( Q ) Quantifications of lymph gland size upon loss of TH ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 34 ) and Ddc ( domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 56 ) when compared to stage matched control ( domeMESO - Gal4 , UAS - GFP / + , n = 90 ) . Data information : The quantifications in Q represent mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(Q) Quantifications of lymph gland size upon loss of TH (domeMESO-Gal4, UAS-GFP; THRNAi, n= 34) and Ddc (domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 56) when compared to stage matched control (domeMESO-Gal4, UAS-GFP/+, n=90). Data information: The quantifications in Q represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( R ) Quantifications of the proportions of % Dome ( green bars ) , % negative ( blue bar ) , % overlap ( Dome and P1 , yellow bars ) and % P1 ( red bars ) . For Dome positive area ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 and domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) , negative and overlap areas ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) and P1 positive area ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 15 and domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 ) . Data information : The quantifications in R represents mean with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in R is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(R) Quantifications of the proportions of %Dome (green bars), %negative (blue bar), %overlap (Dome and P1, yellow bars) and %P1 (red bars). For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18), negative and overlap areas (domeMESO-Gal4, UAS-GFP; THRNAi, n= 15, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18) and P1 positive area (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n= 15 and domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 18). Data information: The quantifications in R represents mean with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in R is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( S ) Quantification of the number of PPO positive crystal cells upon dopamine synthesis perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 23 ) . Data information : The quantifications in S represent mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in S is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(S) Quantification of the number of PPO positive crystal cells upon dopamine synthesis perturbation (domeMESO-Gal4, UAS-GFP/+, n=29, domeMESO-Gal4, UAS-GFP; THRNAi, n= 21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n= 23). Data information: The quantifications in S represent mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in S is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A - C ' ) Expression profile of dopamine activation based receptor sensor ( DA1m ) in the progenitors using domeMESO - Gal4 driver in a temporal manner - 48h , ( A - A ' ) , 72h , ( B - B ' ) and 96h AEL , ( C - C ' ) . ( D ) Quantification of the number DA1m + cells , % DA1m positive cells ( domeMESO - Gal4 ; DA1m , n = 10 at 96h , n = 16 at 72h and n = 10 at 48h AEL ) . Data information : DNA is stained with Hoechst in blue , lifeAct - RFP to mark progenitors in red , Dopamine receptor - based reporter ( DA1m ) in green , All image panels show a 20µm scale bar . AEL indicates After Egg Laying . The quantification in D represents the median with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line .

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

(A-C') Expression profile of dopamine activation based receptor sensor (DA1m) in the progenitors using domeMESO-Gal4 driver in a temporal manner- 48h, (A-A'), 72h, (B-B') and 96h AEL, (C-C'). (D) Quantification of the number DA1m+ cells, %DA1m positive cells (domeMESO-Gal4; DA1m, n=10 at 96h, n=16 at 72h and n=10 at 48h AEL). Data information: DNA is stained with Hoechst in blue, lifeAct-RFP to mark progenitors in red, Dopamine receptor-based reporter (DA1m) in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in D represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( E - G ' ) Temporal expression analysis of dopamine transporter , DAT . ( H ) A line plot representing the trend of DAT expression at 96h AEL . The distance ( x ) axis indicates 0µm as a line beginning from the core region to the periphery . Data information : DNA is stained with Hoechst in blue , Dopamine transporter ( DAT ) in red , domeMESO ( representative of progenitors ) expression in green , All image panels show a 20µm scale bar . AEL indicates After Egg Laying . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line .

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

(E-G') Temporal expression analysis of dopamine transporter, DAT. (H) A line plot representing the trend of DAT expression at 96h AEL. The distance (x) axis indicates 0µm as a line beginning from the core region to the periphery. Data information: DNA is stained with Hoechst in blue, Dopamine transporter (DAT) in red, domeMESO (representative of progenitors) expression in green, All image panels show a 20µm scale bar. AEL indicates After Egg Laying. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines while the MZ is outlined in yellow dashed line.


( I - K ) The loss of progenitor Dop2R and DAT function using domeMESO - Gal4 , UAS - GFP analysed at wandering 3rd instar , 120h AEL . Data information : DNA is stained with Hoechst in blue domeMESO ( representative of progenitors ) expression in green , P1 ( representative of mature plasmatocytes ) in red . All image panels show a 20µm scale bar . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed

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

(I-K) The loss of progenitor Dop2R and DAT function using domeMESO-Gal4, UAS-GFP analysed at wandering 3rd instar, 120h AEL. Data information: DNA is stained with Hoechst in blue domeMESO (representative of progenitors) expression in green, P1 (representative of mature plasmatocytes) in red. All image panels show a 20µm scale bar. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines


( L ) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors ( domeMESO - Gal4 , UAS - GFP / + , n = 46 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 44 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 37 ) . Data information : The quantification in L represents mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in L is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(L) Quantifications of lymph gland size upon loss of Dop2R and DAT in the progenitors (domeMESO-Gal4, UAS-GFP/+, n= 46, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 44, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 37). Data information: The quantification in L represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( M ) Quantifications of the proportions of % Dome ( green bar ) , % negative ( blue bar ) , % overlap ( Dome and P1 , yellow bar ) and % P1 ( red bars ) upon loss of Dop2R and DAT in the progenitors . For Dome positive area ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , negative area ( domeMESO - Gal4 , UAS - GFP / + , n = 28 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) , overlap area ( domeMESO - Gal4 , UAS - GFP / + , n = 26 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) and P1 area ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 23 ) . Data information : The quantification in M represents mean , with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in M is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(M) Quantifications of the proportions of %Dome (green bar), %negative (blue bar), %overlap (Dome and P1, yellow bar) and %P1 (red bars) upon loss of Dop2R and DAT in the progenitors. For Dome positive area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), negative area (domeMESO-Gal4, UAS-GFP/+, n= 28, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23), overlap area (domeMESO-Gal4, UAS-GFP/+, n= 26, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25) and P1 area (domeMESO-Gal4, UAS-GFP/+, n= 27, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 16, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 23). Data information: The quantification in M represents mean, with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in M is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( N ) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation ( domeMESO - Gal4 , UAS - GFP / + , n = 29 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 26 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 25 ) . Data information : The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in N is One - way ANOVA with Tukey ' s multiple comparisons test ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

(N) Quantification of the number of PPO positive crystal cells upon dopamine sensing perturbation (domeMESO-Gal4, UAS-GFP/+, n= 29, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n= 26, domeMESO-Gal4, UAS-GFP; DATRNAi, n= 25). Data information: The quantification in N represents mean with standard deviation with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in N is One-way ANOVA with Tukey's multiple comparisons test 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A ) A temporal analysis of total number of nuclei across development in control background ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 31 at 72h , n = 19 at 96h and n = 23 at 120h AEL ) . The loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 19 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 33 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 18 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 and domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 31 ) . Data information : The quantification represents median with whiskers representing maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis is Two - way Anova with Tukey ' s multiple comparisons test while the control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

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

(A) A temporal analysis of total number of nuclei across development in control background (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 31 at 72h, n= 19 at 96h and n= 23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=19, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=33 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=18, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=25, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25 and domeMESO-Gal4, UAS-GFP; DATRNAi, n=31). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying


( B ) Quantification of total Dome + nuclei counts of control animals ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 19 at 96h and n = 23 at 120h AEL ) . The loss of dopamine modules at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . ( C ) A temporal analysis of Dome - nuclei counts in the control animals ( domeMESO - Gal4 , UAS - GFP / + , n = 37 at 48h , n = 28 at 72h , n = 18 at 96h , n = 23 , at 120h AEL ) . The perturbation of the dopamine modules in the progenitors at 48h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 21 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 22 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 32 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) , at 96h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) and at 120h AEL ( domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 24 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 25 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 30 ) . Data information : The quantification represents median with whiskers representing maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test while the control comparison , domeMESO - Gal4 , UAS - GFP / + , for different time points was done by One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . AEL indicates After Egg

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total Dome+ nuclei counts of control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 19 at 96h and n=23 at 120h AEL). The loss of dopamine modules at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). (C) A temporal analysis of Dome- nuclei counts in the control animals (domeMESO-Gal4, UAS-GFP/+, n= 37 at 48h, n= 28 at 72h, n= 18 at 96h, n= 23, at 120h AEL). The perturbation of the dopamine modules in the progenitors at 48h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=21), at 72h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=22, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=24, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=32, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27), at 96h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22) and at 120h AEL (domeMESO-Gal4, UAS-GFP; THRNAi, n=24, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=30, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=25, domeMESO-Gal4, UAS-GFP; DATRNAi, n=30). Data information: The quantification represents median with whiskers representing maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test while the control comparison, domeMESO-Gal4, UAS-GFP/+, for different time points was done by One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. AEL indicates After Egg Laying


( A ) Quantification of the Dome + BrdU index normalised to Dome + nuclei in a time course manner with dopamine modules perturbed in the progenitors using domeMESO - Gal4 , UAS - GFP driver . At 48h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 11 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 13 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 11 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 5 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 34 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 31 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 15 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) and at 96h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 17 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 28 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) . ( B ) Quantification of the Dome + mitotic index normalised to Dome + nuclei in a time course manner with loss of dopamine modules in the progenitors using domeMESO - Gal4 , UAS - GFP driver . At 48h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 35 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 16 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 30 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 20 ) , at 72h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 27 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 23 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 31 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 27 ) and at 96h AEL ( domeMESO - Gal4 , UAS - GFP / + , n = 19 , domeMESO - Gal4 , UAS - GFP ; THRNAi , n = 21 , domeMESO - Gal4 , UAS - GFP ; DdcRNAi , n = 17 , domeMESO - Gal4 , UAS - GFP ; Dop2RRNAi , n = 20 , domeMESO - Gal4 , UAS - GFP ; DATRNAi , n = 22 ) . Data information : AEL indicates After Egg Laying . The quantifications in A and B represent median with whiskers indicating maximum and minimum value , box indicates the lower and upper quartiles The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(A) Quantification of the Dome+ BrdU index normalised to Dome+ nuclei in a time course manner with dopamine modules perturbed in the progenitors using domeMESO-Gal4, UAS-GFP driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=11, domeMESO-Gal4, UAS-GFP; THRNAi, n=13, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=11, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP; DATRNAi, n=5), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=34, domeMESO-Gal4, UAS-GFP; THRNAi, n=31, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=15, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=17, domeMESO-Gal4, UAS-GFP; THRNAi, n=28, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=20, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=17, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). (B) Quantification of the Dome+ mitotic index normalised to Dome+ nuclei in a time course manner with loss of dopamine modules in the progenitors using domeMESO-Gal4, UAS-GFP driver. At 48h AEL (domeMESO-Gal4, UAS-GFP/+, n=35, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=16, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=30, domeMESO-Gal4, UAS-GFP; DATRNAi, n=20), at 72h AEL (domeMESO-Gal4, UAS-GFP/+, n=27, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=23, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=31, domeMESO-Gal4, UAS-GFP; DATRNAi, n=27) and at 96h AEL (domeMESO-Gal4, UAS-GFP/+, n=19, domeMESO-Gal4, UAS-GFP; THRNAi, n=21, domeMESO-Gal4, UAS-GFP; DdcRNAi, n=17, domeMESO-Gal4, UAS-GFP; Dop2RRNAi, n=20, domeMESO-Gal4, UAS-GFP; DATRNAi, n=22). Data information: AEL indicates After Egg Laying. The quantifications in A and B represent median with whiskers indicating maximum and minimum value, box indicates the lower and upper quartiles The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( C ) Quantifications of % G1 , % S and % G2 / M at different time points , in control and dopamine perturbed backgrounds , color coding scheme to represent the cell cycle phase as shown through FUCCI reporter is below the graph . Across development , at 48h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 34 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 13 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 15 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , n = 19 , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 17 ) , at 72h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 38 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 22 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 29 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , n = 47 , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 29 ) and at 96h AEL ( domeMESO - Gal4 ; UAS - FUCCI / + , n = 55 , domeMESO - Gal4 ; UAS - FUCCI ; THRNAi , n = 14 , domeMESO - Gal4 ; UAS - FUCCI ; DdcRNAi , n = 19 , domeMESO - Gal4 ; UAS - FUCCI ; Dop2RRNAi , domeMESO - Gal4 ; UAS - FUCCI ; DATRNAi , n = 19 ) Data information : FUCCI ( fluorescent ubiquitination - based cell cycle indicator ) reporter shows G1 phase cells in green , S phase cells in red and G2 / M phase cells in yellow . AEL indicates After Egg Laying . The quantifications in in C represents mean . The statistical analysis applied is Two - way Anova with Tukey ' s multiple comparisons test . The domeMESO - Gal4 ; UAS - FUCCI control comparison temporally in C is analysed using One - way ANOVA with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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

(C) Quantifications of %G1, %S and %G2/M at different time points, in control and dopamine perturbed backgrounds, color coding scheme to represent the cell cycle phase as shown through FUCCI reporter is below the graph. Across development, at 48h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=34, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 13, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 15, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 17), at 72h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=38, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 22, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 29, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, n= 47, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 29) and at 96h AEL (domeMESO-Gal4; UAS-FUCCI/+, n=55, domeMESO-Gal4; UAS-FUCCI; THRNAi, n= 14, domeMESO-Gal4; UAS-FUCCI; DdcRNAi, n= 19, domeMESO-Gal4; UAS-FUCCI; Dop2RRNAi, domeMESO-Gal4; UAS-FUCCI; DATRNAi, n= 19) Data information: FUCCI (fluorescent ubiquitination-based cell cycle indicator) reporter shows G1 phase cells in green, S phase cells in red and G2/M phase cells in yellow. AEL indicates After Egg Laying. The quantifications in in C represents mean. The statistical analysis applied is Two-way Anova with Tukey's multiple comparisons test. The domeMESO-Gal4; UAS-FUCCI control comparison temporally in C is analysed using One-way ANOVA with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


( A - I ' ' ) Representative lymph gland images at 48 , 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland . ( A - C ' ' ) The control profile of lymph gland and PSC iDopamine levels at 48h ( A - A ' ' ) , 72h ( B - B ' ' ) and 96h ( C - C ' ' ) . ( D - I ' ' ) Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h ( D - D ' ' and G - G ' ' ) , 72h AEL ( E - E ' ' and H - H ' ' ) and at 96h AEL ( F - F ' ' and I - I ' ' ) . ( J ) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 33 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 8 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 14 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 26 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 25 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 20 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 17 ) . ( K ) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 28 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 18 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 25 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 24 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 22 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 16 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 18 ) . Data information : DNA is stained with Hoechst in blue , Antp - Gal4 ; UAS - GFP ( representative of Posterior Signaling Center , PSC ) in green , iDopamine in red . All image panels show a 20µm scale bar . AEL indicates After Egg Laying . The quantification in J , K represents the median with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in J , K is Two - way Anova with Tukey ' s multiple comparisons test . The control animals Antp - Gal4 ; UAS - GFP / + were analysed by One - way Anova with Tukey ' s multiple comparisons test across time points . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 . For better representation , the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line .

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(A-I'') Representative lymph gland images at 48, 72 and 96h AEL depicting the lymph gland growth trajectory along with dopamine level status in the PSC and remaining lymph gland. (A-C'') The control profile of lymph gland and PSC iDopamine levels at 48h (A-A''), 72h (B-B'') and 96h (C-C''). (D-I'') Downregulation of the dopamine synthesising enzymes TH and Ddc in the PSC at 48h (D-D'' and G-G''), 72h AEL (E-E'' and H-H'') and at 96h AEL (F-F'' and I-I''). (J) Quantification of mean dopamine intensity in the PSC at upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=33, Antp-Gal4; UAS-GFP; THRNAi, n=8, Antp-Gal4; UAS-GFP; DdcRNAi, n=14), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=26, Antp-Gal4; UAS-GFP; THRNAi, n=25, Antp-Gal4; UAS-GFP; DdcRNAi, n=20) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17). (K) Quantification of mean dopamine levels in the lymph gland upon PSC perturbation of dopamine synthesis at 48h AEL (Antp-Gal4; UAS-GFP/+, n=28, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=18), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=25, Antp-Gal4; UAS-GFP; THRNAi, n=24, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=16, Antp-Gal4; UAS-GFP; DdcRNAi, n=18). Data information: DNA is stained with Hoechst in blue, Antp-Gal4; UAS-GFP (representative of Posterior Signaling Center, PSC) in green, iDopamine in red. All image panels show a 20µm scale bar. AEL indicates After Egg Laying. The quantification in J, K represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in J, K is Two-way Anova with Tukey's multiple comparisons test. The control animals Antp-Gal4; UAS-GFP/+ were analysed by One-way Anova with Tukey's multiple comparisons test across time points. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. For better representation, the primary lobe of the lymph gland has been represented and outlined in white dashed lines and the PSC in yellow dashed line.


( L ) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 19 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 13 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 17 ) , at 72h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 17 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 18 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 22 ) and 96h AEL ( Antp - Gal4 ; UAS - GFP / + , n = 15 , Antp - Gal4 ; UAS - GFP ; THRNAi , n = 14 , Antp - Gal4 ; UAS - GFP ; DdcRNAi , n = 15 ) Data information : The quantification in L represents the median with whiskers indicating maximum and minimum values , box indicates the lower and upper quartiles . The statistical analysis applied in L is Two - way Anova with Tukey ' s multiple comparisons test . ' n ' represents the number of lymph gland lobes analyzed , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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(L) Analysis of the total cell counts upon loss of TH and Ddc in the PSC at 48h AEL (Antp-Gal4; UAS-GFP/+, n=19, Antp-Gal4; UAS-GFP; THRNAi, n=13, Antp-Gal4; UAS-GFP; DdcRNAi, n=17), at 72h AEL (Antp-Gal4; UAS-GFP/+, n=17, Antp-Gal4; UAS-GFP; THRNAi, n=18, Antp-Gal4; UAS-GFP; DdcRNAi, n=22) and 96h AEL (Antp-Gal4; UAS-GFP/+, n=15, Antp-Gal4; UAS-GFP; THRNAi, n=14, Antp-Gal4; UAS-GFP; DdcRNAi, n=15) Data information: The quantification in L represents the median with whiskers indicating maximum and minimum values, box indicates the lower and upper quartiles. The statistical analysis applied in L is Two-way Anova with Tukey's multiple comparisons test. 'n' represents the number of lymph gland lobes analyzed, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


A Immunofluorescence staining of HK2 with an AlexaFluor488 - conjugated antibody in HeLa cells expressing mitochondria - targeted RFP . Yellow signals in the merge analysis indicate mitochondrial localization of HK2 . Scale bar : 15 µm .

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A Immunofluorescence staining of HK2 with an AlexaFluor488-conjugated antibody in HeLa cells expressing mitochondria-targeted RFP. Yellow signals in the merge analysis indicate mitochondrial localization of HK2. Scale bar: 15 µm.


B Immunofluorescence staining of HK2 with a secondary AlexaFluor555 - conjugated antibody in HeLa cells expressing both mitochondria - targeted YFP and ER - targeted CFP . The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right ( n = 24 ) . Image magnifications are shown in the lower part of the panel ; arrows indicate HK2 dots in mito - ER contact sites . Scale bar : 15 µm .

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B Immunofluorescence staining of HK2 with a secondary AlexaFluor555-conjugated antibody in HeLa cells expressing both mitochondria-targeted YFP and ER-targeted CFP. The merged white signal indicates MAM localization of HK2 and is quantified in the bar graph on the right (n=24). Image magnifications are shown in the lower part of the panel; arrows indicate HK2 dots in mito-ER contact sites. Scale bar: 15 µm.


C Fluorescence co - staining of HK2 and split - GFP - based probe for ER - mitochondria contacts ( SPLICSL ) on HeLa cells ; HK2 is revealed with a secondary AlexaFluor555 - conjugated antibody , the merged signal is white . Scale bar : 15 µm .

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C Fluorescence co-staining of HK2 and split-GFP-based probe for ER-mitochondria contacts (SPLICSL) on HeLa cells; HK2 is revealed with a secondary AlexaFluor555-conjugated antibody, the merged signal is white. Scale bar: 15 µm.


D Quantification experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells ( n = 24 cells analyzed from three independent experiments ; mean ± SD ) .

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D Quantification experiment showing the percentage of HK2 dots that merge with MAMs in HeLa cells (n=24 cells analyzed from three independent experiments; mean±SD).


E Quantification experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells ( n = 10 cells analyzed from three independent experiments ; mean ± SD ) .

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E Quantification experiment showing the percentage of SPLICSL dots positive for HK2 in HeLa cells (n=10 cells analyzed from three independent experiments; mean±SD).


F Percentage of HK2 dots positive for SPLICSL in HeLa , COLO 741 , MDA - MB - 231 , PN 04 . 4 and S462 cells ( n ≥ 6 cells for each cell line ; mean ± SD ) .

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F Percentage of HK2 dots positive for SPLICSL in HeLa, COLO 741, MDA-MB-231, PN 04.4 and S462 cells (n≥6 cells for each cell line; mean±SD).


H HK2 displacement from HeLa MAMs after a 2 min treatment with cl - HK2pep is shown by loss of merging signal analyzed as in ( C ) . cl - SCRpep is used as a negative control and Pearson ' s co - localization coefficient is indicated in figure ( cl - SCRpep n = 26 cells ; cl - HK2pep n = 24 cells ; p < 0 . 001 with a Student ' s t test ) . Scale bar : 10 µm .

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H HK2 displacement from HeLa MAMs after a 2 min treatment with cl-HK2pep is shown by loss of merging signal analyzed as in (C). cl-SCRpep is used as a negative control and Pearson's co-localization coefficient is indicated in figure (cl-SCRpep n=26 cells; cl-HK2pep n=24 cells; p<0.001 with a Student's t test). Scale bar: 10 µm.


Effect of cl - HK2pep on glucose phosphorylation by human recombinant HK2 ( I ) where both total hexokinase activity and HK1 / HK2 specific activities are measured .

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Effect of cl-HK2pep on glucose phosphorylation by human recombinant HK2 (I) where both total hexokinase activity and HK1/HK2 specific activities are measured.


Effect of cl - HK2pep on glucose phosphorylation in 4T1 cell extracts ( J ) , where both total hexokinase activity and HK1 / HK2 specific activities are measured .

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Effect of cl-HK2pep on glucose phosphorylation in 4T1 cell extracts (J), where both total hexokinase activity and HK1/HK2 specific activities are measured.


Effect of cl - HK2pep on cellular Ca2 + dynamics ER Ca2 + levels are measured by the FRET - based , D4ER fluorescent probe expressed in the lumen of ER ( A ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

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Effect of cl-HK2pep on cellular Ca2+ dynamics ER Ca2+ levels are measured by the FRET-based, D4ER fluorescent probe expressed in the lumen of ER (A) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep on IP3 levels . IP3 levels are assessed with the GFP - PHD probe ; histamine ( 100μM ) is used as a positive control for IP3 generation ; data are reported as mean of fluorescent signals ± SEM ( n = 3 independent experiments ; B ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at the 5 μM concentration used here [

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

Effect of cl-HK2pep on IP3 levels. IP3 levels are assessed with the GFP-PHD probe; histamine (100μM) is used as a positive control for IP3 generation; data are reported as mean of fluorescent signals±SEM (n=3 independent experiments; B) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [


Effect of cl - HK2pep on cellular Ca2 + dynamics changes in mitochondrial Ca2 + levels are recorded ( C ; scale bar : 20µm ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

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Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded (C; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep on cellular Ca2 + dynamics changes in mitochondrial Ca2 + levels are recorded and quantified using the GCAMP6f sensor ( in D , as mean of 475 / 410 nm ratio ; signal ± SEM of at least 5 independent experiments and more than 20 cells analized ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at the 5 μM concentration used

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

Effect of cl-HK2pep on cellular Ca2+ dynamics changes in mitochondrial Ca2+ levels are recorded and quantified using the GCAMP6f sensor (in D, as mean of 475/410 nm ratio; signal±SEM of at least 5 independent experiments and more than 20 cells analized) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here


Effect of cl - HK2pep on cellular Ca2 + dynamics ( E ) as percentage of cells with increased Ca2 + in mitochondria , with a threshold 475 / 410 ratio for positivity > 3 ; baseline mean ratio = 1 . 84 ± 0 . 54 ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Xe - C is Xestospongin C , which selectively inhibits IP3R at the 5 μM concentration used here [

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

Effect of cl-HK2pep on cellular Ca2+ dynamics (E) as percentage of cells with increased Ca2+ in mitochondria, with a threshold 475/410 ratio for positivity >3; baseline mean ratio=1.84±0.54) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here [


Effect of cl - HK2pep on cellular Ca2 + dynamics with mitochondria - targeted aequorin ( F , where data are reported as mean of [ Ca2 + ] ± SEM of 3 independent experimental days ) . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) .

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Effect of cl-HK2pep on cellular Ca2+ dynamics with mitochondria-targeted aequorin (F, where data are reported as mean of [Ca2+]±SEM of 3 independent experimental days). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each).


Effect of cl - HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe . Kinetic experiments ( G , single cell analysis , n = 216 cells analyzed in at least 10 independent

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Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments (G, single cell analysis, n=216 cells analyzed in at least 10 independent experiments


Effect of cl - HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe . Kinetic experiments H , representative field ; scale bar : 20µm ) Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Where indicated , cells are kept in Ca2 + free medium plus 500 µM EGTA with or without 10 µM BAPTA -

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

Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. Kinetic experiments H, representative field; scale bar: 20µm) Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM


Effect of cl - HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe . quantified ( I and J ; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point , with a depolarization threshold placed at 40 % of initial value ) . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) . Where indicated , cells are kept in Ca2 + free medium plus 500 µM EGTA with or without 10 µM BAPTA - AM ; Xe - C is Xestospongin C , which selectively inhibits IP3R at the 5 μM concentration used

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

Effect of cl-HK2pep treatment on mitochondrial membrane potential assessed with the TMRM probe. quantified (I and J; TMRM fluorescence is normalized to initial value and expressed in percentage for each time point, with a depolarization threshold placed at 40% of initial value). Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each). Where indicated, cells are kept in Ca2+ free medium plus 500 µM EGTA with or without 10 µM BAPTA-AM; Xe-C is Xestospongin C, which selectively inhibits IP3R at the 5 μM concentration used here


Effects of the PTP desensitizers CsA or C63 ( 5 µM each , 1 h preincubation ) on mitochondrial Ca2 + levels recorded with GCAMP6f ( A ) in cells treated with cl - HK2pep . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

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

Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial Ca2+ levels recorded with GCAMP6f (A) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


Effects of the PTP desensitizers CsA or C63 ( 5 µM each , 1 h preincubation ) on mitochondrial membrane potential assessed with TMRM ( B ) in cells treated with cl - HK2pep . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

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

Effects of the PTP desensitizers CsA or C63 (5 µM each, 1 h preincubation) on mitochondrial membrane potential assessed with TMRM (B) in cells treated with cl-HK2pep. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


cell death induction by cl - HK2pep ; in the cytofluorimetric analyses viable cells are double negative for Annexin V - FITC and 7 - AAD staining and measured 15 min after peptide treatment data are presented as mean ± SD and they were obtained from 3 different experiments or more ; 3 technical replicates in all analyzed experiment . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) data are presented as mean ± SD of at least 3 independent experiments . * * * p < 0 . 001 with a Student ' s t

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) data are presented as mean±SD of at least 3 independent experiments. *** p<0.001 with a Student's t test


cell death induction by cl - HK2pep ; in the cytofluorimetric analyses in the kinetic experiment shown in ( D ; scale bar : 50µm ) viable cells are double negative for Annexin V - FITC and 7 - AAD and TMRM positive . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each )

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses in the kinetic experiment shown in (D; scale bar: 50µm) viable cells are double negative for Annexin V-FITC and 7-AAD and TMRM positive. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each)


cell death induction by cl - HK2pep ; in the cytofluorimetric analyses viable cells are double negative for Annexin V - FITC and 7 - AAD staining and measured 15 min after peptide treatment Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated , the caspase inhibitor Z - VAD - fmk or the calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment .

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated, the caspase inhibitor Z-VAD-fmk or the calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.


cell death induction by cl - HK2pep ; in the cytofluorimetric analyses viable cells are double negative for Annexin V - FITC and 7 - AAD staining and measured 15 min after peptide treatment data are presented as mean ± SD and they were obtained from 3 different experiments or more ; 3 technical replicates in all analyzed experiment . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment . data are presented as mean ± SD of at least 3 independent experiments . p < 0 . 0001 with a two - way ANOVA ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; Bonferroni post - test in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep and cl - HK2pep vs PD150606 + cl - HK2pep ) .

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

cell death induction by cl-HK2pep; in the cytofluorimetric analyses viable cells are double negative for Annexin V-FITC and 7-AAD staining and measured 15 min after peptide treatment data are presented as mean±SD and they were obtained from 3 different experiments or more; 3 technical replicates in all analyzed experiment. Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each) where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).


Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM ( G , normalized fluorescence signal mean ± SD ; 3 different experiments including 3 technical replicates each ) in cells treated with cl - HK2pep Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment . data are presented as mean ± SD of at least 3 independent experiments . p < 0 . 0001 with a two - way ANOVA ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; Bonferroni post - test in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep and cl - HK2pep vs PD150606 + cl - HK2pep ) .

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

Effect of calpain inhibition on mitochondrial membrane potential assessed with TMRM (G, normalized fluorescence signal mean±SD; 3 different experiments including 3 technical replicates each) in cells treated with cl-HK2pep Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment. data are presented as mean±SD of at least 3 independent experiments. p<0.0001 with a two-way ANOVA (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); Bonferroni post-test in graph ***p<0.001 (cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep).


Effect of calpain inhibition on mitochondrial Ca2 + levels recorded with GCAMP6f ( H ) in cells treated with cl - HK2pep . Data information : Experiments throughout the Figure are carried out on HeLa cells ; cl - SCRpep , negative control of cl - HK2pep ( 2 μM each ) ; where indicated calpain inhibitor PD150606 ( 50 µM each ) are pre - incubated 1 h before peptide treatment .

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

Effect of calpain inhibition on mitochondrial Ca2+ levels recorded with GCAMP6f (H) in cells treated with cl-HK2pep . Data information: Experiments throughout the Figure are carried out on HeLa cells; cl-SCRpep, negative control of cl-HK2pep (2 μM each); where indicated calpain inhibitor PD150606 (50 µM each) are pre-incubated 1 h before peptide treatment.


A Representative IHCs in human reactive lymphoid tissue , where HK2 expression is mainly restricted to proliferating blasts within germinal centers ( center ) , and in patient B - CLL cells . Scale bar 100 µm .

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

A Representative IHCs in human reactive lymphoid tissue, where HK2 expression is mainly restricted to proliferating blasts within germinal centers (center), and in patient B-CLL cells. Scale bar 100 µm.


Effect of cl - HK2pep on human B - CLL cells freshly isolated from patients . HK2 - expressing B - CLL cells ( B ; GAPDH and respiratory complex III subunit UQCRC2 are cytosol and mitochondrial loading controls , respectively ) Cells are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 patients and on CD19 + , primary B lymphocytes from 5 healthy controls .

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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells (B; GAPDH and respiratory complex III subunit UQCRC2 are cytosol and mitochondrial loading controls, respectively) Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.


Effect of cl - HK2pep on human B - CLL cells freshly isolated from patients . HK2 - expressing B - CLL cells undergo mitochondrial depolarization assessed by TMRM staining ( C ) Experiments are carried out on samples from at least 10 patients and on CD19 + , primary B lymphocytes from 5 healthy controls . data are presented as mean ± SEM ; all data were obtained from 3 technical replicates in all analyzed patient samples . two - way ANOVA : p < 0 . 0001 ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) Student ' s t test ; * * * p < 0 . 01 , * p < 0 . 05 .

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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. HK2-expressing B-CLL cells undergo mitochondrial depolarization assessed by TMRM staining (C) Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. two-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments) Student's t test; ***p<0.01, *p<0.05.


ffect of cl - HK2pep on human B - CLL cells freshly isolated from patients . cell death , measured by cytofluorimetric analysis of Annexin V - FITC and 7 - AAD staining Cells are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 patients and on CD19 + , primary B lymphocytes from 5 healthy controls . In C , D , F and G data are presented as mean ± SEM ; all data were obtained from 3 technical replicates in all analyzed patient samples . wo - way ANOVA : p < 0 . 0001 ( cl - HK2pep treatment vs cl - HK2pep + PD150606 or cl - SCRpep treatments ) ; tudent ' s t test ; * * * p < 0 . 01 , * p < 0 . 05 .

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

ffect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. In C, D, F and G data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. wo-way ANOVA: p<0.0001 (cl-HK2pep treatment vs cl-HK2pep+PD150606 or cl-SCRpep treatments); tudent's t test; ***p<0.01, *p<0.05.


Effect of cl - HK2pep on human B - CLL cells freshly isolated from patients . cell death , measured by cytofluorimetric analysis of Annexin V - FITC and 7 - AAD staining Cells are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 patients and on CD19 + , primary B lymphocytes from 5 healthy controls .

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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls.


Effect of cl - HK2pep on human B - CLL cells freshly isolated from patients . cell death , measured by cytofluorimetric analysis of Annexin V - FITC and 7 - AAD staining Cells are treated with 5 µM cl - HK2pep ; PD150606 ( 50 μM ) is pre - incubated for 1 h . Experiments are carried out on samples from at least 10 patients and on CD19 + , primary B lymphocytes from 5 healthy controls . data are presented as mean ± SEM ; all data were obtained from 3 technical replicates in all analyzed patient samples . Bonferroni post - test in graph * * * p < 0 . 001 ( cl - HK2pep vs cl - SCRpep and cl - HK2pep vs PD150606 + cl - HK2pep ) Student ' s t test ; * * * p < 0 . 01 , * p < 0 . 05 .

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

Effect of cl-HK2pep on human B-CLL cells freshly isolated from patients. cell death, measured by cytofluorimetric analysis of Annexin V-FITC and 7-AAD staining Cells are treated with 5 µM cl-HK2pep; PD150606 (50 μM) is pre-incubated for 1 h. Experiments are carried out on samples from at least 10 patients and on CD19+, primary B lymphocytes from 5 healthy controls. data are presented as mean±SEM; all data were obtained from 3 technical replicates in all analyzed patient samples. Bonferroni post-test in graph ***p<0.001(cl-HK2pep vs cl-SCRpep and cl-HK2pep vs PD150606+cl-HK2pep) Student's t test; ***p<0.01, *p<0.05.


Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 and shHK2 - 2 ) in CT26 colon cancer cells ( A ) Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells (A) Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . HK2 - silencing with two different shRNAs ( shHK2 - 1 and shHK2 - 2 ) in CT26 colon cancer cells inhibits growth in soft agar ( B ; colony area ± SEM ; n = 6 replicates obtained from 3 independent experiments ; Student ' s t test * * * p < 0 . 001 ; A . U . , arbitrary units ) . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. HK2-silencing with two different shRNAs (shHK2-1 and shHK2-2) in CT26 colon cancer cells inhibits growth in soft agar (B; colony area±SEM; n=6 replicates obtained from 3 independent experiments; Student's t test ***p<0.001; A.U., arbitrary units). Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with cl - HK2pep ( 50 µM , 6 h ; C , scale bar : 100 µm ) reverts formation of foci ; dead cells are highlighted by Trypan Blue staining . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with cl-HK2pep (50 µM, 6 h; C, scale bar: 100 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 targeting on in vitro tumorigenicity . Treatment with uncleaved HK2pep ( 2 µM , 48 h ; D , scale bar : 200 µm ) reverts formation of foci ; dead cells are highlighted by Trypan Blue staining . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2 targeting on in vitro tumorigenicity. Treatment with uncleaved HK2pep (2 µM, 48 h; D, scale bar: 200 µm) reverts formation of foci; dead cells are highlighted by Trypan Blue staining. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . HK2 - and MMP - 2 - expressing CT26 cells ( E , scale bar : 100 µm ) are allografted in Balb / c male mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control .

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

Effect of HK2-targeting on in vivo neoplastic growth. HK2- and MMP-2-expressing CT26 cells (E, scale bar: 100 µm) are allografted in Balb/c male mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control.


Effect of HK2 - targeting on in vivo neoplastic growth . Intratumor administration ( 5 injections of 2 nmol peptide every 12 h ) of either cl - HK2pep or HK2pep ( F ; CNT n = 4 ; cl - HK2pep n = 5 ; HK2pep n = 5 mice ) in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . In F , mean tumor volume ± SD is reported ; Student ' s t test * * * p < 0 .

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

Effect of HK2-targeting on in vivo neoplastic growth. Intratumor administration (5 injections of 2 nmol peptide every 12 h) of either cl-HK2pep or HK2pep (F; CNT n=4; cl-HK2pep n=5; HK2pep n=5 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In F, mean tumor volume±SD is reported; Student's t test ***p<0.001


Effect of HK2 - targeting on in vivo neoplastic growth . intraperitoneal administration of HK2pep ( G ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 6 ; HK2pep n = 7 mice ) reduce neoplastic growth in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . In G , representative ultrasound inspections and 3D reconstructions of tumor masses are shown ( light grey : SCRpep treated animal ; light blue : HK2pep treated animal ) . normalized volume ± SEM ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - test in the graphs * p < 0 . 05 ; * * * p < 0 . 001 .

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

Effect of HK2-targeting on in vivo neoplastic growth. intraperitoneal administration of HK2pep (G; 5 injections of 60 nmol peptide every 12 h; SCRpep n=6; HK2pep n=7 mice) reduce neoplastic growth in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. In G, representative ultrasound inspections and 3D reconstructions of tumor masses are shown (light grey: SCRpep treated animal; light blue: HK2pep treated animal). normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


Effect of HK2 - targeting on in vivo neoplastic growth . the same effect is observed on allografts of breast carcinoma 4T1 cells ( H ; 5 injections of 60 nmol peptide every 12 h ; SCRpep n = 5 ; HK2pep n = 7 mice ) in Balb / c female mice . Data information : Throughout the Figure , cl - SCRpep or SCRpep are used as peptide negative control . normalized volume ± SEM ; a two - way ANOVA is performed ( p < 0 . 001 - HK2pep vs SCRpep ) ; Bonferroni post - test in the graphs * p < 0 . 05 ; * * * p < 0 . 001 .

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

Effect of HK2-targeting on in vivo neoplastic growth. the same effect is observed on allografts of breast carcinoma 4T1 cells (H; 5 injections of 60 nmol peptide every 12 h; SCRpep n=5; HK2pep n=7 mice) in Balb/c female mice. Data information: Throughout the Figure, cl-SCRpep or SCRpep are used as peptide negative control. normalized volume±SEM; a two-way ANOVA is performed (p<0.001 - HK2pep vs SCRpep); Bonferroni post-test in the graphs *p<0.05; ***p<0.001.


( B - C ) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote the mean , and error bars denote s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) The phospholipases pla2g4aa and pla2g4ab are highly expressed in neutrophils. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( D - E - F ) The expression of cyclooxygenases cox1 , cox2a and cox2b is enriched in macrophages . ( G - H ) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs . ( I - O ) The prostaglandin receptors ptger1a , ptger1b , ptger2a , ptger3 , ptger4a are mostly expressed in HSPCs . ( P - Q ) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs . Data information : For each panel , data represents biological triplicates plated in technical duplicates . Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 ; * * * * P < . 0001 . Center values denote the mean , and error bars denote s . e . m .

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

(D-E-F) The expression of cyclooxygenases cox1, cox2a and cox2b is enriched in macrophages. (G-H) The prostaglandin synthases ptges3a and ptges3b are only expressed by ECs. (I-O) The prostaglandin receptors ptger1a, ptger1b, ptger2a, ptger3, ptger4a are mostly expressed in HSPCs. (P-Q) The prostaglandin transporters slco2b1 and abcc4 are specifically expressed in ECs. Data information: For each panel, data represents biological triplicates plated in technical duplicates. Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001; ****P<.0001. Center values denote the mean, and error bars denote s.e.m.


( A ) Schematic indicating the imaging area in the tail at 72hpf , as indicated by the black box ; fluorescence imaging of the CHT in double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2 . ( B ) Quantification of runx1 : mcherry positive - cells ( C ) Quantification of cd45 : CFP positive - cells in double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2 . Data information : Statistical analysis was completed using one - way ANOVA , multiple comparison test . * P < . 05 ; * * P < . 01 ; * * * P < . 001 * * * * P < . 0001 . Scale bar 200μm ( A ) .

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

(A) Schematic indicating the imaging area in the tail at 72hpf, as indicated by the black box; fluorescence imaging of the CHT in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. (B) Quantification of runx1:mcherry positive-cells (C) Quantification of cd45:CFP positive-cells in double transgenic cd45:CFP-NTR/runx1:mcherry embryos in DMSO and after treatment with MTZ and PGH2 or PGE2. Data information: Statistical analysis was completed using one-way ANOVA, multiple comparison test. * P<.05; **P<.01; ***P<.001 ****P<.0001. Scale bar 200μm (A).


( A ) WISH for cmyb expression at 60hpf in wild type and slco2b1 - / - embryos . ( B ) Quantification of cmyb - expressing cells . Each n represents the number of cmyb - expressing cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote the mean , and error values denote s . e . m . The statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 Scale bar 500μm ( A )

(A) WISH for cmyb expression at 60hpf in wild type and slco2b1-/- embryos. (B) Quantification of cmyb-expressing cells. Each n represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 500μm (A)


( C ) Schematic indicating the imaging area in the tail at 60hpf , as indicated by the black box ; fluorescence imaging in the CHT of kdrl : mCherry ; cmyb : GFP embryos injected with control - and slco2b1 - Mos . ( D ) Quantification of HSPCs associated to ECs . Each n represents the number of yellow spots for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information : Centre values denote the mean , and error values denote s . e . m . The statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 Scale bar 200μm ( C )

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

(C) Schematic indicating the imaging area in the tail at 60hpf, as indicated by the black box; fluorescence imaging in the CHT of kdrl:mCherry;cmyb:GFP embryos injected with control- and slco2b1-Mos. (D) Quantification of HSPCs associated to ECs. Each n represents the number of yellow spots for each embryo (biological replicates). Each experiment has been repeated three independent times. Data information: Centre values denote the mean, and error values denote s.e.m. The statistical analysis was completed using an unpaired two tailed t test. ***P < .001 Scale bar 200μm (C)


( E ) Experimental outline and quantification of time - lapse live imaging in controls and slco2b1 - morphants cmyb : GFP + cells , using Imaris software . Data information : The statistical analysis was completed using an unpaired two tailed t test . * * * P < .

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

(E) Experimental outline and quantification of time-lapse live imaging in controls and slco2b1-morphants cmyb:GFP+ cells, using Imaris software. Data information: The statistical analysis was completed using an unpaired two tailed t test. ***P < .001


( A ) Anti - GFP and pH3 immunostainings of either controls ( Ctrl - Mo ) or slco2b1 - morphants ( slco2b1 - MO ) cmyb : GFP embryos . ( B ) Quantification of the number of the pH3 + HSCs in controls or slco2b1 - morphants . Centre values denote the mean , and error values denote s . e . m , statistical analysis was completed using an unpaired two tailed t test . * * * P < . 001 . Data information : Scale bar 50μm ( A )

(A) Anti-GFP and pH3 immunostainings of either controls (Ctrl-Mo) or slco2b1-morphants (slco2b1-MO) cmyb:GFP embryos. (B) Quantification of the number of the pH3+ HSCs in controls or slco2b1-morphants. Centre values denote the mean, and error values denote s.e.m, statistical analysis was completed using an unpaired two tailed t test. ***P < .001. Data information: Scale bar 50μm (A)


( D ) Quantification of PGE2 concentration in control and slco2b1 morphants . The statistical analysis was completed using an unpaired two tailed t test * P < . 01 . Centre values denote the mean , and error bars denote s . e . m .

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

(D) Quantification of PGE2 concentration in control and slco2b1 morphants. The statistical analysis was completed using an unpaired two tailed t test *P < .01. Centre values denote the mean, and error bars denote s.e.m.


( E ) Fluorescence imaging in the CHT of cmyb : GFP embryos injected with control - and slco2b1 - MOs and treated with PGE2 . ( F ) Quantification of GFP positive cells . Statistical analysis : one - way ANOVA , multiple comparison , * P < . 01 ; * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m . Data information : Scale bar 200μm ( E ) .

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

(E) Fluorescence imaging in the CHT of cmyb:GFP embryos injected with control- and slco2b1-MOs and treated with PGE2. (F) Quantification of GFP positive cells. Statistical analysis: one-way ANOVA, multiple comparison, *P < .01; ****P < .0001. Centre values denote the mean, and error bars denote s.e.m. Data information: Scale bar 200μm (E).


( A ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO . ( B ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ . ( C ) Fluorescence imaging of the CHT of double transgenic cd45 : CFP - NTR / runx1 : mcherry embryos injected with control - or slco2b1 - morpholinos , after treatment with DMSO + MTZ + PGH2 . ( D ) Quantification of runx1 : mcherry positive - cells . Each dot represents the number of red cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . ( E ) Quantification of cd45 : CFP positive - cells . Each dot represents the number of blue cells for each embryo ( biological replicates ) . Each experiment has been repeated three independent times . Data information for ( D ) and ( E ) : Statistical analysis was completed using one - way ANOVA , multiple comparison test . * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m Scale bar 200μm ( A - C ) .

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

(A) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO. (B) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ. (C) Fluorescence imaging of the CHT of double transgenic cd45:CFP-NTR/runx1:mcherry embryos injected with control- or slco2b1-morpholinos, after treatment with DMSO+MTZ+PGH2. (D) Quantification of runx1:mcherry positive-cells. Each dot represents the number of red cells for each embryo (biological replicates). Each experiment has been repeated three independent times. (E) Quantification of cd45:CFP positive-cells. Each dot represents the number of blue cells for each embryo (biological replicates). Each experiment has been repeated three independent times.Data information for (D) and (E): Statistical analysis was completed using one-way ANOVA, multiple comparison test. ****P<.0001. Centre values denote the mean, and error bars denote s.e.m Scale bar 200μm (A-C).


( A ) WISH for cmyb at 60hpf in kdrl : Gal4 + embryos injected with control - and slco2b1 - morpholinos and / or co - injected with the Tol2 - UAS : slco2b1 vector and tol2 mRNA . The region indicated by the dashed bas been magnified and shows in ( B ) . ( C ) Quantification and statistical analysis were completed using one - way ANOVA , multiple comparison test . * * * * P < . 0001 . Centre values denote the mean , and error bars denote s . e . m . Each dot represents the number of cmyb - expressing cells for each embryo ( biological replicates ) . Each experiment has been repeat three independent times .

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

(A) WISH for cmyb at 60hpf in kdrl:Gal4+ embryos injected with control- and slco2b1- morpholinos and/or co-injected with the Tol2-UAS:slco2b1 vector and tol2 mRNA. The region indicated by the dashed bas been magnified and shows in (B). (C) Quantification and statistical analysis were completed using one-way ANOVA, multiple comparison test. ****P< .0001. Centre values denote the mean, and error bars denote s.e.m. Each dot represents the number of cmyb-expressing cells for each embryo (biological replicates). Each experiment has been repeat three independent times.


( A ) Immunohistochemistry on paraffin sections for cKit ( HSPCs marker ) and CD31 ( endothelial cell marker ) at E13 and E16 stages of mouse fetal liver . ( B ) Distance distribution between cKithigh cells and endothelial cells ( n = 79 from 3 E13 fetal livers ; n = 92 from 3 E16 fetal livers ) , binned into 20μm intervals . Statistical analysis was completed using t - test . * P < . 01 ; * * P < . 001 ; * * * P < . 0001 . Centre values denote the mean , and error values denote s . e . m All nuclei marked with DAPI . Scale bar

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

(A) Immunohistochemistry on paraffin sections for cKit (HSPCs marker) and CD31 (endothelial cell marker) at E13 and E16 stages of mouse fetal liver. (B) Distance distribution between cKithigh cells and endothelial cells (n=79 from 3 E13 fetal livers; n=92 from 3 E16 fetal livers), binned into 20μm intervals. Statistical analysis was completed using t-test. *P<.01; **P<.001; ***P<.0001. Centre values denote the mean, and error values denote s.e.m All nuclei marked with DAPI. Scale bar 20μm