Figure 1B . Representative 2D projection of confocal images showing caspase activation at the dissection time point ( red channel ) and in the recent past ( green channel , arrows ) within putative escort somatic cells via DBS - S - QF sensor ; TUNEL staining indicates apoptosis ( grey , arrowhead ) ; DAPI labels the nuclei in the entire Figure . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; ; QUAS - Gal4 / + ( BL83123 ) . C . Representative 2D projection of confocal images showing escort and follicular somatic cells permanently labelled with DBS - S - QF sensor ( green channel , arrows ) ; the arrowhead indicates the presence of germline cells positive for TUNEL staining ( red , arrowhead ) . Notice the lack of TUNEL signal and therefore apoptosis in somatic cells permanently labelled with DBS - S - QF sensor ( green ) . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; QUAS - flippase ( BL30126 ) / + ; Actin5C FRT - stop - FRT lacZ - nls / + ( BL6355 ) Data information : Scale bars represents 10 µm in the entire Figure . D . Graph showing the percentage of germaria permanently labelled with DBS - S - QF overtime at 25 ° C ( N = 4 biological replicates ; n = 520 and n = 536 germaria were inspected at 7 and 14 days post adult eclosion , respectively ) and 29 ° C ( N = 7 , n = 799 at 7 days and n = 588 at 14 days ) . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotype : Actin DBS - S - QF , UAS - mCD8 - GFP , QUAS - tomato - HA / + ; QUAS - flippase ( BL30126 ) / + ; Actin5C FRT - stop - FRT lacZ - nls / + ( BL6355 ) . Data information : The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets . E . Percentage of germaria showing permanently labelled with DBS - S - QF the ECD over time at 25 ° C and 29 ° C . FasIII immunostaining was used to locate the DBS - S - QF labelling in the inspected germaria . The N and n numbers of these experiments are indicated in D . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotypes are shown in D . F . Percentage of germaria showing permanently labelled with DBS - S - QF the ECD over time at 25 ° C and 29 ° C . FasIII immunostaining was used to locate the DBS - S - QF labelling in the inspected germaria . The N and n numbers of these experiments are indicated in D . A two - way ANOVA and Tukey ' s multiple comparison tests were used to determine statistical significance ( n . s = not significant ; * * * * p < 0 . 0001 ) . Genotypes are shown in D . Data information : The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets .

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

Figure 1B. Representative 2D projection of confocal images showing caspase activation at the dissection time point (red channel) and in the recent past (green channel, arrows) within putative escort somatic cells via DBS-S-QF sensor; TUNEL staining indicates apoptosis (grey, arrowhead); DAPI labels the nuclei in the entire Figure. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; ;QUAS-Gal4 /+ (BL83123). C. Representative 2D projection of confocal images showing escort and follicular somatic cells permanently labelled with DBS-S-QF sensor (green channel, arrows); the arrowhead indicates the presence of germline cells positive for TUNEL staining (red, arrowhead). Notice the lack of TUNEL signal and therefore apoptosis in somatic cells permanently labelled with DBS-S-QF sensor (green). Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection. Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355) Data information: Scale bars represents 10 µm in the entire Figure.D. Graph showing the percentage of germaria permanently labelled with DBS-S-QF overtime at 25 °C (N=4 biological replicates; n=520 and n=536 germaria were inspected at 7 and 14 days post adult eclosion, respectively) and 29 °C (N=7, n=799 at 7 days and n=588 at 14 days). A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotype: Actin DBS-S-QF, UAS-mCD8-GFP, QUAS-tomato-HA/+; QUAS-flippase (BL30126)/+; Actin5C FRT-stop-FRT lacZ-nls/+ (BL6355). Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.E. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. F. Percentage of germaria showing permanently labelled with DBS-S-QF the ECD over time at 25 °C and 29 °C. FasIII immunostaining was used to locate the DBS-S-QF labelling in the inspected germaria. The N and n numbers of these experiments are indicated in D. A two-way ANOVA and Tukey's multiple comparison tests were used to determine statistical significance (n.s = not significant; **** p<0.0001). Genotypes are shown in D. Data information: The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets.


Figure 2A - B . Representative 2D projection of confocal images showing the expression of the cell identity Castor in follicular cells control with Dronc expression ( A : 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + ) and without Dronc ( B : 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ) . Notice the reduction in the number of Castor - expressing cells in the FCD and the reduction in size of this region in B . Nuclei are labelled with DAPI ( blue ) ; Castor ( green ) and FasIII ( grey ) label the follicular cells ; UAS - Histone - RFP labels the nuclei of Gal4 - expressing cells ( red ) . Data information : Scale bars represents 10 µm . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . C . Quantification of total number of follicular cells ( left ) and Castor - expressing cells ( right ) within the FCD ( FasIII positive region ) in either heterozygous or homozygous Dronc mutant conditions : Genotypes : CTRL = 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + ; Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . CTRL = ptc - Gal4 ( BL2017 ) / + ; UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . Dronc - / - = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . N = 2 ; the n number for each column in order of appearance n = 16 , n = 14 , n = 20 , n = 17 , n = 19 , n = 18 . Statistical significance was determined by using an unpaired parametric Welch ' s t - test ( * * * * p ≤ 0 . 001 ) . The median and quartiles are indicated in the violin plots . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . E . Percentage of germaria showing TUNEL staining in different cell types of the germarium ( somatic cells and germline cells ) upon altering Dronc expression in all of the escort cells and follicular stem cells . N = 3 biological replicates ; the n number of inspected germaria per condition is indicated close to the genotype . The genotypes in the graph are : White triangle ( n = 378 germaria ) = ptc - Gal4 ( BL2017 ) / + ; + / + . Yellow circle ( n = 381 ) = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . Red square ( n = 196 ) = ptc - Gal4 ( BL2017 ) / + ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - suntag - HA . Blue circle ( n = 184 ) = ptc - Gal4 ( BL2017 ) / UAS - Dronc ( BL56198 ) ; DroncKO UAS - Histone - RFP ( BL56555 ) Tub - G80ts ( BL7019 ) / + . An ordinary one - way ANOVA and Holm - Sidak ' s multiple comparison test was used to determine statistical significance ( n . s = not significant ; * * p ≤ 0 . 01 ; * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . The graph shows the mean and standard deviations . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . J - K . Representative 2D projection of confocal images of the genotypes quantified in I . Nuclei are labelled with DAPI ( blue ) . Red and green nuclei show the expression of different Fucci markers . FasIII ( grey ) was used to locate the FCD . Notice the reduction of follicular cells and limited overlap between the green and red fluorescent signals ( white arrowheads ) in K . Data information : Scale bars represents 10 µm . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2A-B. Representative 2D projection of confocal images showing the expression of the cell identity Castor in follicular cells control with Dronc expression (A: 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+) and without Dronc (B: 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF). Notice the reduction in the number of Castor-expressing cells in the FCD and the reduction in size of this region in B. Nuclei are labelled with DAPI (blue); Castor (green) and FasIII (grey) label the follicular cells; UAS-Histone-RFP labels the nuclei of Gal4-expressing cells (red). Data information: Scale bars represents 10 µm. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).C. Quantification of total number of follicular cells (left) and Castor-expressing cells (right) within the FCD (FasIII positive region) in either heterozygous or homozygous Dronc mutant conditions: Genotypes: CTRL= 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+; Dronc -/- = 109-30-Gal4 (BL7023)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF.CTRL=ptc-Gal4 (BL2017)/+; UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+. Dronc -/- = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF. N=2; the n number for each column in order of appearance n=16, n=14, n=20, n=17, n=19, n=18. Statistical significance was determined by using an unpaired parametric Welch's t-test (****p≤0.001). The median and quartiles are indicated in the violin plots. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).E. Percentage of germaria showing TUNEL staining in different cell types of the germarium (somatic cells and germline cells) upon altering Dronc expression in all of the escort cells and follicular stem cells. N=3 biological replicates; the n number of inspected germaria per condition is indicated close to the genotype. The genotypes in the graph are: White triangle (n=378 germaria) = ptc-Gal4 (BL2017)/+; +/+. Yellow circle (n=381) = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ +. Red square (n=196) = ptc-Gal4 (BL2017)/+; DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-suntag-HA. Blue circle (n=184)= ptc-Gal4 (BL2017)/UAS-Dronc (BL56198); DroncKO UAS-Histone-RFP (BL56555) Tub-G80ts (BL7019)/+. An ordinary one-way ANOVA and Holm-Sidak's multiple comparison test was used to determine statistical significance (n.s = not significant; ** p≤0.01; *** p≤0.001; ****p≤0.0001). The graph shows the mean and standard deviations. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).J-K. Representative 2D projection of confocal images of the genotypes quantified in I. Nuclei are labelled with DAPI (blue). Red and green nuclei show the expression of different Fucci markers. FasIII (grey) was used to locate the FCD. Notice the reduction of follicular cells and limited overlap between the green and red fluorescent signals (white arrowheads) in K. Data information: Scale bars represents 10 µm. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).


Figure 3A . Quantification of total number of follicular cells in germaria with follicular stem cells expressing normal levels of Dronc ( DroncKO / + ) , without Dronc expression ( DroncKO / DroncKO - Suntag - HA - Cherry ) , and expressing a catalytically inactive form of Dronc ( DroncKO / DroncKO - FL - CAEA - Suntag - HA - Cherry ) . The genotypes are as follows : 109 - 30 - Gal4 ( BL7023 ) / + ( n = 13 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT Dronc - GFP - Apex FRT - Suntag - HA - Cherry ( n = 15 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; DroncKO Tub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT Dronc - GFP - Apex FRT - Dronc FL - CAEA - Suntag - HA - Cherry ( n = 14 ) . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . B . Quantification of total Castor - expressing cells within the FasIII cellular domain in the genotypes shown in A . n numbers are shown in A . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * * p ≤ 0 . 0001 ) . C . Percentage of Castor - expressing cells versus the total number of Follicular cells in germaria of the genotypes indicated in A . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . D . Quantification of total number of follicular cells within the FasIII cellular domain in the following genotypes : 109 - 30 - Gal4 / + ; Tub - G80ts ( BL7019 ) / + ( n = 16 ) . 109 - 30 - Gal4 ( BL7023 ) / UAS - DriceRNAi UAS - DecayRNAi ( a gift from Pascal Meier ) ; UAS - DammRNAi , UAS - Dcp1RNAi ( a gift from Pascal Meier ) ( n = 15 ) . 109 - 30 - Gal4 ( BL7023 ) / + ; Tub - G80ts ( BL7019 ) / UAS - Dcp1RNAi ( BL28909 ) ( n = 25 ) 109 - 30 - Gal4 ( BL7023 ) / + ; Tub - G80ts ( BL7019 ) / UAS - DriceRNAi ( BL32403 ) ( n = 16 ) 109 - 30 - Gal4 ( BL7023 ) / UAS - Dark - sh ; Tub - G80ts ( BL7019 ) / + ( a gift from M . Miura ) ( n = 18 ) . 109 - 30 - Gal4 ( BL7023 ) / UAS - microRNA - RHG ; Tub - G80ts ( BL7019 ) / + ( a gift from Iswar Hariharan ) ( n = 10 ) . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . E . Quantification of total number of Castor - expressing cells in germaria of the genotypes indicated in D . n numbers are shown in D . A one - way ANOVA and Dunnett ' s multiple comparison post - test was used to determine statistical significance ( * * * p ≤ 0 . 001 ; * * * * p ≤ 0 . 0001 ) . F . Percentage of Castor - expressing cells versus the total number of Follicular cells ( FasIII + cells ) in germaria of the genotypes indicated in A . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant , * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . The box plots show the median , first quartile , and third quartile of datasets . The whiskers illustrate the range between the maximum and minimum values of datasets . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

Figure 3A. Quantification of total number of follicular cells in germaria with follicular stem cells expressing normal levels of Dronc (DroncKO/+), without Dronc expression (DroncKO/DroncKO- Suntag-HA-Cherry), and expressing a catalytically inactive form of Dronc (DroncKO/DroncKO- FL-CAEA-Suntag-HA-Cherry ). The genotypes are as follows: 109-30-Gal4 (BL7023)/+ (n=13). 109-30-Gal4 (BL7023)/+; DroncKO Tub-G80ts (BL7019) / UAS-flippase (BL8209) DroncKO-FRT Dronc-GFP-Apex FRT-Suntag-HA-Cherry (n=15). 109-30-Gal4 (BL7023)/+; DroncKO Tub-G80ts (BL7019) / UAS-flippase (BL8209) DroncKO-FRT Dronc-GFP-Apex FRT-Dronc FL-CAEA-Suntag-HA-Cherry (n=14). A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.B. Quantification of total Castor-expressing cells within the FasIII cellular domain in the genotypes shown in A. n numbers are shown in A. A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (****p≤0.0001). C. Percentage of Castor-expressing cells versus the total number of Follicular cells in germaria of the genotypes indicated in A. A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.D. Quantification of total number of follicular cells within the FasIII cellular domain in the following genotypes: 109-30-Gal4/+; Tub-G80ts (BL7019)/+ (n=16). 109-30-Gal4 (BL7023)/UAS-DriceRNAi UAS-DecayRNAi (a gift from Pascal Meier); UAS-DammRNAi, UAS-Dcp1RNAi (a gift from Pascal Meier) (n=15). 109-30-Gal4 (BL7023)/+; Tub-G80ts (BL7019)/ UAS-Dcp1RNAi (BL28909) (n=25) 109-30-Gal4 (BL7023)/+; Tub-G80ts (BL7019)/ UAS-DriceRNAi (BL32403) (n=16) 109-30-Gal4 (BL7023)/UAS-Dark-sh; Tub-G80ts (BL7019)/+ (a gift from M. Miura) (n=18). 109-30-Gal4 (BL7023)/UAS-microRNA-RHG; Tub-G80ts (BL7019)/+ (a gift from Iswar Hariharan) (n=10). A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.E. Quantification of total number of Castor-expressing cells in germaria of the genotypes indicated in D. n numbers are shown in D. A one-way ANOVA and Dunnett's multiple comparison post-test was used to determine statistical significance (***p≤0.001; ****p≤0.0001). F. Percentage of Castor-expressing cells versus the total number of Follicular cells (FasIII+ cells) in germaria of the genotypes indicated in A. A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (n.s.= not significant, *p≤0.05, **p≤0.01, ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. The box plots show the median, first quartile, and third quartile of datasets. The whiskers illustrate the range between the maximum and minimum values of datasets. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


Figure 4A - B . Representative 2D projections of confocal images showing the expression of Ci - 155 ( blue and grey channels ) , ptc - GFP ( green and grey ; ptc - GFP is a bona - fide transcriptional read out of Hh - pathway and weak hypomorph allele and Castor ( red and grey ) in germaria with either normal ( A ) or deficient Dronc expression ( B ) . Genotypes : 109 - 30 - Gal4 ( BL7023 ) / ptc - GFPCB02030 ( a gift from Isabel Guerrero ) ( A ) and 109 - 30 - Gal4 ( BL7023 ) / ptc - GFPCB02030 ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase DroncKO - FRT - Dronc - GFP - APEX - FRT - QF . Data information : Scale bars represents 10 µm in the entire figure . Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . D . Western blot showing Caspase - 9 expression ( upper lane ) and actin ( bottom lane , loading control ) in either control or Caspase - 9 deficient OVCAR - 3 cells ( 24h and 72h post - transfection of an shRNA against Caspase - 9 ) . Notice the strong downregulation of Caspase - 9 72h after siRNA treatment . H . Quantification of total number of follicular cells ( left ) or Castor - expressing cells ( right ) within the FasIII cellular domain in the following genotypes from left to right : Dronc - / - = CTRL = 109 - 30 - Gal4 ( BL7023 ) / ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 16 ) . CTRL = 109 - 30 - Gal4 ( BL7023 ) / UAS - smoAct ( BL44621 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 11 ) . Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / UAS - smoAct ( BL44621 ) ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 8 ) . CTRL = 109 - 30 - Gal4 ( BL7023 ) / UAS - Ci ( BL28984 ) ; DroncKOTub - G80ts ( BL7019 ) / + . ( n = 7 ) . Dronc - / - = 109 - 30 - Gal4 ( BL7023 ) / UAS - UAS - Ci ( BL28984 ) ; DroncKOTub - G80ts ( BL7019 ) / UAS - flippase ( BL8209 ) DroncKO - FRT - Dronc - GFP - APEX - FRT - QF ( n = 21 ) . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant ; * * * * p ≤ 0 . 0001 ; ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . Unless otherwise indicated , all experimental data presented were obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . J . Quantification of total number of follicular cells ( left ) or Castor - expressing cells ( right ) within the FasIII cellular domain in the following genotypes from left to right : Dronc + / + = ptc - Gal4 ( BL2017 ) / + ; Tub - G80ts ( BL7019 ) ( n = 19 ) . Dronc + / - = ptc - Gal4 ( BL2017 ) / + ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 23 ) . Dronc + / - UAS - Dronc = ptc - Gal4 ( BL2017 ) / + ; DroncKOTub - G80ts ( BL7019 ) / UAS - Dronc ( BL56198 ) ( n = 14 ) . A one - way ordinary ANOVA and Welch ' s correction were used to establish statistical significance ( n . s . = not significant ; * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . Unless otherwise indicated , all experimental data presented were obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-B. Representative 2D projections of confocal images showing the expression of Ci-155 (blue and grey channels), ptc-GFP (green and grey; ptc-GFP is a bona-fide transcriptional read out of Hh-pathway and weak hypomorph allele and Castor (red and grey) in germaria with either normal (A) or deficient Dronc expression (B). Genotypes: 109-30-Gal4 (BL7023)/ptc-GFPCB02030 (a gift from Isabel Guerrero) (A) and 109-30-Gal4 (BL7023)/ptc-GFPCB02030; DroncKOTub-G80ts (BL7019)/ UAS-flippase DroncKO-FRT-Dronc-GFP-APEX-FRT-QF. Data information: Scale bars represents 10 µm in the entire figure. Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels).D. Western blot showing Caspase-9 expression (upper lane) and actin (bottom lane, loading control) in either control or Caspase-9 deficient OVCAR-3 cells (24h and 72h post-transfection of an shRNA against Caspase-9). Notice the strong downregulation of Caspase-9 72h after siRNA treatment.H. Quantification of total number of follicular cells (left) or Castor-expressing cells (right) within the FasIII cellular domain in the following genotypes from left to right: Dronc -/- = CTRL= 109-30-Gal4 (BL7023)/; DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=16). CTRL= 109-30-Gal4 (BL7023)/UAS-smoAct (BL44621); DroncKOTub-G80ts (BL7019)/+ (n=11). Dronc -/- = 109-30-Gal4 (BL7023)/UAS-smoAct (BL44621); DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=8). CTRL= 109-30-Gal4 (BL7023)/UAS-Ci (BL28984); DroncKOTub-G80ts (BL7019)/+. (n=7). Dronc -/- = 109-30-Gal4 (BL7023)/UAS- UAS-Ci (BL28984); DroncKOTub-G80ts (BL7019)/ UAS-flippase (BL8209) DroncKO-FRT-Dronc-GFP-APEX-FRT-QF (n=21). A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (n.s.= not significant; ****p≤0.0001;). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). Unless otherwise indicated, all experimental data presented were obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.J. Quantification of total number of follicular cells (left) or Castor-expressing cells (right) within the FasIII cellular domain in the following genotypes from left to right: Dronc +/+ = ptc-Gal4 (BL2017)/+; Tub-G80ts (BL7019) (n=19). Dronc +/- = ptc-Gal4 (BL2017)/+; DroncKOTub-G80ts (BL7019)/+ (n=23). Dronc +/- UAS-Dronc = ptc-Gal4 (BL2017)/+; DroncKOTub-G80ts (BL7019)/UAS-Dronc (BL56198) (n=14). A one-way ordinary ANOVA and Welch's correction were used to establish statistical significance (n.s.= not significant; ****p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). Unless otherwise indicated, all experimental data presented were obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


Figure 5B . Relative number of Ptc - positive puncta per germaria of the following genotypes , from left to right : DroncKO Tub - G80ts / + ( n = 10 ) . ptc - Gal4 / + ; Tub - G80ts / + ( n = 9 ) . ptc - Gal4 / + ; DroncKO Tub - G80ts / + ( n = 10 ) . A Kruskal - Wallis test and Dunn ' s multiple comparison post - test were used to determine statistical significance ( * * * p ≤ 0 . 001 Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . C . Western blot showing Ptc ( upper lane ) and actin ( bottom lane , loading control ) expression in ovaries of the genotypes shown in A . Notice the Ptc accumulation in double heterozygous germaria ( ptc - Gal4 / + ; DroncKO Tub - G80ts / + ) . E . Pearson colocalization index between Ptc and Atg8 in germaria of the following genotypes : ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ( n = 12 ) . ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 18 ) . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * p ≤ 0 . 01 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . F . Fraction of Ptc positive that overlaps with Atg8 in germaria of the genotypes shown in E . n number is indicated in E . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * * p ≤ 0 . 001 ) . The Y axis in the graph shows the Manders ' Coefficient M1 - M2 . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . G . Fraction of Atg8 + autophagosomes that overlaps with Ptc in germaria of the genotypes shown in E . n number is indicated in E . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * * p ≤ 0 . 01 ) . The Y axis in the graph shows the Manders ' Coefficient M2 - M1 . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

Figure 5B. Relative number of Ptc-positive puncta per germaria of the following genotypes, from left to right: DroncKO Tub-G80ts /+ (n=10). ptc-Gal4 /+; Tub-G80ts/+ (n=9). ptc-Gal4 /+; DroncKO Tub-G80ts /+ (n=10). A Kruskal-Wallis test and Dunn's multiple comparison post-test were used to determine statistical significance (***p≤0.001 Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.C. Western blot showing Ptc (upper lane) and actin (bottom lane, loading control) expression in ovaries of the genotypes shown in A. Notice the Ptc accumulation in double heterozygous germaria (ptc-Gal4 /+; DroncKO Tub-G80ts /+).E. Pearson colocalization index between Ptc and Atg8 in germaria of the following genotypes: ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=12). ptc-Gal4 (BL2017)/UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=18). An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.F. Fraction of Ptc positive that overlaps with Atg8 in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (*** p≤0.001). The Y axis in the graph shows the Manders' Coefficient M1-M2. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.G. Fraction of Atg8+ autophagosomes that overlaps with Ptc in germaria of the genotypes shown in E. n number is indicated in E. An unpaired parametric Welch's t-test was used to establish the statistical significance (** p≤0.01). The Y axis in the graph shows the Manders' Coefficient M2-M1. Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


Figure 6B . Total number of Atg8 + autophagosomes in germaria of the following genotypes ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ( n = 21 ) . ptc - Gal4 ( BL2017 ) / UAS - GFP - mCherry - Atg8 ( BL37749 ) ; DroncKOTub - G80ts ( BL7019 ) / + ( n = 28 ) . An unpaired parametric Welch ' s t - test was used to establish the statistical significance ( * p ≤ 0 . 05 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . C . Volume of Atg8 + autophagosomes in germaria of the genotypes described in B . n number is indicated in B . A non - parametric Mann - Whitney t - test was used to determine statistical significance ( * * * p < 0 . 001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells . H . Western blot showing the expression levels of the autophagy marker p62 ( upper lane ) , Caspase - 9 ( middle lane ) and Actin ( bottom lane , loading control ) in either scrambled or Caspase - 9 deficient OVCAR - 3 cells ; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions , in cell culture media containing EtOH ( 0 . 2 % ) , and in cell culture media containing EtOH ( 0 . 2 % ) + bafilomycin A1 ( 400nM ) . I . Quantification of p62 protein levels in the experimental conditions described in H . one sample T Wilcoxon test was used to calculate statistical significance , * p ≤ 0 . 05 , N ≥ 3 . Bars indicate value of the mean while error bars represent the standard deviation . K . Quantification of the total number of Castor - expressing cells within the FasIII cellular domain in the genotypes described in J . n number is indicated also in J . An ordinary one - way ANOVA and Dunnett ' s multiple comparison post - test were used to determine statistical significance ( n . s . = not significant , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Data information : Experimental flies were kept after eclosion from the pupae for 14 days at 29 ° C prior to dissection ( applicable to all panels ) . All the experimental data shown have been obtained from N ≥ 2 biological replicates . The median and quartiles are indicated in the violin plots . All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells .

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

Figure 6B. Total number of Atg8+ autophagosomes in germaria of the following genotypes ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749) (n=21). ptc-Gal4 (BL2017)/ UAS-GFP-mCherry-Atg8 (BL37749); DroncKOTub-G80ts (BL7019)/+ (n=28). An unpaired parametric Welch's t-test was used to establish the statistical significance (* p≤0.05). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.C. Volume of Atg8+ autophagosomes in germaria of the genotypes described in B. n number is indicated in B. A non-parametric Mann-Whitney t-test was used to determine statistical significance (*** p<0.001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.H. Western blot showing the expression levels of the autophagy marker p62 (upper lane), Caspase-9 (middle lane) and Actin (bottom lane, loading control) in either scrambled or Caspase-9 deficient OVCAR-3 cells; the protein levels of the different read outs were measured at 72h after siRNA treatment in cells grown during the last 4 h before sample processing in our standard cell culture conditions, in cell culture media containing EtOH (0.2%), and in cell culture media containing EtOH (0.2%) + bafilomycin A1 (400nM).I. Quantification of p62 protein levels in the experimental conditions described in H. one sample T Wilcoxon test was used to calculate statistical significance, * p≤0.05, N≥3. Bars indicate value of the mean while error bars represent the standard deviation.K. Quantification of the total number of Castor-expressing cells within the FasIII cellular domain in the genotypes described in J. n number is indicated also in J. An ordinary one-way ANOVA and Dunnett's multiple comparison post-test were used to determine statistical significance (n.s.= not significant, ** p≤0.01, **** p≤0.0001). Data information: Experimental flies were kept after eclosion from the pupae for 14 days at 29°C prior to dissection (applicable to all panels). All the experimental data shown have been obtained from N ≥ 2 biological replicates. The median and quartiles are indicated in the violin plots. All the quantifications were made in germaria containing one single group of germline cells wrapped by follicular cells.


Figure 1 ( C , D Confocal micrographs of HeLa cells expressing GFP - TECPR1 and treated with osmotic shock assay or LLOMe ( C ) , or infected with the bacteria indicated ( D ) and fixed at 1 h ( S . Typhimurium and L . monocytogenes ) or 0 . 5 h ( S . flexneri ) post - infection . Cells were stained for Galectin - 8 and DNA ( DAPI ) . Scale bar , 20 μm . ( F ) Percentage of bacteria positive for mCherry - TECPR1 in ATG5 knockout ( KO ) or ATG5 - complemented MEF cells at 1 h ( S . Typhimurium ) or 0 . 5 h ( S . flexneri ) post - infection . Mean + / - SD of four ( S . Typhimurium ) or two ( S . flexneri ) independent experiments . n > 100 bacteria per coverslip . ( G ) Percentage of S . Typhimurium positive for GFP - TECPR1 at 1 h post - infection in HeLa cells pretreated or not with 100 nM Wortmannin . Mean + / - SD of two independent experiments . n > 100 bacteria per coverslip . H ) Confocal micrographs of HeLa cells expressing GFP - TECPR1 either alone or together with FLAG - tagged neutral sphingomyelinase 2 ( nSMase2 ; ( H ) ) Cells were stained for Galectin - 8 and DNA ( DAPI ) . Scale bar , 20 μm . ( I ) Percentage of S . Typhimurium positive for GFP - TECPR1 or endogenous Galectin - 8 at 1 h post - infection in HeLa cells expressing FLAG - nSMase2 or not . Mean + / - SD of two ( TECPR1 ) or three ( Gal8 ) independent experiments . n > 100 bacteria per coverslip .

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 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 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 1(C, D Confocal micrographs of HeLa cells expressing GFP-TECPR1 and treated with osmotic shock assay or LLOMe (C), or infected with the bacteria indicated (D) and fixed at 1 h (S. Typhimurium and L. monocytogenes) or 0.5 h (S. flexneri) post-infection. Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm.(F) Percentage of bacteria positive for mCherry-TECPR1 in ATG5 knockout (KO) or ATG5-complemented MEF cells at 1 h (S. Typhimurium) or 0.5 h (S. flexneri) post-infection. Mean +/- SD of four (S. Typhimurium) or two (S. flexneri) independent experiments. n > 100 bacteria per coverslip. (G) Percentage of S. Typhimurium positive for GFP-TECPR1 at 1 h post-infection in HeLa cells pretreated or not with 100 nM Wortmannin. Mean +/- SD of two independent experiments. n > 100 bacteria per coverslip.H) Confocal micrographs of HeLa cells expressing GFP-TECPR1 either alone or together with FLAG-tagged neutral sphingomyelinase 2 (nSMase2; (H)) Cells were stained for Galectin-8 and DNA (DAPI). Scale bar, 20 μm. (I) Percentage of S. Typhimurium positive for GFP-TECPR1 or endogenous Galectin-8 at 1 h post-infection in HeLa cells expressing FLAG-nSMase2 or not. Mean +/- SD of two (TECPR1) or three (Gal8) independent experiments. n > 100 bacteria per coverslip.


Figure 2 ( C ) Percentage of S . Typhimurium positive for the indicated constructs in HeLa cells , fixed at the indicated timepoints post - infection . Mean + / - SEM of 3 - 4 independent experiments performed in duplicate . n > 100 bacteria per coverslip . ( E ) Confocal micrographs of HeLa cells expressing TECPR1 N ' DysF - GFP with or without FLAG - nSMase2 , infected with S . Typhimurium , fixed at 30 min post - infection and stained with anti - Galectin 8 antibody . DNA stained with DAPI . Scale bar , 20 μm . ( F ) Percentage of S . Typhimurium positive for N ' DysF - GFP in Hela cells , expressing FLAG - nSMase or not , fixed at 30 min post - infection . Mean + / - SEM of three independent experiments performed in duplicate . ( G and H ) Confocal micrographs of HeLa cells expressing the indicated GFP - tagged DysF domains , infected with mCherry - S . Typhimurium and fixed at 30 min post - infection ( G ) or treated with osmotic shock assay , fixed and stained with anti - Galectin 8 antibody ( H ) . The bottom row in H depicts an enlarged version of the boxed region shown above . Scale bar , 20 μm .

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Figure 2(C) Percentage of S. Typhimurium positive for the indicated constructs in HeLa cells, fixed at the indicated timepoints post-infection. Mean +/- SEM of 3-4 independent experiments performed in duplicate. n > 100 bacteria per coverslip.(E) Confocal micrographs of HeLa cells expressing TECPR1 N' DysF-GFP with or without FLAG-nSMase2, infected with S. Typhimurium, fixed at 30 min post-infection and stained with anti-Galectin 8 antibody. DNA stained with DAPI. Scale bar, 20 μm.(F) Percentage of S. Typhimurium positive for N' DysF-GFP in Hela cells, expressing FLAG-nSMase or not, fixed at 30 min post-infection. Mean +/- SEM of three independent experiments performed in duplicate.(G and H) Confocal micrographs of HeLa cells expressing the indicated GFP-tagged DysF domains, infected with mCherry-S. Typhimurium and fixed at 30 min post-infection (G) or treated with osmotic shock assay, fixed and stained with anti-Galectin 8 antibody (H). The bottom row in H depicts an enlarged version of the boxed region shown above. Scale bar, 20 μm.


Figure 3 ( B Percentage of S . Typhimurium positive for the indicated GFP - tagged N - terminal DysF constructs at 30 min post - infection . Mean + / - SD of two ( EK134 , 135AA ) or three independent experiments . n > 100 bacteria per coverslip . p value from one - way ANOVA with Dunnett ' s multiple comparisons test . C ) Percentage of S . Typhimurium positive for the indicated GFP - tagged N - terminal DysF constructs at 30 min post - infection . Mean + / - SD of three independent experiments . n > 100 bacteria per coverslip . p value from one - way ANOVA with Dunnett ' s multiple comparisons test . ( F ) Confocal micrographs of HeLa cells expressing either GFP - TECPR1 WT or W154A and treated with LLOMe or DMSO vehicle control for 10 min and fixed . Scale bar , 20 μm . ( G ) Number of GFP - positive puncta per cell were enumerated in samples depicted in ( F ) . Data from 50 cells from a single experiment , representative of two . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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 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 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

Figure 3(B Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of two (EK134,135AA) or three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.C) Percentage of S. Typhimurium positive for the indicated GFP-tagged N-terminal DysF constructs at 30 min post-infection. Mean +/- SD of three independent experiments. n > 100 bacteria per coverslip. p value from one-way ANOVA with Dunnett's multiple comparisons test.(F) Confocal micrographs of HeLa cells expressing either GFP-TECPR1 WT or W154A and treated with LLOMe or DMSO vehicle control for 10 min and fixed. Scale bar, 20 μm. (G) Number of GFP-positive puncta per cell were enumerated in samples depicted in (F). Data from 50 cells from a single experiment, representative of two. p value from one-way ANOVA with Tukey's multiple comparisons test.


Figure 4 ( C - E ) Liposome sedimentation assay . The indicated proteins were incubated with either liposomes ( containing sphingomyelin or not ) or buffer only , pelleted by centrifugation and equal portions of supernatant and pellet fractions analysed by SDS - PAGE and Coomassie staining . Representative gels depicted ( C ) . Fraction of TECPR1 ( D ) or DysF WT or W154A ( E ) proteins present in pellet under the indicated conditions , expressed as % binding . Note pelleting in the absence of liposomes likely due to affinity of proteins for tube walls . Mean + / - SD of three independent experiments . p value from one - way ANOVA with Tukey ' s multiple comparisons test . ( F ) In vitro LC3 lipid conjugation assay . Liposomes containing sphingomyelin or not were incubated with LC3 lipid conjugation machinery : ATG7 ( E1 ) , ATG3 ( E2 ) , ATG5 - ATG12 ( E3 ) , LC3B , MgCl2 , ATP and the indicated TECPR1 proteins . Control reactions without ATG3 or ATP were included . Mixes were incubated at 37 C for 3 h , centrifuged and equal fractions of both supernatant and pellet analysed by SDS - PAGE and Coomassie staining . Lipidated LC3B - II appears in pellet fraction whereas both soluble LC3B - I and AMPylated LC3B are in the supernatant , the latter of the same apparent molecular weight as LC3B - II . ( G ) Fraction of LC3B protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1 proteins in either absence or presence of sphingomyelin . Mean + / - SD from three independent experiments . p value from one - way ANOVA with Tukey ' s multiple comparisons test .

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

Figure 4(C-E) Liposome sedimentation assay. The indicated proteins were incubated with either liposomes (containing sphingomyelin or not) or buffer only, pelleted by centrifugation and equal portions of supernatant and pellet fractions analysed by SDS-PAGE and Coomassie staining. Representative gels depicted (C). Fraction of TECPR1 (D) or DysF WT or W154A (E) proteins present in pellet under the indicated conditions, expressed as % binding. Note pelleting in the absence of liposomes likely due to affinity of proteins for tube walls. Mean +/- SD of three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.(F) In vitro LC3 lipid conjugation assay. Liposomes containing sphingomyelin or not were incubated with LC3 lipid conjugation machinery: ATG7 (E1), ATG3 (E2), ATG5-ATG12 (E3), LC3B, MgCl2, ATP and the indicated TECPR1 proteins. Control reactions without ATG3 or ATP were included. Mixes were incubated at 37 C for 3 h, centrifuged and equal fractions of both supernatant and pellet analysed by SDS-PAGE and Coomassie staining. Lipidated LC3B-II appears in pellet fraction whereas both soluble LC3B-I and AMPylated LC3B are in the supernatant, the latter of the same apparent molecular weight as LC3B-II.(G) Fraction of LC3B protein present in pellet from complete in vitro LC3 lipid conjugation assay with the indicated TECPR1 proteins in either absence or presence of sphingomyelin. Mean +/- SD from three independent experiments. p value from one-way ANOVA with Tukey's multiple comparisons test.


Figure 5 ( A Confocal micrographs of indicated MEF cell lines infected with mCherry - expressing S . Typhimurium , fixed at 30 min post - infection and stained with anti - ATG5 antibody ( A ) Scale bar , 20 μm . C ) Confocal micrographs of indicated MEF cell lines infected with mCherry - expressing S . Typhimurium , fixed at 30 min post - infection and stained with anti - LC3 antibody ( C ) . Scale bar , 20 μm . ( E ) Percentage of S . Typhimurium positive for endogenous LC3 in indicated MEF cells treated with 100 nM Wortmannin or DMSO vehicle control , fixed at 1 h post - infection . Mean + / - SEM of three independent experiments performed in duplicate . n > 100 bacteria per coverslip . p value from one - way ANOVA with Tukey ' s multiple comparisons test . ( F ) Confocal micrographs of either control of ATG16L1 - deficient MEF cells , transfected with either control or TECPR1 siRNA ( siRNA no . 66 depicted ) , infected with mCherry - expressing S . Typhimurium for 30 min , fixed and stained with anti - LC3 antibody . Scale bar , 20 μm .

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Figure 5(A Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-ATG5 antibody (A) Scale bar, 20 μm.C) Confocal micrographs of indicated MEF cell lines infected with mCherry-expressing S. Typhimurium, fixed at 30 min post-infection and stained with anti-LC3 antibody (C). Scale bar, 20 μm.(E) Percentage of S. Typhimurium positive for endogenous LC3 in indicated MEF cells treated with 100 nM Wortmannin or DMSO vehicle control, fixed at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. p value from one-way ANOVA with Tukey's multiple comparisons test.(F) Confocal micrographs of either control of ATG16L1-deficient MEF cells, transfected with either control or TECPR1 siRNA (siRNA no. 66 depicted), infected with mCherry-expressing S. Typhimurium for 30 min, fixed and stained with anti-LC3 antibody. Scale bar, 20 μm.


Figure 6 ( A ) Percentage of S . Typhimurium positive for Galectin - 8 and / or GFP - TECPR1 in either control Hela cells or those expressing GFP - TECPR1 at 1 h post - infection . Mean + / - SEM of three independent experiments performed in duplicate . n > 100 bacteria per coverslip . ( B ) Confocal micrographs of HeLa cells expressing the indicated GFP - TECPR1 constructs or not ( control ) , infected with S . Typhimurium for 30 min , fixed and stained with anti - Galectin - 8 and DAPI . Scale bar , 20 μm . Arrowheads indicate bacteria positive for Galectin 8 , of which more are present in cells expressing GFP - TECPR1 WT but not W154A . Arrow indicates bacterium to which GFP - TECPR1 but not Galectin 8 has been recruited . ( C ) Percentage of S . Typhimurium positive for Galectin - 8 in Hela cells expressing the indicated GFP - TECPR1 constructs or not ( control ) and infected with S . Typhimurium for 1 h . Mean + / - SEM of five independent experiments performed in triplicate . p value from one - way ANOVA with Tukey ' s multiple comparisons test . ( D ) Fold intracellular proliferation of S . Typhimurium in HeLa cells expressing the indicated GFP - TECPR1 constructs or not ( control ) as assessed by colony forming unit assay . Mean + / - SEM of four ( ΔPH ) or five ( control , WT , W154A and ΔAIR ) independent experiments . p value from one - way ANOVA with Dunnett ' s multiple comparisons test .

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Figure 6(A) Percentage of S. Typhimurium positive for Galectin-8 and/or GFP-TECPR1 in either control Hela cells or those expressing GFP-TECPR1 at 1 h post-infection. Mean +/- SEM of three independent experiments performed in duplicate. n > 100 bacteria per coverslip. (B) Confocal micrographs of HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control), infected with S. Typhimurium for 30 min, fixed and stained with anti-Galectin-8 and DAPI. Scale bar, 20 μm. Arrowheads indicate bacteria positive for Galectin 8, of which more are present in cells expressing GFP-TECPR1 WT but not W154A. Arrow indicates bacterium to which GFP-TECPR1 but not Galectin 8 has been recruited.(C) Percentage of S. Typhimurium positive for Galectin-8 in Hela cells expressing the indicated GFP-TECPR1 constructs or not (control) and infected with S. Typhimurium for 1 h. Mean +/- SEM of five independent experiments performed in triplicate. p value from one-way ANOVA with Tukey's multiple comparisons test.(D) Fold intracellular proliferation of S. Typhimurium in HeLa cells expressing the indicated GFP-TECPR1 constructs or not (control) as assessed by colony forming unit assay. Mean +/- SEM of four (ΔPH) or five (control, WT, W154A and ΔAIR) independent experiments. p value from one-way ANOVA with Dunnett's multiple comparisons test.


Figure 1 ( A - B ) Capsids for WT , K158A , K158A / T8I and T8I were classified into the indicated categories . Representative tomograms of K158A mutant ( A ) and the percentage ( B ) for each category are shown . The margin of error was calculated Images and quantification are from the same samples . ( C ) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour . Mean data represent n = 2 technical replicates . Scale bar 10 μm . ( F ) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP - 1 cell lines stimulated for 24 hours with LPS ( 1 ng / ml ) normalised to mock . Mean data n = 2 biological replicates . ( H ) Quantification of the indicated chemokine transcripts by qPCR from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants normalised to mock infected . Mean data represent n = 3 biological replicates . ( I - J ) Protein quantification of CXCL10 ( I ) and TNFa ( J ) by Luminex ™ from supernatant of PMA - treated THP - 1 / SAMHD1 KO cells infected with the indicated HIV - 1 mutants . Data were normalised to mock . Mean data represent n = 2 biological replicates .

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

Figure 1(A-B) Capsids for WT, K158A, K158A/T8I and T8I were classified into the indicated categories. Representative tomograms of K158A mutant (A) and the percentage (B) for each category are shown. The margin of error was calculated Images and quantification are from the same samples.(C) Virion stability assay using TIRF microscopy to quantify the number of viral particles in presence of SLO and IP6 for the indicated mutant after 1 hour. Mean data represent n=2 technical replicates. Scale bar 10 μm.(F) Quantification of CXCL10 and IFIT1 transcripts by qPCR of the indicated THP-1 cell lines stimulated for 24 hours with LPS (1 ng/ml) normalised to mock. Mean data n=2 biological replicates.(H) Quantification of the indicated chemokine transcripts by qPCR from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants normalised to mock infected. Mean data represent n=3 biological replicates.(I-J) Protein quantification of CXCL10 (I) and TNFa (J) by Luminex™ from supernatant of PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants. Data were normalised to mock. Mean data represent n=2 biological replicates.


Figure 2 ( D ) qPCR quantification of the indicated chemokine transcripts from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants . ( E ) qPCR quantification of the indicated chemokine transcripts level from PMA - treated THP - 1 / SAMHD1 KO cells transduced for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants . ( F ) Protein quantification of IL - 6 by Luminex ™ from PMA - treated THP - 1 / SAMHD1 KO cells infected with the indicated HIV - 1 mutants .

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Figure 2(D) qPCR quantification of the indicated chemokine transcripts from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with the indicated VLPs mutants. (E) qPCR quantification of the indicated chemokine transcripts level from PMA-treated THP-1/SAMHD1 KO cells transduced for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants.(F) Protein quantification of IL-6 by Luminex™ from PMA-treated THP-1/SAMHD1 KO cells infected with the indicated HIV-1 mutants.


Figure 3IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection with either poly I : C ( 0 . 5 μg / ml ) ( B ) mean data represent n = 2 biological replicatesIFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection with HT - DNA ( 0 . 1 μg / ml ) ( C ) mean data represent n = 2 biological replicatesIFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants ( D ) . IFIT1 - Luc reporter activity from PMA - treated THP - 1 / SAMHD1 KO infected with for 24 hours with 50ng / RT of the indicated HIV - 1 GFP capsids mutants in the presence of DMSO or 0 . 5 μg / ml H151 ( E )

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Figure 3IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with either poly I:C (0.5 μg/ml) (B) mean data represent n=2 biological replicatesIFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection with HT-DNA (0.1 μg/ml) (C) mean data represent n=2 biological replicatesIFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO cells stimulated by transfection infected for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants (D).IFIT1-Luc reporter activity from PMA-treated THP-1/SAMHD1 KO infected with for 24 hours with 50ng/RT of the indicated HIV-1 GFP capsids mutants in the presence of DMSO or 0.5 μg/ml H151 (E)


Figure 4 ( B - D ) Protein quantification of CXCL10 ( B ) , IL - 6 ( C ) and TNFa ( D ) by Luminex ™ from HMDMs infected with the indicated HIV - 1 mutants . ( F - H ) Protein quantification from SupT1 cells as in ( B - D ) ) . Protein quantification from infected Jurkat T cells as in ( B - D ) .

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Figure 4(B-D) Protein quantification of CXCL10 (B), IL-6 (C) and TNFa (D) by Luminex™ from HMDMs infected with the indicated HIV-1 mutants.(F-H) Protein quantification from SupT1 cells as in (B-D)).Protein quantification from infected Jurkat T cells as in (B-D).


Figure 2B ) Volcano plot for the F test on differential expression between WT and mdx mice . C ) Volcano plot for the F test on differential expression between WT and mdx + + mice . D ) Volcano plot for the F test on differential expression between WT and mdx + - mice . E , F ) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT , mdx , mdx + + and mdx + - mice groups . The central band of the boxplot denotes the median ; the box denotes the first and third quartile ; the whiskers are computed as min ( max ( x ) , Q3 + 1 . 5 IQR ) and max ( min ( x ) , Q1 - 1 . 5 * IQR ) , where min ( x ) and max ( x ) denote the minimum and maximum of the distribution , Q1 and Q3 the first and third quartile , and IQR the interquartile range . G ) Volcano plot for the F tests on differential expression between mdx + + and mdx + - mice . Scatter plots comparing the estimated logFCs across mice groups . Panel H : mdx vs mdx + + . Scatter plots comparing the estimated logFCs across mice groups . + + . Panel I : mdx vs mdx + - . Panel J : mdx + + vs mdx + - .

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 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 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 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

Figure 2B) Volcano plot for the F test on differential expression between WT and mdx mice. C) Volcano plot for the F test on differential expression between WT and mdx++ mice. D) Volcano plot for the F test on differential expression between WT and mdx+- mice.E, F) Boxplot comparing the distribution of the Dmd and Prune2 genes in the WT, mdx, mdx++ and mdx+- mice groups. The central band of the boxplot denotes the median; the box denotes the first and third quartile; the whiskers are computed as min(max(x), Q3 + 1.5 IQR) and max(min(x), Q1 - 1.5 * IQR), where min(x) and max(x) denote the minimum and maximum of the distribution, Q1 and Q3 the first and third quartile, and IQR the interquartile range.G) Volcano plot for the F tests on differential expression between mdx++ and mdx+- mice.Scatter plots comparing the estimated logFCs across mice groups. Panel H: mdx vs mdx++.Scatter plots comparing the estimated logFCs across mice groups. ++. Panel I: mdx vs mdx+-. Panel J: mdx++ vs mdx+-.


Figure 3A ) Histogram of the likelihood ratio p - values for the test on differences between WT and mdx at any time point . B ) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6 , 12 , 18 , 24 and 30 for the 1532 genes with significant differences between WT and mdx . D ) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice . E ) Trajectory plots comparing the trajectories of Cd59a , Chordc1 , Hpgd , Ldhb , Lincred1 , Myo6 , Slc39a11 and Tlr1 in WT , mdx + + and mdx + - mice . F ) Scatter plots comparing the estimated logFC of mdx + + vs WT ( x axis ) and of mdx + - vs WT ( y axis ) at weeks 6 , 12 , 18 , 24 and 30 . H ) upstream regulator analysis ( H ) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice .

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Figure 3A) Histogram of the likelihood ratio p-values for the test on differences between WT and mdx at any time point.B) Distribution of the estimated logFCs for mdx versus WT mice at weeks 6, 12, 18, 24 and 30 for the 1532 genes with significant differences between WT and mdx.D) Trajectory plots comparing the trajectories of Chordc1 and Psat1 in WT and mdx mice.E) Trajectory plots comparing the trajectories of Cd59a, Chordc1, Hpgd, Ldhb, Lincred1, Myo6, Slc39a11 and Tlr1 in WT, mdx++ and mdx+- mice.F) Scatter plots comparing the estimated logFC of mdx++ vs WT (x axis) and of mdx+- vs WT (y axis) at weeks 6, 12, 18, 24 and 30.H) upstream regulator analysis (H) performed by Ingenuity Pathway Analysis of the comparison between mdx and wt mice.


Figure 4A ) Barplot showing the percentage of significant genes in the muscle modules . Next to each module name we report the total number of genes that it comprises . B ) Barplot showing the percentage of significant genes in the blood modules . Next to each module name we report the total number of genes that it comprises . C ) Matrix with the values of the cross - correlation between blood and muscle module eigengenes . To enhance visibility , only correlations with absolute value above 0 . 5 are shown .

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Figure 4A) Barplot showing the percentage of significant genes in the muscle modules. Next to each module name we report the total number of genes that it comprises. B) Barplot showing the percentage of significant genes in the blood modules. Next to each module name we report the total number of genes that it comprises. C) Matrix with the values of the cross-correlation between blood and muscle module eigengenes. To enhance visibility, only correlations with absolute value above 0.5 are shown.


Figure 5A ) RT - PCR ( 30 cycles ) showing skipping of exon 23 for PMO and PS49 oligos . No skipping was visible in saline treated mice . B ) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration . M is molecular ladder . WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle . NT is saline treated mdx mice . C ) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset ( logFC WT vs mdx at week 12 ) . D , E ) Histograms comparing the distribution of selected genes across different mice groups ( PMO , PS49 and saline in the MoTreat dataset , WT and mdx at week 12 in the MoLong dataset ) .

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Figure 5A) RT-PCR (30 cycles) showing skipping of exon 23 for PMO and PS49 oligos. No skipping was visible in saline treated mice.B) Western blot showing the percentage of restoration of dystrophin protein in mdx mice following antisense oligonucleotides administration. M is molecular ladder. WT gastrocnemius represents the standard curve using protein extracts from gastrocnemius muscle. NT is saline treated mdx mice.C) Barplot comparing the estimated logFC for the PS49 and PMO treatments in the MoTreat dataset to the desired drug effect obtained from the MoLong dataset (logFC WT vs mdx at week 12).D, E) Histograms comparing the distribution of selected genes across different mice groups (PMO, PS49 and saline in the MoTreat dataset, WT and mdx at week 12 in the MoLong dataset).


Figure 6B ) Volcano plot for the F test on differential expression between healthy individuals and DMD patients . C ) Scatter plot showing the group effect at baseline ( x - axis ) and the effect of the interaction between age and group ( y - axis ) . Black dots represent non differentially expressed genes , while blue dots represent differentially expressed genes . E ) Volcano plot for the F test on differential expression between treated and untreated DMD patients . F ) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al . ( 2012 ) . G ) Scatter plot showing genes for which a significant effect of treatment with steroids . The group effects at baseline are plotted on the x - axis , while the effects of steroids are plotted on the y - axis .

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Figure 6B) Volcano plot for the F test on differential expression between healthy individuals and DMD patients.C) Scatter plot showing the group effect at baseline (x-axis) and the effect of the interaction between age and group (y-axis). Black dots represent non differentially expressed genes, while blue dots represent differentially expressed genes.E) Volcano plot for the F test on differential expression between treated and untreated DMD patients.F) Barplot comparing the logFC of the genes associated with steroid treatment estimated in this study to the estimates of Liu et al. (2012).G) Scatter plot showing genes for which a significant effect of treatment with steroids. The group effects at baseline are plotted on the x-axis, while the effects of steroids are plotted on the y-axis.


Figure 1 ( A - C ) Representative images of Cellular localization of TLR2 - p and scrTLR2 - p peptides in BM - derived macrophages were observed using confocal microscopy . ( A ) Cells were probed with anti TLR2 antibody following by staining with APC - labeled secondary antibody or ( B ) with TLR1 antibody following by staining with FITC - labeled secondary antibody ( left panels , red ) . For membrane staining , cells were labelled with lipid dye , DiD ( C ) . Then rhodamine - labeled fluorescent peptide was added to the cells ( middle panels , green ) . Merged images are shown in the right panel . Scale bars 10µm . The mean Pearson correlation coefficient observed of TLR2 - p or scrTLR2 - p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S . EM ( n = 19 - 61 ) .

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Figure 1(A-C) Representative images of Cellular localization of TLR2-p and scrTLR2-p peptides in BM-derived macrophages were observed using confocal microscopy. (A) Cells were probed with anti TLR2 antibody following by staining with APC-labeled secondary antibody or (B) with TLR1 antibody following by staining with FITC-labeled secondary antibody (left panels, red). For membrane staining, cells were labelled with lipid dye, DiD (C). Then rhodamine-labeled fluorescent peptide was added to the cells (middle panels, green). Merged images are shown in the right panel. Scale bars 10µm. The mean Pearson correlation coefficient observed of TLR2-p or scrTLR2-p peptides with TLR2 TLR1 and DiD presents as mean of 2 independent experiments ± S.EM (n=19-61).


Figure 2 ( A ) Biochemical analyses of peptide interaction with TLR . BM - derived macrophages were incubated with 1µM fluorescently labeled rhodamine ( Rho ) - TLR2 - p or its mutant Rho - ScrTLR2 - p peptide . After 1h of incubation at 370C the cells were lysated . Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies . Proteins samples were resolved by SDS - PAGE and the presence of the labeled peptide was detected with a fluorescent scanner . Subsequently , the gel was transferred to a membrane and subjected to Western blotting for TLR6 , 1 , 2 and 4 in the appropriate samples . Equal loading was measured by detecting of anti - tubulin in the cell lysate . The results are presented as the mean of fluorescent intensity from two independent experiments . Non - specific binding of the peptides to G protein beads was subtracted . The results are mean ± S . E . M of at least two independent experiments . ( B ) Proteins analysis of secreted pro - inflammatory cytokines , IL - 6 and TNF - α from primary CD115 + cells . Cells were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed and incubated with LTA ( 5μg / ml ) , PAM3CSK ( 0 . 5μg / ml ) or LPS ( 0 . 2μg / ml ) at 370C for 5h ( for TNF - α detection ) and 22h ( for IL - 6 detection ) . The results are mean ± S . E . M of two independent experiments ( n = 8 ) .

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Figure 2(A) Biochemical analyses of peptide interaction with TLR. BM-derived macrophages were incubated with 1µM fluorescently labeled rhodamine (Rho)-TLR2-p or its mutant Rho-ScrTLR2-p peptide. After 1h of incubation at 370C the cells were lysated. Soluble fraction was used for immunoprecipitation with the indicated TLR antibodies. Proteins samples were resolved by SDS-PAGE and the presence of the labeled peptide was detected with a fluorescent scanner. Subsequently, the gel was transferred to a membrane and subjected to Western blotting for TLR6,1,2 and 4 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. The results are presented as the mean of fluorescent intensity from two independent experiments. Non-specific binding of the peptides to G protein beads was subtracted. The results are mean± S.E.M of at least two independent experiments.(B) Proteins analysis of secreted pro-inflammatory cytokines, IL-6 and TNF-α from primary CD115+ cells. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with LTA (5μg/ml), PAM3CSK (0.5μg/ml) or LPS (0.2μg/ml) at 370C for 5h (for TNF-α detection) and 22h (for IL-6 detection). The results are mean± S.E.M of two independent experiments (n=8).


Figure 3 ( B - C ) Represented images of cellular interaction between TLR2 and TLR6 ( A ) and between F4 / 80 and TLR2 ( B ) in BM - derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer ( FRET ) using ImageStreamX . Scale bars 10μm . Cells were incubated with 20µM of TLR2 - p or scrTLR2 - p peptide for 0 . 5h , then washed and incubated with 500ng / ml LTA for another 0 . 5h at 370C . Cells were probed with anti TLR6 - PE or anti F4 / 80 - PE conjugated antibody ( donor ) and anti TLR2 antibody following by staining with APC - labeled secondary antibody ( acceptor ) . PE intensity ( middle panel ) and FRET intensity ( right panel ) were measured via ImageStreanX imaging flow cytometer . The FRET intensity shown are mean ± S . E . M of two independent experiments ( n = 2579 - 15567 ) . ( D ) Represented image of ERK1 , 2 phosphorylation levels in CD115 + BM monocytes cells upon LTA exposure . Cells were pre - treated for 0 . 5h with 20µM of TLR2 - p peptide , scrTLR2 - p peptide or untreated , then washed and incubated with 500ng / ml LTA for the indicated times . ERK1 , 2 phosphorylation levels and total ERK1 , 2 levels were detected by western blotting . Equal loading was detected by measuring anti tubulin . Results are representative data of 2 independent experiments . The bands intensity was quantified for p - ERK levels after 15min of LTA exposure . Results are normalized to tubulin levels and are the mean of 2 experiments ± S . D . M .

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 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

Figure 3(B-C) Represented images of cellular interaction between TLR2 and TLR6 (A) and between F4/80 and TLR2 (B) in BM-derived macrophages with the indicated treatments were observed by Fluorescence Resonance Energy Transfer (FRET) using ImageStreamX. Scale bars 10μm. Cells were incubated with 20µM of TLR2-p or scrTLR2-p peptide for 0.5h, then washed and incubated with 500ng/ml LTA for another 0.5h at 370C. Cells were probed with anti TLR6-PE or anti F4/80-PE conjugated antibody (donor) and anti TLR2 antibody following by staining with APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured via ImageStreanX imaging flow cytometer. The FRET intensity shown are mean ± S.E.M of two independent experiments (n=2579-15567).(D) Represented image of ERK1,2 phosphorylation levels in CD115+BM monocytes cells upon LTA exposure. Cells were pre-treated for 0.5h with 20µM of TLR2-p peptide, scrTLR2-p peptide or untreated, then washed and incubated with 500ng/ml LTA for the indicated times. ERK1,2 phosphorylation levels and total ERK1,2 levels were detected by western blotting. Equal loading was detected by measuring anti tubulin. Results are representative data of 2 independent experiments. The bands intensity was quantified for p-ERK levels after 15min of LTA exposure. Results are normalized to tubulin levels and are the mean of 2 experiments ± S.D.M.


Figure 4 ( B ) Representative colonoscopy images of the indicated treatments ( day 7 ) . C57BL mice were treated with 2 % of dextran sulfate sodium ( DSS ) in drinking water for 7 days . An amount of 5mg / Kg of TLR2 - p and scrTLR2 - p peptides were injected IP every following day from day one . ( C ) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only , DSS with TLR2 - p peptide , DSS with scrTLR2 - p peptide and untreated mice ( n = 19 , 21 , 22 and 19 respectively ) . Results represent 3 independent experiments , scale bars 100µm and 1000μm . ( D ) A graphical summary of changes in body weight ( day 6 ) of the indicated groups . ( E ) Representative H & amp ; amp ; E histological images for distal colon section ( day 7 ) of 9 - 10 sections from 2 independent experiments . Black asterisk indicates on large collection of blood in the lumen , arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa . ( F ) A graphical summary of histological severity score of the indicated treatments .

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 B-PANEL_START 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

Figure 4(B) Representative colonoscopy images of the indicated treatments (day 7). C57BL mice were treated with 2% of dextran sulfate sodium (DSS) in drinking water for 7 days. An amount of 5mg/Kg of TLR2-p and scrTLR2-p peptides were injected IP every following day from day one.(C) A graphical summary of endoscopic colitis grades assessed on day 7 after treated with DSS only, DSS with TLR2-p peptide, DSS with scrTLR2-p peptide and untreated mice (n= 19, 21, 22 and 19 respectively). Results represent 3 independent experiments, scale bars 100µm and 1000μm.(D) A graphical summary of changes in body weight (day 6) of the indicated groups.(E) Representative H&amp;E histological images for distal colon section (day 7) of 9-10 sections from 2 independent experiments. Black asterisk indicates on large collection of blood in the lumen, arrowheads indicate on widespread mucosal collapse and ulceration and arrows indicates on edema in the sub mucosa.(F) A graphical summary of histological severity score of the indicated treatments.