Gcn5 is expressed in all compartments of the Drosophila larval LG

Cartoon of the third instar larval LG, indicating the anterior primary lobes and the posterior lobes (A). The primary lobe of the LG depicting different subpopulations: the Posterior Signaling Centre (PSC: blue) functioning as the niche, Medullary zone (MZ: green) houses the progenitors, Intermediate progenitor zone (IZ: yellow) which contains the cells transitioning from progenitors to specific lineages, and the Cortical zone (CZ: brown) containing the differentiated hemocytes population – plasmatocytes and crystal cells (B). Gcn5 expression in different compartments of lymph gland (C-F’). Gcn5 (red) expression in the - Posterior Signaling Centre (PSC) using Collier-GFP (green) (C-C’, E-E’), Medullary zone prohemocytes using Dome-GFP (green) (D-D’, F-F’). Nuclei are stained with DAPI (Blue). Scale Bar: 50µm (C-D’), 30µm (E-F’).

Whole animal gcn5 mutants show defective LG homeostasis.

Posterior Signaling Center (PSC) cell numbers marked by Antennapedia (red) in gcn5[C137Y/C137Y] (B-B’), gcn5[E333st/E333st] (C-C’) and gcn5[E333st/C137Y] (D-D’’) as compared to wildtype (A-A’). Graphical representation of PSC cell numbers of the gcn5 mutants as compared to the wildtype (E), n=24 for wildtype, n=37 for gcn5[C137Y/C137Y], n=29 for gcn5[E333st/E333st] and n=20 for gcn5[E333st/C137Y] for PSC cell number quantification. Plasmatocyte differentiation was marked by P1 (red) in gcn5[C137Y/C137Y] (G-G’), gcn5[E333st/E333st] (H-H’) and gcn5[E333st/C137Y] (I-I’) as compared to the wildtype (F-F’). Graphical representation of plasmatocyte differentiation index of the gcn5 mutants compared to wildtype (J), n=20 for wildtype, n=29 for gcn5[C137Y/C137Y], n=33 for gcn5[E333st/E333st] and n=26 for gcn5[E333st/C137Y] for quantification. Crystal cell differentiation marked by Hnt (red) in gcn5[C137Y/C137Y] (L-L’), gcn5[E333st/E333st] (M-M’) and gcn5[E333st/C137Y] (N-N’) as compared to wildtype (K-K’). Graphical representation of crystal cell differentiation index for gcn5 mutants compared to wildtype (O), n=22 for wildtype, n=26 for gcn5[C137Y/C137Y], n=33 for gcn5[E333st/E333st] and n=31 for gcn5[E333st/C137Y] for quantification. Cells undergoing DNA damage marked by γH2AX (red) in gcn5[C137Y/C137Y] (Q-Q’), gcn5[E333st/E333st] (R-R’) and gcn5[E333st/C137Y] (S-S’) as compared to wildtype (P-P’). Graphical representation of γH2AX positive cells undergoing DNA damage in the gcn5 mutants compared to the wildtype (T), n=24 for wildtype, n=54 for gcn5[C137YC137Y], n=41 for gcn5[E333st/E333st]and n=31 for gcn5[E333st/C137Y] for quantification. Nuclei are stained with DAPI (Blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-S’). Nuclei are stained with DAPI (blue). Scale Bar: 50µm (A-S’).

Modulation of Gcn5 levels in the prohemocytes alters blood cell homeostasis in the lymph gland

Posterior Signalling Center (PSC) population marked by Antennapedia (red) upon Dome-Gal4 mediated knockdown (B-B’) or over-expression (C-C’) of Gcn5 as compared to wildtype (A- A’). Plasmatocyte differentiation marked by P1 (red) upon Dome-Gal4 mediated knockdown (E-E’) or over-expression (F-F’) of Gcn5 as compared to wildtype (D-D’). Crystal cell differentiation marked by Hnt (red) upon Dome-Gal4 mediated knockdown (H-H’) or over- expression (I-I’) of Gcn5 as compared to wild type (G-G’). Cells undergoing DNA damage marked by γH2AX (red) upon Dome-Gal4 mediated knockdown (K-K’) or over-expression (L- L’) of Gcn5 as compared to wild type (J-J’). Graphical representation of PSC numbers upon modulation of Gcn5 levels in the Dome positive population compared to wildtype (M), n=28 for wildtype, n=28 for Gcn5 knockdown, and n=20 for Gcn5 over-expression. Graphical representation of prohemocyte index upon modulation of Gcn5 levels in dome population compared to wildtype (N), n=30 for wildtype, n=30 for Gcn5 knockdown, and n=30 for Gcn5 over-expression. Graphical representation of plasmatocyte differentiation index upon modulation of Gcn5 levels in dome population compared to wildtype (O), n=35 for wildtype, n=23 for Gcn5 knockdown, and n=25 for Gcn5 over-expression. Graphical representation of Crystal cell differentiation index upon modulation of Gcn5 levels in the dome population compared to wildtype (P), n=37 for wildtype, n=21 for Gcn5 knockdown, and n=33 for Gcn5 over-expression. Graphical representation of γH2AX positive cells upon modulation of Gcn5 levels in the dome population compared to wildtype (Q), n=33 for wildtype, n=27 for Gcn5 knockdown, and n=38 for Gcn5 over-expression. Nuclei are stained with DAPI (blue). Dome>GFP positive population (Green) indicates the expression domain of Dome-Gal4 activity in the LG. n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the Lymph Gland. Values are mean ± SD, and asterisk marks statistically significant differences (**p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-L’).

Prohemocyte-specific expression of Gcn5 domain deletion constructs affects LG homeostasis.

Schematic of the SAGA HAT complex containing different modules – Sgf29, Ada2, Gcn5, and Ada3. Gcn5 is an 813 aa protein in Drosophila containing the Pcaf homology domain, HAT domain, Ada domain, and Bromodomain (A). Posterior Signalling Center (PSC) numbers marked by Antennapedia (red) upon expression of different Gcn5 domain deletion constructs in the tep-GFP (green) population using tep4-Gal4 (C-F), compared to wildtype (B). Progenitor index marked by tep (green) upon expression of different Gcn5 domain deletion constructs (C’- F’), compared to wildtype (B’). Plasmatocyte differentiation marked by P1 (red) upon expression of different Gcn5 domain deletion constructs in the tep-GFP population using tep4- Gal4 (green) (C”-F”), compared to wildtype (B”). Crystal cell differentiation marked by Hnt (red) upon expression of different Gcn5 domain deleted constructs in the tep-GFP population using tep4-Gal4 (green) (C”’-F”’), compared to wildtype (B”’). Graphical representation of PSC numbers upon expression of Gcn5 domain deletion constructs, compared to wildtype (G), n=40 for wildtype, n=29 for Gcn5ΔHAT expression, n=35 for Gcn5ΔPcaf expression, n=33 for Gcn5ΔBromo expression, and n=26 for Gcn5ΔAda expression in the tep population. Graphical representation of Prohemocyte index upon expression of Gcn5 domain deletion constructs, compared to wildtype (H), n=24 for wildtype, n=24 for Gcn5ΔHAT expression, n=34 for Gcn5ΔPcaf expression, n=24 for Gcn5ΔBromo expression, and n=24 for Gcn5ΔAda expression in the tep population. Graphical representation of Plasmatocyte differentiation index upon expression of Gcn5 domain deleted constructs, compared to wildtype (I), n=38 for wildtype, n=30 for Gcn5ΔHAT expression, n=29 for Gcn5ΔPcaf expression, n=27 for Gcn5ΔBromo expression, and n=27 for Gcn5ΔAda expression in the tep population. Graphical representation of Crystal cell numbers upon expression of Gcn5 domain deleted constructs, compared to wildtype (J), n=23 for wildtype, n=35 for Gcn5ΔHAT expression, n=26 for Gcn5ΔPcaf expression, n=26 for Gcn5ΔBromo expression, and n=26 for Gcn5ΔAda expression in the tep population. Nuclei are stained with DAPI (blue). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (B-F’’’).

Autophagic flux in Drosophila blood cells is negatively regulated by Gcn5

p62 (red) labeling the autophagy adaptor protein upon Hml-Gal4 mediated Gcn5 knockdown (B), or overexpression (C) as compared to wildtype (A). Atg8 (red), the autophagosomal marker upon Hml-Gal4 mediated Gcn5 knockdown (B’) or over-expression (C’) as compared to wildtype (A’). Nuclei were stained with DAPI (blue). Graphical representation of the p62 positive puncta/cell (D). Graphical representation of Atg8 positive puncta/cell (E). Values are mean ± SD, and asterisk marks statistically significant differences (***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 20µm (A-E’).

Genetic and chemical ablation of autophagy leads to aberrant blood cell differentiation.

Plasmatocyte differentiation marked by P1 (red) upon tep4-Gal4 mediated knockdown of TFEB (C-C’), Atg8a (E-E’), Atg5 (G-G’), or Atg18 (I-I’) in the tep population (green) as compared to wildtype (A-A’). Crystal cell differentiation marked by Hnt (red) upon tep4-Gal4 mediated knockdown of TFEB (D-D’), Atg8a (F-F’), Atg5 (H-H’), or Atg18 (J-J’) in the tep population (green) as compared to wildtype (B-B’). Graphical representation of Plasmatocyte differentiation index upon knockdown of TFEB (n=33), Atg8a (n=28), Atg5 (n=24), or Atg18 (n=22) in the tep population, compared to the wildtype (n=22) (K). Graphical representation of Crystal cell numbers upon knockdown of TFEB (n=21), Atg8a (n=44), Atg5 (n=30), or Atg18 (n=58) in the tep population, compared to the wildtype (n=28) (L). Plasmatocyte differentiation (red) upon Chloroquine treatment (N) as compared to control (M). Crystal cell differentiation upon Chloroquine treatment (P) as compared to control (O). Graphical representation of Plasmatocyte Differentiation Index upon Chloroquine treatment (n=30), compared to control (n=25) (Q). Graphical representation of Crystal Cell numbers upon Chloroquine treatment (n=25), compared to control (n=31) (R). Nuclei are stained with DAPI (blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, Student’s t- test with Welch’s correction). Scale Bar: 50µm (A-P).

Modulation of mTORC1 activity regulates blood cell differentiation in the Lymph gland.

Plasmatocyte differentiation marked by P1 (red) upon 3BDO treatment (B-B’) as compared to control (A-A’). Crystal cell differentiation marked by Hnt (red) upon 3BDO treatment (D-D’) compared to control (C-C’). Graphical representation of Plasmatocyte Differentiation Index upon 3BDO treatment (n=28) compared to control (n=24) (E). Graphical representation of Crystal cell numbers upon 3BDO treatment (n=27) compared to control (n=24) (F). Plasmatocyte differentiation marked by P1 (red) upon Rapamycin treatment (H-H’) compared to control (G-G’). Crystal cell differentiation marked by Hnt (red) upon Rapamycin treatment (J-J’) compared to control (I-I’). Graphical representation of Plasmatocyte Differentiation Index upon Rapamycin treatment (n=28) compared to control (n=20) (K). Graphical representation of Crystal cell numbers upon Rapamycin treatment (n=25) compared to control (n=20) (L). Immunoblot showing GCN5 (93 kDa) protein levels in different diet conditions – fed, starved, and high-fat diet (M). Immunoblot showing GCN5 (93 kDa) protein levels upon Rapamycin treatment compared to wildtype (N). β-actin (42kDa) was used as the loading control. Nuclei are stained with DAPI (blue). Quantification of GCN5 protein levels in the fed, starved and High Fat diet scenario (O). Quantification of GCN5 protein levels in the wildtype as compared to rapamycin treatment (P). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-J’).

mTORC1 can override the effect of Gcn5 level modulation over autophagy.

Plasmatocyte differentiation marked by P1 (red) upon 3BDO treatment in tep4-Gal4 mediated (green) Gcn5 knockdown background (B-B’) compared to control treatment in the same genetic background (A-A’). Crystal cell differentiation marked by Hnt (red) upon 3BDO treatment in the tep-mediated (green) Gcn5 knockdown background (D-D’) compared to control treatment in the same genetic background (C-C’). Plasmatocyte differentiation marked by P1 (red) upon Rapamycin treatment in Dome-mediated (green) Gcn5 over-expression background (F-F’) compared to control treatment in the same genetic background (E-E’). Crystal cell differentiation marked by Hnt (red) upon Rapamycin treatment in Dome-Gal4 mediated (green) Gcn5 over-expression background (H-H’) as compared to control treatment in the same genetic background (G-G’). Graphical representation of Plasmatocyte Differentiation Index upon 3BDO treatment (n=21) and control treatment (n=20) in the tep- mediated Gcn5 knockdown background (I). Graphical representation of Crystal cell numbers upon 3BDO treatment (n=20) and control treatment (n=24) in the tep-mediated Gcn5 knockdown background (J). Graphical representation of Plasmatocyte Differentiation Index upon Rapamycin treatment (n=27) and control treatment (n=21) in the Dome-mediated Gcn5 over-expression background (K). Graphical representation of Crystal Cell numbers upon Rapamycin treatment (n=42) and control treatment (n=24) in the Dome-mediated Gcn5 over- expression background (L). Nuclei are stained with DAPI (blue). n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (**p<0.01; ***p<0.001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-H’).

Gcn5-mTORC1-TFEB signaling axis regulates autophagy to control blood cell homeostasis.

A cartoon summarizing how Gcn5-mTORC1-TFEB signaling axis regulates autophagy to control blood cell homeostasis in the Drosophila LG.

Blood cell homeostasis is affected in the whole animal gcn5 heterozygous mutants.

PSC cell numbers marked by Antennapedia (red) in gcn5[C137Y/+] (B-B’) or gcn5[E333st/+] (C-C’) as compared to wildtype (A-A’). Graphical representation of PSC cell numbers of the gcn5 mutants compared to the wildtype (D), n=24 for wildtype, n=33 for gcn5[C137Y/+], and n=41 for gcn5[E333st/+] for PSC cell numbers quantification. Plasmatocyte differentiation was marked by P1 (red) in gcn5[C137Y/+] (F-F’) or gcn5[E333st/+] (G-G’) compared to the wildtype (E-E’). Graphical representation of plasmatocyte differentiation index of the gcn5 heterozygous mutants compared to wildtype (H), n=24 for wildtype, n=20 for gcn5[C137Y/+], and n=24 for gcn5[E333st/+] for quantification. Crystal cell differentiation marked by Hnt (red) in gcn5[C137Y/+] (J-J’) or gcn5[E333st/+] (K-K’) compared to wildtype (I-I’). Graphical representation of the number of Crystal cells for gcn5 heterozygous mutants compared to wildtype (L), n=26 for wildtype, n=26 for gcn5[C137Y/+], and n=23 for gcn5[E333st/+] for quantification. Cells undergoing DNA damage marked by γH2AX (red) in gcn5[C137Y/+] (N-N’) or gcn5[E333st/+] (O-O’) as compared to wildtype (M-M’). Graphical representation of γH2AX marked DNA damage of the heterozygous gcn5 mutants compared to the wildtype (P), n=31 for wildtype, n=42 for gcn5[C137Y/+], and n=28 for gcn5[E333st/+] for quantification. Nuclei are stained with DAPI (Blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-O’).

Validation of Gcn5 knockdown and over-expression constructs

Gcn5 (red) expression upon Hml-Gal4 mediated Gcn5 knockdown (B-B’) or over-expression (C-C’) in the hml population, compared to wildtype (A-A’). FLAG (red) expression upon Gcn5 over-expression (E-E’) in the hml population, compared to wildtype (D-D’). Nuclei are stained with DAPI (Blue). Scale Bar: 50µm (A-E’).

Collier mediated modulation of Gcn5 levels in the PSC alters LG homeostasis.

PSC cell population marked by Antennapedia (red) upon knockdown (B-B’) or over-expression (C-C’) of Gcn5 in the Collier population (green) compared to wildtype (A-A’). Plasmatocyte differentiation marked by P1 (red) upon knockdown (E-E’) or over-expression (F-F’) of Gcn5 in the Collier population (green) compared to wildtype (D-D’). Crystal cell differentiation marked by Hnt (red) upon knockdown (H-H’) or over-expression (I-I’) of Gcn5 in the Collier population (green) compared to wild type (G-G’). Cells undergoing DNA damage marked by γH2AX (red) upon knockdown (K-K’) or over-expression (L-L’) of Gcn5 in the Collier population (green) compared to wild type (J-J’). Graphical representation of PSC cell numbers upon modulation of Gcn5 levels in the Collier population compared to wildtype (M), n=21 for wildtype, n=29 for Gcn5 knockdown, and n=22 for Gcn5 over-expression. Graphical representation of plasmatocyte differentiation Index upon modulation of Gcn5 levels in Collier population compared to wildtype (N), n=23 for wildtype, n=23 for Gcn5 knockdown, and n=20 for Gcn5 over-expression. Graphical representation of numbers of Crystal cells upon modulation of Gcn5 levels in the Collier population compared to wildtype (O), n=22 for wildtype, n=21 for Gcn5 knockdown, and n=24 for Gcn5 over-expression. Graphical representation of γH2AX positive cells upon modulation of Gcn5 levels in the Collier population compared to wildtype (P), n=29 for wildtype, n=27 for Gcn5 knockdown, and n=21 for Gcn5 over-expression. Nuclei are stained with DAPI (blue). PSC cell specific Gcn5 modulation was performed using Collier-Gal4. n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; **p<0.01; ***p<0.001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-L’)

HmlΔ mediated modulation of Gcn5 levels in the differentiated blood cell population results in altered hematopoiesis.

PSC cell population marked by Antennapedia (red) upon knockdown (B-B’) or over-expression (C-C’) of Gcn5 in the HmlΔ population (green) compared to wildtype (A-A’). Plasmatocyte differentiation marked by P1 (red) upon knockdown (E-E’) or over-expression (F-F’) of Gcn5 in the HmlΔ population (green) compared to wildtype (D-D’). Crystal cell differentiation marked by Hnt (red) upon knockdown (H-H’) or over-expression (I-I’) of Gcn5 in the HmlΔ population (green) compared to wildtype (G-G’). Cells undergoing DNA damage marked by γH2AX (red) upon knockdown (K-K’) or over-expression (L-L’) of Gcn5 in the HmlΔ population (green) compared to wildtype (J-J’). Graphical representation of PSC cell numbers upon modulation of Gcn5 levels in the HmlΔ population compared to wildtype (M), n=24 for wildtype, n=25 for Gcn5 knockdown, and n=36 for Gcn5 over-expression. Graphical representation of plasmatocyte differentiation Index upon modulation of Gcn5 levels in HmlΔ population compared to wildtype (N), n=20 for wildtype, n=20 for Gcn5 knockdown, and n=21 for Gcn5 over-expression. Graphical representation of numbers of Crystal cells upon modulation of Gcn5 levels in the HmlΔ population compared to wildtype (O), n=25 for wildtype, n=21 for Gcn5 knockdown, and n=22 for Gcn5 over-expression. Graphical representation of γH2AX positive cells upon modulation of Gcn5 levels in the HmlΔ population compared to wildtype (P), n=23 for wildtype, n=29 for Gcn5 knockdown, and n=27 for Gcn5 over-expression. Nuclei are stained with DAPI (blue). HmlΔ-Gal4 was used for differentiated blood cells specific Gcn5 modulation. n represent the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-L’).

GCN5 positively regulates crystal cell differentiation cell-autonomously

GCN5 (magenta) expression in lz-GFP (Green) positive crystal cells in larval LGs where lz-Gal4 drives UAS-GFP in wild type genetic background (A-B). Crystal cell differentiation marked by Hindsight (Hnt, magenta) upon GCN5 over-expression using lz- Gal4GFP (D) as compared to control (C) and quantitated as total number of crystal cells per LG lobe represented by n, where n=31 for wildtype and n=36 for GCN5 overexpression. (G). GCN5 (magenta) expression in posterior LG lobes marked by GFP (Green) driven by tep4- Gal4 (E-F). Nuclei = DAPI (Blue) and GFP driven by either lz-Gal4 or tep4-Gal4. Values are mean ± SD, and asterisk marks statistically significant differences with *** as p<0.001 analyzed by Student’s t- test with Welch’s correction. Scale Bar: 30µm (A-B), 50 µm (C-F).

Prohemocyte-specific expression of Gcn5 domain deletion constructs leads to increased DNA damage.

Cells undergoing DNA damage marked by γH2AX (red) upon expression of different Gcn5 domain deletion constructs in the tep-GFP population using tep4-Gal4 (green) (B-E’) as compared to wildtype (A, A’). Graphical representation of Cells undergoing DNA damage marked by γH2AX (F), where n=27 for wildtype, n=26 for Gcn5ΔHAT expression, n=27 for Gcn5ΔPcaf expression, n=23 for Gcn5ΔBromo expression, and n=25 for Gcn5ΔAda expression in the tep population. Nuclei are stained with DAPI (blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (**p<0.01; ***p<0.001; ****p<0.0001, Student’s t-test with Welch’s correction). Scale Bar: 50µm (A-E’)

Gcn5 over-expression leads to suppression of autophagy-related genes

Transcript levels of different autophagy effector genes in Hml-Gal4 mediated Gcn5 over-expression genetic background as compared to the Hml-Gal4 X wt control. rp49 was used as an endogenous control to normalize the mRNA expression.

Prohemocyte- specific genetic abrogation of autophagy affects the niche cell numbers and induces DNA damage.

PSC/niche cells marked by Antennapedia (red) upon tep4-Gal4 mediated knockdown of TFEB (C-C’), Atg8a (E-E’), Atg5 (G-G’), or Atg18 (I-I’) in the tep population (green) as compared to wildtype (A-A’). DNA damage marked by Gamma-H2Ax (red) upon tep4-Gal4 mediated knockdown of TFEB (D-D’), Atg8a (F-F’), Atg5 (H-H’), or Atg18 (J-J’) in the tep population (green) as compared to wildtype (B-B’). Graphical representation of niche cell numbers upon knockdown of TFEB (n=40), Atg8a (n=36), Atg5 (n=39), Atg18 (n=41) in the tep population, compared to the wildtype (n=33) (K). Graphical representation of Gamma-H2Ax positive foci upon knockdown of TFEB (n=37), Atg8a (n=34 , Atg5 (n=33), or Atg18 (n=36) in the tep population, compared to the wildtype (n=35 ) (L). Graphical representation of prohemocyte index upon knockdown of TFEB (n=30), Atg8a (n=30), Atg5 (n=30), Atg18 (n=30) in the tep population, compared to the wildtype (n=33) (M). Nuclei are stained with DAPI (blue). n represents the number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, Student’s t- test with Welch’s correction). Scale Bar: 50µm (A-J’)

Autophagy levels in the LG hemocytes are affected upon systemic Chloroquine treatment

Chloroquine treated tep4-Gal4GFP X wt larvae expressing GFP (Green) driven by tep4-Gal4 showing p62 or Atg8 positive puncta (magenta) in LG hemocytes (B-B’, D-D’) as compared to vehicle treated (A-A’, C-C’) and represented as number of p62 or Atg8 positive puncta per cell (n),where n= 255 cells for vehicle treated and n=300 cells for chloroquine treated p62 positive cells and n= 360 cells for vehicle treated and n= 390 cells for chloroquine treated Atg8 positive cells respectively.(E and F). Nuclei = DAPI (Blue) and GFP driven by tep4-Gal4. Values are mean ± SD, and asterisk marks statistically significant differences with **** as p<0.0001 analyzed by Student’s t- test with Welch’s correction. Scale Bar: 30µm (A-D’)

Genetic modulation of mTORC1 activity in the hematopoietic progenitors regulates blood cell differentiation

Plasmatocyte differentiation marked by P1 (magenta) upon tep4-Gal4 mediated depletion of tor (B) or raptor (C) or over-expression of Rheb (D) as compared to the wild type control (A) quantitated as plasmatocyte differentiation index per LG lobe where n represents the number of primary lobes of LG, n= 45 for wildtype, n= 39 for tor knockdown, n= 43 for raptor knockdown and n=44 for Rheb overexpression. (I). Crystal cell differentiation marked by Hindsight (Hnt, magenta) upon tep4-Gal4 mediated depletion of tor (F) or raptor (G) or over-expression of Rheb (H) as compared to the wild type control (E) quantitated as crystal cell differentiation index per LG lobe(n), where n represents the number of primary lobes of LG, n= 31 for wildtype, n= 42 for tor knockdown, n=30 for raptor knockdown, n=28 for Rheb overexpression. (J). Nuclei = DAPI (Blue) and GFP is driven by tep4-Gal4. n = number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, ns = non-significant. Student’s t- test with Welch’s correction). Scale Bar: 50µm (A-H)

Genetic modulation of mTORC1 activity in the hematopoietic progenitors alters niche cell numbers and causes DNA damage

PSC/niche cell numbers marked by Antennapedia (Antp, magenta) upon tep4-Gal4 mediated depletion of tor (B) or raptor (C) or over-expression of Rheb (D) as compared to the wild type control (A) quantitated as Antp positive niche cell numbers per LG lobe represented by n, where n= 38 for wildtype, n= 37 for tor knockdown, n=42 for raptor knockdown, n=37 for Rheb overexpression. (I). γ-H2Ax foci positive cells showing DNA damage (magenta) upon tep4-Gal4 mediated depletion of tor (F) or raptor (G) or over-expression of Rheb (H) as compared to the wild type control (E) quantitated as total number of γ-H2Ax positive foci per LG lobe represented by n, where n= 40 for wildtype, n= 43 for tor knockdown, n=34 for raptor knockdown, n=37 for Rheb overexpression. (J). Nuclei = DAPI (Blue) and GFP is driven by tep4-Gal4. n = number of individual primary lobes of the LG. Individual data points in the graphs represent individual primary lobes of the LG. Values are mean ± SD, and asterisk marks statistically significant differences (*p<0.05; ***p<0.001; ****p<0.0001, ns = non-significant. Student’s t- test with Welch’s correction). Scale Bar: 50µm (A-H)