicated two - drug combinations in the SeAx and Myla cells after 48 h treatment . In each graph the most synergistic area ( MSA ) is highlighted , which represents the most synergistic 3 - by - 3 dose - window with the respective MSA score . The Zero interaction potency model was applied to quantify the degree of synergy , according to which an MSA score below - 10 indicates that drugs are antagonistic ( green ) , a score between - 10 and 10 indicates that 2 drugs are additive ( white ) , while a score above 10 indicates a synergistic effect ( red ) . ( D ) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs . One representative out of two independent experiments is shown . E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L - CTCL patients with JPX - 0750 , IQDMA and FRAx597 . One experiment performed in triplicates using CellTiter - Glo viability assays upon 48h drug treatment is shown . The relative STAT3 / 5 log2 ratio value is depicted in grey . F - H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug .

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Figure 6A Inhibitor treatment was carried out with increased dose escalation for 24h. SeAx cells were collected 2h after 7,5 ng/ml IL-2 and IL-4 cytokine addition. STAT3/5 target gene products MYC, PIM1, MCL-1, and AKT1/2/3, as well as proteins regulating the cell cycle, such as CYCLIN D2 and D3, and CDK6 were probed by Western blotting. HSC70 served as loading control. The normalized protein levels, quantified by densitometry, are shown below the respective blots. One representative out of three independent experiments is shown.Synergy analysis of the indicated two-drug combinations in the SeAx and Myla cells after 48 h treatment. In each graph the most synergistic area (MSA) is highlighted, which represents the most synergistic 3-by-3 dose-window with the respective MSA score. The Zero interaction potency model was applied to quantify the degree of synergy, according to which an MSA score below -10 indicates that drugs are antagonistic (green), a score between -10 and 10 indicates that 2 drugs are additive (white), while a score above 10 indicates a synergistic effect (red).(D) Heatmap showing MSA scores of the combination of FRAx597 with the listed drugs. One representative out of two independent experiments is shown.E Heatmap showing the IC50 values upon treatment of primary PBMCs isolated from L-CTCL patients with JPX-0750, IQDMA and FRAx597. One experiment performed in triplicates using CellTiter-Glo viability assays upon 48h drug treatment is shown. The relative STAT3/5 log2 ratio value is depicted in grey.F-H Spearman correlation analysis using CNA log2 ratios and the IC50 values for the respective drug.


Figure 7B Growth rate of Hut78 , Myla and H9 - derived tumors measured on the respective day post - injection . The mean of the respective tumor volumes for H9 group and for the vehicle - treated groups for Hut78 and Myla are shown . Tumor volume = ( length x width2 ) / 2 . Error bars represent mean + / - SEM . n represents the total number of tumors that engrafted per group . C - D Myla - derived tumor weight upon experiment termination , after 20 days of treatment with 5 mg / kg of ( C ) JPX - 0750 ( n = 5 ) and IQDMA ( n = 6 ) , or vehicle ( n = 7 ) , and ( D ) FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , with n representing the number of analyzed tumors per group . Statistical significance was calculated using two - tailed paired t - test . P - value : < 0 . 05 ( * ) . Error bars represent mean + / - SEM . H & E analysis of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , and FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( E , F ) H & E with n representing the number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 and 25 µm . Violin plots show the perimeter of the annotated tumor cell - infiltration / expansion region into the intradermal and subcutaneous region of the skin Statistical significance was calculated using two - tailed paired t - test with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range . IHC analysis of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , and FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( G , H ) Ki67 , as a marker for proliferation with n representing the number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 and 25 µm . Violin plots show the quantification of the percentage of Ki67 + cells in the tissue . Statistical significance was calculated using two - tailed paired t - test with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range . IHC analysis of Myla - derived tumors treated with JPX - 0750 ( n = 6 ) , IQDMA ( n = 6 ) or vehicle ( n = 6 ) , and FRAx597 ( n = 5 ) or vehicle ( n = 4 ) , stained with ( I , J ) CD31 , as a tumor vessel marker , with n representing the number of analyzed tumors per group . Pictures shown are from contiguous sections . Dotted rectangles indicate magnified areas . Scale bars , 200 , 100 and 25 µm . CD31 staining was quantified as raw counts of vessels with lumen . Statistical significance was calculated using two - tailed paired t - test with Welch ´ s correction . P - value : < 0 . 05 ( * ) , < 0 . 01 ( * * ) , < 0 . 001 ( * * * ) . The bold dashed line in the middle of the violin plot denotes the median value , while the thin dotted lines denote the interquartile range .

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Figure 7B Growth rate of Hut78, Myla and H9-derived tumors measured on the respective day post-injection. The mean of the respective tumor volumes for H9 group and for the vehicle-treated groups for Hut78 and Myla are shown. Tumor volume = (length x width2)/2. Error bars represent mean +/-SEM. n represents the total number of tumors that engrafted per group.C-D Myla-derived tumor weight upon experiment termination, after 20 days of treatment with 5 mg/kg of (C) JPX-0750 (n = 5) and IQDMA (n = 6), or vehicle (n = 7), and (D) FRAx597 (n = 5) or vehicle (n = 4), with n representing the number of analyzed tumors per group. Statistical significance was calculated using two-tailed paired t-test. P- value: <0.05 (*). Error bars represent mean +/-SEM.H&E analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (E, F) H&E with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the perimeter of the annotated tumor cell-infiltration/expansion region into the intradermal and subcutaneous region of the skin Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (G, H) Ki67, as a marker for proliferation with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. Violin plots show the quantification of the percentage of Ki67+ cells in the tissue. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.IHC analysis of Myla-derived tumors treated with JPX-0750 (n = 6), IQDMA (n = 6) or vehicle (n = 6), and FRAx597 (n = 5) or vehicle (n = 4), stained with (I, J) CD31, as a tumor vessel marker, with n representing the number of analyzed tumors per group. Pictures shown are from contiguous sections. Dotted rectangles indicate magnified areas. Scale bars, 200, 100 and 25 µm. CD31 staining was quantified as raw counts of vessels with lumen. Statistical significance was calculated using two-tailed paired t-test with Welch´s correction. P-value: <0.05 (*), <0.01 (**), <0.001 (***). The bold dashed line in the middle of the violin plot denotes the median value, while the thin dotted lines denote the interquartile range.


Figure 1RT - qPCR analysis of METTL3 and METTL14 expression ( A ) in shRNA knockdown METTL3 cells . I HA - METTL14 Co - IP with FLAG - tagged METTL3 domain - deletion mutants . β - actin was used as the loading control .

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Figure 1RT-qPCR analysis of METTL3 and METTL14 expression (A) in shRNA knockdown METTL3 cells.I HA-METTL14 Co-IP with FLAG-tagged METTL3 domain-deletion mutants. β-actin was used as the loading control.


Figure 2F Immunoblot analysis of FLAG - METTL14 , LC3 - II , and β - actin in HEK293T cells exposed to the autophagy inhibitor Baf - A1 and activator Torin 1 , respectively . The FLAG - METTL14 or LC3 - II / β - actin densitometric ratio was recorded by ImageJ . G Dot blots of m6A levels in the presence of MG132 in 293T , RBE , SK - Cha - 1 , and SK - N - SH cells .

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Figure 2F Immunoblot analysis of FLAG-METTL14, LC3-II, and β-actin in HEK293T cells exposed to the autophagy inhibitor Baf-A1 and activator Torin 1, respectively. The FLAG-METTL14 or LC3-II /β-actin densitometric ratio was recorded by ImageJ.G Dot blots of m6A levels in the presence of MG132 in 293T, RBE, SK-Cha-1, and SK-N-SH cells.


Figure 3G , H HA - METTL14 Co - IP with FLAG - tagged STUB1 - domain - deletion mutants . L Immunoblots showing CHX assay in HEK293T cells . Negative control ( NC ; no transfected cells ) and STUB1 - FLAG plasmid - transfected HEK293T cells were exposed to 100 μg / ml CHX for 0 , 4 , 8 , or 12 hours . β - actin was used as the loading control . The METTL14 / β - actin densitometric ratio was recorded by ImageJ . M , N Dot blot assays showing that enforced expression of wild - type STUB1 , but not the STUB1H260Q mutant , significantly decreases total m6A levels .

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Figure 3G, H HA-METTL14 Co-IP with FLAG-tagged STUB1-domain-deletion mutants.L Immunoblots showing CHX assay in HEK293T cells. Negative control (NC; no transfected cells) and STUB1-FLAG plasmid-transfected HEK293T cells were exposed to 100 μg/ml CHX for 0, 4, 8, or 12 hours. β-actin was used as the loading control. The METTL14/β-actin densitometric ratio was recorded by ImageJ.M, N Dot blot assays showing that enforced expression of wild-type STUB1, but not the STUB1H260Q mutant, significantly decreases total m6A levels.


Figure 4B Immunoblots showing the abundance of METTL14 truncated mutants with or without 10 µM MG132 treatment . Histone H4 was used as the loading control . The HA - METTL14 / β - actin densitometric ratio was recorded by ImageJ . F Immunoblots showing the levels of METTL14 protein after transfection with METTL14 - 3KR mutant plasmids with or without 10 µM MG132 . β - actin was used as the loading control . The HA - METTL14 / β - actin densitometric ratio was recorded by ImageJ . G Immunoblots showing the protein level of METTL14 after transfection with METTL14 - 3KR mutant plasmids under STUB1 knockdown . β - actin was used as the loading control . The HA - METTL14 / β - actin densitometric ratio was recorded by ImageJ .

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Figure 4B Immunoblots showing the abundance of METTL14 truncated mutants with or without 10 µM MG132 treatment. Histone H4 was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.F Immunoblots showing the levels of METTL14 protein after transfection with METTL14-3KR mutant plasmids with or without 10 µM MG132. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ. G Immunoblots showing the protein level of METTL14 after transfection with METTL14-3KR mutant plasmids under STUB1 knockdown. β-actin was used as the loading control. The HA-METTL14/β-actin densitometric ratio was recorded by ImageJ.


Figure 5B METTL14 ubiquitination , as detected by IP of METTL3 truncated mutants with anti - HA antibody . The accumulation of Ub and METTL14 was confirmed in whole - cell lysates . β - actin was used as the loading control . C Immunoblots showing METTL14 protein levels , as detected by IP in response to METTL3 truncated mutants and STUB1 - FLAG . β - actin was used as the loading control .

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Figure 5B METTL14 ubiquitination, as detected by IP of METTL3 truncated mutants with anti-HA antibody. The accumulation of Ub and METTL14 was confirmed in whole-cell lysates. β-actin was used as the loading control.C Immunoblots showing METTL14 protein levels, as detected by IP in response to METTL3 truncated mutants and STUB1-FLAG. β-actin was used as the loading control.


Figure 6E - G Following the subcutaneous inoculation of SK - Cha - 1 - NC ( left ) and SK - Cha - 1 - STUB1 - FLAG ( right ) cells into the flanks of male NOD - SCID mice ( E ) , overexpression of STUB1 - FLAG inhibited malignant cell proliferation ( F ) and reduced subsequent tumor size and growth ( G ) in vivo . Data are means ± SEM . Statistical significance was analyzed by two - way ANOVA ( F ) or two - tailed unpaired t - test ( G ) ( Five mice , * p < 0 . 05 ; * * p < 0 . 01 ) . H - J Following the subcutaneous inoculation of SK - Cha - 1 - sh - NC ( left ) and SK - Cha - 1 - sh - STUB1 - 1 ( right ) cells into the flanks of male nude mice ( H ) , STUB1 knockdown promoted malignant cell proliferation ( I ) and increased subsequent tumor size and growth ( J ) . Data are means ± SEM . Statistical significance was analyzed by two - way ANOVA ( I ) or two - tailed unpaired t - test ( J ) . ( Six mice , * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ) . Immunoblots ( K ) showing METTL14 protein levels STUB1 - FLAG overexpression in five pairs of mouse tumors . β - actin was used as the loading control . The METTL14 or STUB1 - FLAG / β - actin densitometric ratio was recorded by ImageJ .

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Figure 6E-G Following the subcutaneous inoculation of SK-Cha-1-NC (left) and SK-Cha-1-STUB1-FLAG (right) cells into the flanks of male NOD-SCID mice (E), overexpression of STUB1-FLAG inhibited malignant cell proliferation (F) and reduced subsequent tumor size and growth (G) in vivo. Data are means ± SEM. Statistical significance was analyzed by two-way ANOVA (F) or two-tailed unpaired t-test (G) (Five mice, *p < 0.05; **p < 0.01). H-J Following the subcutaneous inoculation of SK-Cha-1-sh-NC (left) and SK-Cha-1-sh-STUB1-1 (right) cells into the flanks of male nude mice (H), STUB1 knockdown promoted malignant cell proliferation (I) and increased subsequent tumor size and growth (J). Data are means ± SEM. Statistical significance was analyzed by two-way ANOVA (I) or two-tailed unpaired t-test (J). (Six mice, *p < 0.05; **p < 0.01; ***p < 0.001).Immunoblots (K) showing METTL14 protein levels STUB1-FLAG overexpression in five pairs of mouse tumors. β-actin was used as the loading control. The METTL14 or STUB1-FLAG /β-actin densitometric ratio was recorded by ImageJ.


Figure 7B Immunoblot analysis showing STUB1 protein levels in eight pairs of CCA patient samples . N , adjacent non - tumor tissue ; T , tumor tissue . β - actin was used as the loading control . The STUB1 / β - actin densitometric ratio was recorded by ImageJ . C The 5 - year disease - free survival of patients with high expression levels of METTL14 ( p = 0 . 0366 ) and low expression levels of STUB1 ( p = 0 . 0803 ) in the Fudan University intrahepatic cholangiocarcinoma ( FU - iCCA ) cohort among patient samples within the 5 - 95 % ranges of STUB1 and METTL14 protein levels .

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Figure 7B Immunoblot analysis showing STUB1 protein levels in eight pairs of CCA patient samples. N, adjacent non-tumor tissue; T, tumor tissue. β-actin was used as the loading control. The STUB1/β-actin densitometric ratio was recorded by ImageJ.C The 5-year disease-free survival of patients with high expression levels of METTL14 (p = 0.0366) and low expression levels of STUB1 (p = 0.0803) in the Fudan University intrahepatic cholangiocarcinoma (FU-iCCA) cohort among patient samples within the 5-95% ranges of STUB1 and METTL14 protein levels.


Figure 1A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A ) A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , metabolic activity was assessed by a WST - 1 assay ( B ) A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment viral protein expression was analyzed by FACS ( C ) A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment ; and initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR ( D ) . E J - Lat 8 . 4 cells were mock - treated or treated with 5 - AzadC ( 400nM ) or TNFα ( 10ng / ml ) as positive control . At 24 h , 48 h or 72 h post - treatment , initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR .

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Figure 1A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A)A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, metabolic activity was assessed by a WST-1 assay (B)A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment viral protein expression was analyzed by FACS (C)A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D).E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.


Figure 2A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B ) A - D J - Lat 8 . 4 cells were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , metabolic activity was assessed by a WST - 1 assay ( C , D ) . Data information : Means and standard errors of the means from three independent biological duplicates ( n = 6 ) are indicated . The result obtained with mock - treated cells was arbitrarily set at a value 100 % ( C , D ) . The selected doses were indicated by " an arrow " . Plasmatic concentrations after usual dosage ( Cmax ) of each drug in human therapy are indicated below the LRA names and by a box in the graph .

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Figure 2A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.


Figure 3A - D J - Lat 8 . 4 cell line was mock - treated or treated with 5 - AzadC and / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . Viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B ) A - D J - Lat 8 . 4 cell line was mock - treated or treated with 5 - AzadC and / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . metabolic activity was assessed by a WST - 1 assay ( C , D ) . Means and standard errors of the means from three independent biological duplicates ( n = 6 ) are indicated . The result obtained with mock - treated cells was arbitrarily set at a value of 100 % ( C , D ) . E From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from 24 HIV + patients presented in Appendix Table S2 , the extracellular HIV - 1 genomic RNA levels for each LRA treatment are represented . One night after cell purification , cells were mock - treated or simultaneously treated with 5 - AzadC ( 1µM ) and / or SAHA ( 1µM ) . Six days after treatment , the concentration of viral RNA in culture supernatants was determined ( in copies / ml ) . The results were reported as the actual HIV RNA copy numbers / ml or as an estimated value calculated as 50 % of the smallest value when HIV RNA was not detected in order to assign a log value . Means are represented .

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

Figure 3A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D).E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


Figure 4A - F J - Lat 8 . 4 ( A , C , E ) and 15 . 4 ( B , D , F ) cell lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested and analysed as follows : viral p24 production in cell supernatant was measured ( A and B ) A - F J - Lat 8 . 4 ( A , C , E ) and 15 . 4 ( B , D , F ) cell lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested and analysed as follows : FACS analyses were performed and the percentages of GFP + cells are presented as histograms ( C and D ) A - F J - Lat 8 . 4 ( A , C , E ) and 15 . 4 ( B , D , F ) cell lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested and analysed as follows : initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR and results were normalized using the β - actin gene primers and are presented as histograms representing fold - inductions compared to the mock - treated condition ( E and F ) . Means and standard errors of the means from three independent biological duplicates ( n = 6 ) are indicated . The result obtained with mock - treated cells was arbitrarily set at a value of 1 ( E , F ) .

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

Figure 4A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B)A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D)A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).


Figure 5One night after cell purification , CD8 + - depleted PBMCs from 12 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added to the cultures . Six days after 5 - AzadC treatment , WST - 1 assay was performed . Mock value was arbitrary fixed at 100 % for each individual .

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

Figure 5One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.


Figure 6Figure 6 : Representation of reactivation status of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + patients . A - C From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + patients ( Table 2 ) , the extracellular HIV - 1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures ( A ) , from patient cell cultures presenting no viral reactivation in mock condition ( C ) , and from patient cell cultures exhibiting reactivation in mock condition ( B ) . One night after cell purification , cells were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . Six days after 5 - AzadC treatment , the concentration of viral RNA in culture supernatants was determined ( in copies / ml ) . The results were reported as the actual HIV RNA copy numbers / ml or as an estimated value calculated as 50 % of the smallest value when HIV RNA was not detected in order to assign a log value . Means are represented .

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

Figure 6Figure 6: Representation of reactivation status of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients.A-C From data of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients (Table 2), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


Figure 7A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + patients ( shown in Appendix Table S2 ) . B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + patients ( shown in Table 2 ) . Data information : The results were reported as the actual HIV RNA copy numbers / ml or as an estimated value calculated as 50 % of the smallest value when HIV RNA was not detected in order to assign a log value . Means are represented by a line .

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

Figure 7A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Appendix Table S2).B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Table 2).Data information: The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented by a line.


Figure 8Figure 8 : Representation of reactivation status of ex vivo cultures of resting CD4 + T cells . A - C From data of ex vivo cultures of resting CD4 + T cells isolated from HIV + patients ( Table 3 ) , the extracellular HIV - 1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures ( A ) , from patient cell cultures presenting no viral reactivation in mock condition ( C ) , and from patient cell cultures exhibiting reactivation in mock condition ( B ) . One night after cell purification , cells were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . Six days after 5 - AzadC treatment , the concentration of viral RNA in culture supernatants was determined ( in copies / ml ) . The results were reported as the actual HIV RNA copy numbers / ml or as an estimated value calculated as 50 % of the smallest value when HIV RNA was not detected in order to assign a log value . Means are represented .

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

Figure 8Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


Figure 9A - H One night after cell purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T cells from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . The activation status of CD4 + T cell subset was assessed 6 days after 5 - AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented . A - H One night after cell purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T cells from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . The activation status of CD4 + T cell subset was assessed 6 days after 5 - AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented . A - H One night after cell purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T cells from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . The activation status of CD4 + T cell subset was assessed 6 days after 5 - AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented . A - H One night after cell purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T cells from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . The activation status of CD4 + T cell subset was assessed 6 days after 5 - AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

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

Figure 9A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.


Figure 10One night after cell purification , HLA DR - CD69 - CD25 - CD4 + T cells from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced and HDACIs were added in the cultures . The median fluorescence intensity of CD4 receptor of viable CD4 + T cell subset was assessed 6 days after 5 - AzadC treatment by flow cytometry analysis relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

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

Figure 10One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.


Figure 1 ( B , C ) SH - SY5Y cells were infected with HSV - 2 ( MOI , 1 ) , for the indicated time intervals , and culture supernatants were isolated for measurement of LDH ( B ) or HMBG1 ( C ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ p ≤ 0 . 01 ; ∗ ∗ ∗ p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ( Mann - Whitney test , two - tailed in ( E ) SH - SY5Y cells were infected with HSV - 2 ( MOI = 1 ) for the indicated time intervals , and cell lysates were immunoblotted for p - MLKL and vinculin . For comparison , we used lysates from THP1 cells treated with Z - VAD ( Z , 20 μM ) , TNFα ( T , 100 ng / ml ) , Birinapant ( B , 100 nM ) , and Necrostain - 1 ( NEC - 1 , 10 μM ) as indicated . ( F , G ) SH - SY5Y cells were infected with HSV - 1 and HSV - 2 ( both MOI = 1 ) for the indicated time intervals . Cell lysates were evaluated for CASP3 activity ( G ) and immunoblotted for cleaved caspase 3 ( CL - CASP3 ) , cleaved PARP ( CL - PARP ) , and vinculin ( F ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ p ≤ 0 . 01 ; ∗ ∗ ∗ p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 . ( Mann - Whitney test , two - tailed in ( J , K ) SH - SY5Y cells were infected with HSV - 1 and HSV - 2 ( both MOI = 1 ) for the indicated time intervals , and cell lysates were immunoblotted for GSDME and vinculin . ( L , M ) SH - SY5Y cells were infected with EMCV and VSV ( both MOI = 1 ) for 16 h , and cell lysates were immunoblotted for GSDME and vinculin .

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

Figure 1(B, C) SH-SY5Y cells were infected with HSV-2 (MOI, 1), for the indicated time intervals, and culture supernatants were isolated for measurement of LDH (B) or HMBG1 (C). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p≤0.001; **** p≤0.0001. (Mann-Whitney test, two-tailed in(E) SH-SY5Y cells were infected with HSV-2 (MOI=1) for the indicated time intervals, and cell lysates were immunoblotted for p-MLKL and vinculin. For comparison, we used lysates from THP1 cells treated with Z-VAD (Z, 20 μM), TNFα (T, 100 ng/ml), Birinapant (B, 100 nM), and Necrostain-1 (NEC-1, 10 μM) as indicated.(F, G) SH-SY5Y cells were infected with HSV-1 and HSV-2 (both MOI=1) for the indicated time intervals. Cell lysates were evaluated for CASP3 activity (G) and immunoblotted for cleaved caspase 3 (CL-CASP3), cleaved PARP (CL-PARP), and vinculin (F). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p≤0.001; **** p≤0.0001. (Mann-Whitney test, two-tailed in(J, K) SH-SY5Y cells were infected with HSV-1 and HSV-2 (both MOI=1) for the indicated time intervals, and cell lysates were immunoblotted for GSDME and vinculin.(L, M) SH-SY5Y cells were infected with EMCV and VSV (both MOI=1) for 16 h, and cell lysates were immunoblotted for GSDME and vinculin.


Figure 2ESC - derived neuron progenitor cells were infected with HSV - 1 and HSV - 2 ( both MOI = 1 ) for the indicated time intervals , and lysates were isolated and analyzed as indicated by ( B ) immunoblottingHuman brain slices were infected with HSV - 1 - GFP ( 1 ˟ 106 PFU / ml ) and HSV - 2 - GFP ( 1 ˟ 106 PFU / ml ) for 72 h , visualized microscopicallyHuman brain slices were infected with HSV - 1 - GFP ( 1 ˟ 106 PFU / ml ) and HSV - 2 - GFP ( 1 ˟ 106 PFU / ml ) for 72 h , and lysed for analysis of GSDME and vinculin . Mouse mixed brain cells were infected with HSV - 2 ( MOI = 10 ) for 36 h and lysed for analysis of GSDME and vinculin by immunoblotting . ( I ) . Mouse mixed brain cells were infected with HSV - 2 ( MOI = 10 ) for 36 h and lysed for analysis of LDH release was measured by ELISA ( J ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ p ≤ 0 . 05 ; ∗ ∗ p ≤ 0 . 01 ( Mann - Whitney test , two - tailed in J ) .

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

Figure 2ESC-derived neuron progenitor cells were infected with HSV-1 and HSV-2 (both MOI=1) for the indicated time intervals, and lysates were isolated and analyzed as indicated by (B) immunoblottingHuman brain slices were infected with HSV-1-GFP (1˟106 PFU/ml) and HSV-2-GFP (1˟106 PFU/ml) for 72 h, visualized microscopicallyHuman brain slices were infected with HSV-1-GFP (1˟106 PFU/ml) and HSV-2-GFP (1˟106 PFU/ml) for 72 h, and lysed for analysis of GSDME and vinculin.Mouse mixed brain cells were infected with HSV-2 (MOI=10) for 36 h and lysed for analysis of GSDME and vinculin by immunoblotting. (I).Mouse mixed brain cells were infected with HSV-2 (MOI=10) for 36 h and lysed for analysis of LDH release was measured by ELISA (J). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗p ≤ 0.05; ∗∗p ≤ 0.01 (Mann-Whitney test, two-tailed in J).


Figure 3 ( A ) Lysates from SH - SY5Y and THP1 cells were immunoblotted for a panel of caspases and vinculin . ( B ) SH - SY5Y cells were pre - treated with CASP2 inhibitor Z - VDVAD ( 20 μM ) for 1 h and infected with HSV - 2 ( MOI = 1 ) . Lysates were isolated 16 hpi for immunoblot analysis . ( C , SH - SY5Y cells were transfected with CASP2 gRNA - Cas9 RNPs or AAVS1 gRNA - Cas9 RNPs and infected with HSV - 2 . Lysates were isolated 16 h post infection and immunoblotted for GSDME , CASP2 , cleaved caspase 3 ( CL - CASP3 ) , cleaved PARP ( CL - PARP ) and vinculin ( C ) . G ) SH - SY5Y cells were infected with HSV - 2 . Supernatant and lysates were isolated for identification of HMGB1 ( G , 16 h ) . ( H ) SH - SY5Y cells transfected with AAVS1 or CASP2 gRNA - Cas9 RNPs and infected with HSV - 2 for 16 h and examined for morphological changes by microscopy . Boxes indicate areas highlighted in the zoomed images . Arrows indicate ballooning or syncytial cells .

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

Figure 3(A) Lysates from SH-SY5Y and THP1 cells were immunoblotted for a panel of caspases and vinculin.(B) SH-SY5Y cells were pre-treated with CASP2 inhibitor Z-VDVAD (20 μM) for 1 h and infected with HSV-2 (MOI=1). Lysates were isolated 16 hpi for immunoblot analysis.(C, SH-SY5Y cells were transfected with CASP2 gRNA-Cas9 RNPs or AAVS1 gRNA-Cas9 RNPs and infected with HSV-2. Lysates were isolated 16 h post infection and immunoblotted for GSDME, CASP2, cleaved caspase 3 (CL-CASP3), cleaved PARP (CL-PARP) and vinculin (C).G) SH-SY5Y cells were infected with HSV-2. Supernatant and lysates were isolated for identification of HMGB1 (G, 16 h).(H) SH-SY5Y cells transfected with AAVS1 or CASP2 gRNA-Cas9 RNPs and infected with HSV-2 for 16 h and examined for morphological changes by microscopy. Boxes indicate areas highlighted in the zoomed images. Arrows indicate ballooning or syncytial cells.


Figure 4 ( B , SH - SY5Y cells transfected with AAVS1 or BID gRNA - Cas9 RNPs were infected with HSV - 2 . Lysates were isolated 16 h post infection and immunoblotted for GSDME , CL - CASP3 and CL - PARP ( B ) C ) SH - SY5Y cells transfected with AAVS1 or BID gRNA - Cas9 RNPs were infected with HSV - 2 . Lysates were isolated 16 h post infection and assayed for CASP3 activity ( C ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ ∗ p ≤ 0 . 001 two - way ANOVA in C ( G ) NADPH reductase activity was measured to evaluate mitochondria function in SH - SY5Y cells infected with HSV - 2 ( MOI , 1 ) for the indicated time interval . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ ∗ p ≤ 0 . 001 ( Mann - Whitney test , two - tailed in GSH - SY5Y cells were treated with ethidium bromide ( 2 μg / ml ) and infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysate were for immunoblotting for cell death markers GSDMESH - SY5Y cells were treated with ethidium bromide ( 2 μg / ml ) and infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysate and supernatant were for immunoblotting for cell death markers HMGB1SH - SY5Y cells were treated with ethidium bromide ( 2 μg / ml ) and infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysate were for immunoblotting for cell death markers GSDME SH - SY5Y cells were transfected with Poly I : C ( 50 μg / ml ) or dsDNA ( 20 μg / ml ) for 24 h ( J ) .

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

Figure 4(B, SH-SY5Y cells transfected with AAVS1 or BID gRNA-Cas9 RNPs were infected with HSV-2. Lysates were isolated 16 h post infection and immunoblotted for GSDME, CL-CASP3 and CL-PARP (B)C) SH-SY5Y cells transfected with AAVS1 or BID gRNA-Cas9 RNPs were infected with HSV-2. Lysates were isolated 16 h post infection and assayed for CASP3 activity (C). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗∗p ≤ 0.001 two-way ANOVA in C(G) NADPH reductase activity was measured to evaluate mitochondria function in SH-SY5Y cells infected with HSV-2 (MOI, 1) for the indicated time interval. Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗∗p ≤ 0.001 (Mann-Whitney test, two-tailed in GSH-SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV-2 (MOI=1, 16 h). Lysate were for immunoblotting for cell death markers GSDMESH-SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV-2 (MOI=1, 16 h). Lysate and supernatant were for immunoblotting for cell death markers HMGB1SH-SY5Y cells were treated with ethidium bromide (2 μg/ml) and infected with HSV-2 (MOI=1, 16 h). Lysate were for immunoblotting for cell death markers GSDME SH-SY5Y cells were transfected with Poly I:C (50 μg/ml) or dsDNA (20 μg/ml) for 24 h (J).


Figure 5 ( A , SH - SY5Y cells were infected with HSV - 2 ( MOI = 1 ) for the indicated time intervals , and immunoblotted for ER stress markers ( BIP , phospho - IRE1α , phospho - eIF2α , cleaved - ATF6 , CHOP ) B ) SH - SY5Y cells were infected with HSV - 2 ( MOI = 1 ) for the indicated time intervals , and analyzed for mRNA levels of the ER stress response genes BIP , ATF6 and CHOP by RT - qPCR ( B ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ p ≤ 0 . 01 ; ∗ ∗ ∗ p ≤ 0 . 001 ; ∗ ∗ ∗ ∗ p ≤ 0 . 0001 ( Mann - Whitney test , two - tailed in B ( C ) Lysate from SH - SY5Y cells with HSV - 2 infection ( MOI = 1 ) were immunoblotted for BCL2 family proteins MCL - 1 , Bcl - XL , and phospho - BAD ( S112 ) . Vinculin was used as loading control . SH - SY5Y cells were pretreated with TUDCA ( 500 ng / ml ) for 1 h and infected with HSV - 2 ( MOI = 1 ) . Lysates were isolated after 16 h of HSV - 2 infection and subjected to immunoblotting for CL - CASP3 , CL - PARP , GSDMESH - SY5Y cells were pretreated with TUDCA ( 500 ng / ml ) for 1 h and infected with HSV - 2 ( MOI = 1 ) . supernatants ( 24 h ) were analysed for LDH release ( H , ELISA ) . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ p ≤ 0 . 01 ; ∗ ∗ ∗ p ≤ 0 . 001 ; ∗ ∗ ∗ ∗ p ≤ 0 . 0001 two - way ANOVA in H ) . ( L ) SH - SY5Y cells transduced with lenti - plasmid BIP or empty vector control were infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysates were isolated and immunoblotted for GSDME and tBID .

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

Figure 5(A, SH-SY5Y cells were infected with HSV-2 (MOI=1) for the indicated time intervals, and immunoblotted for ER stress markers (BIP, phospho-IRE1α, phospho-eIF2α, cleaved-ATF6, CHOP)B) SH-SY5Y cells were infected with HSV-2 (MOI=1) for the indicated time intervals, and analyzed for mRNA levels of the ER stress response genes BIP, ATF6 and CHOP by RT-qPCR (B). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001 (Mann-Whitney test, two-tailed in B(C) Lysate from SH-SY5Y cells with HSV-2 infection (MOI=1) were immunoblotted for BCL2 family proteins MCL-1, Bcl-XL, and phospho-BAD (S112). Vinculin was used as loading control.SH-SY5Y cells were pretreated with TUDCA (500 ng/ml) for 1 h and infected with HSV-2 (MOI=1). Lysates were isolated after 16 h of HSV-2 infection and subjected to immunoblotting for CL-CASP3, CL-PARP, GSDMESH-SY5Y cells were pretreated with TUDCA (500 ng/ml) for 1 h and infected with HSV-2 (MOI=1). supernatants (24 h) were analysed for LDH release (H, ELISA). Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001 two-way ANOVA in H).(L) SH-SY5Y cells transduced with lenti-plasmid BIP or empty vector control were infected with HSV-2 (MOI=1, 16 h). Lysates were isolated and immunoblotted for GSDME and tBID.


Figure 6SH - SY5Y cells were infected with HSV - 2 for the indicated time intervals . Total RNA was analyzed for spliced XBP1 ( XBP1s ) by RT - qPCR Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ p ≤ 0 . 01 ; ∗ ∗ ∗ p ≤ 0 . 001 ( Mann - Whitney test , two - tailed in BSH - SY5Y cells were infected with HSV - 2 for the indicated time intervals . Lysates were immunoblotted for IRE1α phosphor - JNK and XBP1s ( C ) . ( E ) SH - SY5Y cells transfected with AAVS1 or IRE1A gRNA - Cas9 RNPs were infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysates and supernatant were isolated and immunoblotted for IRE1α , GSDME , cytochrome c and HMGB1 release . G ) SH - SY5Y cells were pretreated with APY29 for 1 h and following HSV - 2 infection ( 16 h ) . Lysate were immunoblotted for p - JNK . ( H ) SH - SY5Y cells were pretreated with SP600125 ( 10 μM ) for 1 h infected with HSV - 2 ( MOI = 1 , 16 h ) . Lysates and supernatant were immunoblotted for GSDME , cytochrome c , HMGB1 , and vinculin .

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

Figure 6SH-SY5Y cells were infected with HSV-2 for the indicated time intervals. Total RNA was analyzed for spliced XBP1 (XBP1s) by RT-qPCR Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001 (Mann-Whitney test, two-tailed in BSH-SY5Y cells were infected with HSV-2 for the indicated time intervals. Lysates were immunoblotted for IRE1α phosphor-JNK and XBP1s (C).(E) SH-SY5Y cells transfected with AAVS1 or IRE1A gRNA-Cas9 RNPs were infected with HSV-2 (MOI=1, 16 h). Lysates and supernatant were isolated and immunoblotted for IRE1α, GSDME, cytochrome c and HMGB1 release.G) SH-SY5Y cells were pretreated with APY29 for 1 h and following HSV-2 infection (16 h). Lysate were immunoblotted for p-JNK.(H) SH-SY5Y cells were pretreated with SP600125 (10 μM) for 1 h infected with HSV-2 (MOI=1, 16 h). Lysates and supernatant were immunoblotted for GSDME, cytochrome c, HMGB1, and vinculin.


Figure 7 ( B - D ) iPSC - derived microglia cells were treated with the conditioned medium from HSV - 2 - infected control and GSDME - depleted SH - SY5Y cells for 8 h . Total RNA was isolated and analyzed for expression of IL6 , TNFA , and CXCL10 by RT - qPCR . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ ∗ p ≤ 0 . 001 ; ∗ ∗ ∗ ∗ p ≤ 0 . 0001 ( Two - way ANOVA in B - D ( E - G ) Conditioned medium from SH - SY5Y cells with HSV - 2 infection ( MOI = 1 , 24 h ) was transferred to THP1 cells ( WT , STING - / - , MYD88 - / - , TRIF - / - and MAVS - / - ) . Total RNA was isolated 8h later and analyzed for expression of IL6 ( E ) , TNFA ( F ) , and CXCL10 ( G ) by RT - qPCR . Data information : All data shown are representative of at least 3 independent experiments . Data are presented as mean ± s . d . in all graphs . ∗ ∗ ∗ p ≤ 0 . 001 ; ∗ ∗ ∗ ∗ p ≤ 0 . 0001 Wilcoxon matched - pairs test in E - G ) .

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

Figure 7(B-D) iPSC-derived microglia cells were treated with the conditioned medium from HSV-2-infected control and GSDME-depleted SH-SY5Y cells for 8 h. Total RNA was isolated and analyzed for expression of IL6, TNFA, and CXCL10 by RT-qPCR. Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001 (Two-way ANOVA in B-D(E-G) Conditioned medium from SH-SY5Y cells with HSV-2 infection (MOI=1, 24 h) was transferred to THP1 cells (WT, STING-/-, MYD88-/-, TRIF-/- and MAVS-/-). Total RNA was isolated 8h later and analyzed for expression of IL6 (E), TNFA (F), and CXCL10 (G) by RT-qPCR. Data information: All data shown are representative of at least 3 independent experiments. Data are presented as mean ± s.d. in all graphs. ∗∗∗p ≤ 0.001; ∗∗∗∗p ≤ 0.0001 Wilcoxon matched-pairs test in E-G).


Figure 1 ( A ) Intracellular endogenous Aβ42 ( green ) , Ankyrin - G ( AnkG ; magenta ) and GFP ( blue ) in siBin1 - , siCD2AP - and siControl - treated primary cortical neurons ( neurons ) expressing GFP immunolabelled at 9 DIV with anti - Aβ42 ( clone 12F4 ) and anti - AnkG , analysed by spinning - disk confocal microscopy . The white rectangles indicate the dendrites ( Dd ) and axons ( Ax ) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG ( magenta ) and GFP ( blue ) , respectively . Scale bars , 10 µm . ( B ) Aβ42 line profiles in dendrites ( Dd ) of siControl ( grey line ) , siBin1 ( blue line ) and siCD2AP ( red line ) neurons shown in ( c ) . ( C ) Aβ42 line profiles in axons ( Ax ) of siControl ( grey line ) , siBin1 ( blue line ) and siCD2AP ( red line ) neurons shown in ( c ) . ( D ) Quantification of Aβ42 ( 12F4 ) intensity in cell body ( CB ) , dendrite ( Dd ) and axon ( Ax ) ( n = 5 , NCB = 44 - 53 , NDd = 90 - 120 , NAx = 60 - 74 ; * * * * PCB < 0 . 0001 siBin1 vs . siControl , * * * PCB < 0 . 001 siCD2AP vs . siControl , * * * * PDd < 0 . 0001 siBin1 vs . siControl , * * * * PDd < 0 . 0001 siCD2AP vs . siControl , * * * * PAx < 0 . 0001 siBin1 vs . siControl , t - test , mean ± SEM ) . ( E ) Quantification of extracellular endogenous Aβ40 , Aβ42 and of Aβ42 / Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1 , siCD2AP or siControl neurons ( n = 6 ; * PAβ40 = 0 . 0270 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0378 siBin1 vs . siControl , * PAβ42 / 40 = 0 . 0463 siCD2AP vs . siControl , t - test , mean ± SEM ) . ( F ) Endogenous APP and APP - CTFs levels by western blot with anti - APP antibody ( Y188 ) of siBin1 - , siCD2AP - or siControl - treated neurons at 9 DIV . ( G ) Quantification of APP and APP - CTFs levels normalized to APP ( n = 4 ; * PAPP = 0 . 0355 siBin1 vs . siControl , * * * PAPPCTFs / APP < 0 . 001 siBin1 vs . siControl , * * * * PAPPCTFs / APP < 0 . 0001 siCD2AP vs . siControl , t - test , mean ± SEM ) .

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

Figure 1(A) Intracellular endogenous Aβ42 (green), Ankyrin-G (AnkG; magenta) and GFP (blue) in siBin1- , siCD2AP- and siControl-treated primary cortical neurons (neurons) expressing GFP immunolabelled at 9 DIV with anti-Aβ42 (clone 12F4) and anti-AnkG, analysed by spinning-disk confocal microscopy. The white rectangles indicate the dendrites (Dd) and axons (Ax) magnified below showing Aβ42 in axons and dendrites outlined based on AnkG (magenta) and GFP (blue), respectively. Scale bars, 10 µm.(B) Aβ42 line profiles in dendrites (Dd) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(C) Aβ42 line profiles in axons (Ax) of siControl (grey line), siBin1 (blue line) and siCD2AP (red line) neurons shown in (c).(D) Quantification of Aβ42 (12F4) intensity in cell body (CB), dendrite (Dd) and axon (Ax) (n=5, NCB=44-53, NDd=90-120, NAx=60-74; ****PCB<0.0001 siBin1 vs. siControl, ***PCB<0.001 siCD2AP vs. siControl, ****PDd<0.0001 siBin1 vs. siControl, ****PDd<0.0001 siCD2AP vs. siControl, ****PAx<0.0001 siBin1 vs. siControl, t-test, mean ± SEM).(E) Quantification of extracellular endogenous Aβ40, Aβ42 and of Aβ42/Aβ40 ratio by ELISA analysis of conditioned media of 9 DIV siBin1, siCD2AP or siControl neurons (n=6; *PAβ40=0.0270 siBin1 vs. siControl, *PAβ42/40=0.0378 siBin1 vs. siControl, *PAβ42/40=0.0463 siCD2AP vs. siControl, t-test, mean ± SEM).(F) Endogenous APP and APP-CTFs levels by western blot with anti-APP antibody (Y188) of siBin1-, siCD2AP- or siControl-treated neurons at 9 DIV.(G) Quantification of APP and APP-CTFs levels normalized to APP (n=4; *PAPP=0.0355 siBin1 vs. siControl, ***PAPPCTFs/APP<0.001 siBin1 vs. siControl, ****PAPPCTFs/APP<0.0001 siCD2AP vs. siControl, t-test, mean ± SEM).