Journal: bioRxiv

Article Title: Modulation of cellular ageing regulates Integrated Stress Response signalling to control blood cell homeostasis

doi: 10.1101/2025.01.09.632127

Figure Lengend Snippet: Mean fluorescence intensity of p-eIF2α levels in the core progenitors marked by p-eIF2α antibody (magenta) upon induction of accelerated aging using ( UAS-pirkRNAi or UAS- toll10B ) or decelerated aging ( UAS-atg8 or UAS-foxo ) as compared to wildtype control . Graphical representation of p-eIF2α fluorescence intensity levels upon tepIVGal4 mediated induction of accelerated aging ( UAS-pirkRNAi or UAS-toll10B ) or decelerated aging ( UAS-atg8 or UAS-foxo ) as compared to wildtype control . Relative gcn2 mRNA transcript levels determined by qRT-PCR upon pan-hemocyte-specific (using hmlGal4 ) expression of UAS-atg8GFP , UAS-foxoGFP , UAS-pirkRNAi or UAS-toll10B as compared to wildtype control . The statistical analysis for qRT-PCR was performed using One-way ANOVA (Dunnett test) for comparison of all test genotypes with wildtype control. Plasmatocyte differentiation marked by P1 (magenta) or crystal cell differentiation marked by Hindsight (Hnt, magenta) or lamellocyte differentiation marked by β-integrin- specific Myospheroid (Mys, magenta) upon core progenitor-specific (using tepIVGal4 ) knockdown ( gcn2RNAi, atf4RNAi or eIF2αRNAi) and over-expression ( gcn2 act or crcRA ) of ISR components as compared to wildtype control . Graphical representation of plasmatocyte differentiation index or number of crystal cells or percentage of lamellocyte positive lymph glands upon tepIVGal4 mediated knockdown or over-expression of ISR components as compared to wildtype control. For p-eIF2α levels, tepIVGal4 x UAS-pirkRNAi (N = 14, n = 27), tepIVGal4 x UAS-toll10B (N = 13, n = 26), tepIVGal4 x UAS-atg8GFP (N = 11, n = 22) and tepIVGal4 x UAS-foxoGFP (N = 13, n = 26) were analyzed as compared to tepIVGal4 x wt (N = 12, n = 24) as wildtype control. For plasmatocyte differentiation, tepIVGal4 x UAS-gcn2RNAi (N = 17, n = 34), tepIVGal4 x UAS-eIF2αRNAi (N = 16, n = 32), tepIVGal4 x UAS-atf4RNAi (N = 16, n = 32), tepIVGal4 x UAS-gcn2 act (N = 15, n = 30), tepIVGal4 x UAS-crcRA (N = 18, n = 36) were analyzed as compared to wildtype control, tepIVGal4 x wt (N = 25, n = 49). For crystal cell differentiation, tepIVGal4 x UAS-gcn2RNAi (N = 20, n = 40), tepIVGal4 x UAS-eIF2αRNAi (N = 17, n = 33), tepIVGal4 x UAS-atf4RNAi (N = 13, n = 25), tepIVGal4 x UAS-gcn2 act (N = 18, n = 35), tepIVGal4 x UAS-crcRA (N = 17, n = 33) were analyzed as compared to wildtype control, tepIVGal4 x wt (N = 18, n = 36). For lamellocyte differentiation, tepIVGal4 x UAS-gcn2RNAi (N = 15, n = 30), tepIVGal4 x UAS-eIF2αRNAi (N= 15, n = 30), tepIVGal4 x UAS-atf4RNAi (N = 15, n = 30), tepIVGal4 x UAS-gcn2 act (N = 15, n = 30), tepIVGal4 x UAS-crcRA (N = 15, n = 30) were analyzed as compared to wildtype control, tepIVGal4 x wt (N = 15, n = 30). N denotes the number of larvae & n denotes the number of individual primary lymph gland lobes analyzed per genotype. Individual data points in the graphs represent individual primary lobes of the Lymph gland. GFP expression (green) is driven by tepIVGal4 . Nuclei are stained with DAPI (Blue). Values are mean ± SD, and asterisks denote statistically significant differences (ns denotes not significant, **p < 0.01, *** p < 0.001, ****p < 0.0001). Student’s t-test with Welch’s correction was performed for the statistical analysis. Scale Bar: 50 µm (A-Y).

Article Snippet: Antibodies used were mouse-raised anti-P1 (1:100, kind gift from Dr. Istvan Ando), mouse- raised anti-Hindsight (1:25, 1G9 – DSHB; RRID:Ab_528278), mouse-raised anti-Antp (1:25, 8C11- DSHB; RRID:Ab_528083), mouse-raised anti-γ2AX (1:200, UNC93-5.2.1 - DSHB; RRID:Ab_2618077), mouse-raised anti-Myospheroid (1:25, 6G11 - DSHB; RRID: Ab_528310), rabbit-raised anti-p-eIF2α (1:100, Cell Signaling technologies, RRID:Ab_119A11), CellROX Deep Red Reagent (2.5mM in DMSO, Invitrogen, C10422), PROTEOSTAT Protein detection assay (1:100, Enzo life sciences, CAT No.: ENZ-51023- KP050), rabbit-raised anti-P62/SQSTM1 (1:250, Proteintech, RRID:AB_10694431) and rabbit-raised anti-ATG8 (1:200, Sigma Aldrich, RRID:Ab_2939040) for immunofluorescence based experiments.

Techniques: Fluorescence, Control, Quantitative RT-PCR, Expressing, Comparison, Cell Differentiation, Knockdown, Over Expression, Staining

Journal: bioRxiv

Article Title: Modulation of cellular ageing regulates Integrated Stress Response signalling to control blood cell homeostasis

doi: 10.1101/2025.01.09.632127

Figure Lengend Snippet: Estimation of autophagic flux by evaluating the ratio of p62 positive punctae per cell marked by p62 antibody (magenta) upon core progenitor-specific (using tepIVGal4 ) knockdown of pirk or over-expression of atg8 as compared to wildtype control . Mean fluorescence intensity of ROS levels in the core progenitors marked by Cell-ROX deep red reagent (Red) upon tepIVGal4 mediated expression of UAS-atg8GFP, UAS-foxoGFP or UAS-toll10B as compared to wildtype control . Estimation of protein turnover by estimating the ratio of Proteostat positive punctae per cell marked by PROTEOSTAT detection reagent (magenta) upon tepIVGal4 mediated expression of UAS-atg8GFP, UAS-foxoGFP, UAS-pirkRNAi or UAS-toll10B as compared to wildtype control . Graphical representation of the ratio of p62 positive punctae per cell or Mean fluorescence intensity of ROS levels in the tepIV -positive core progenitors or ratio of Proteostat positive punctae per cell upon expression of UAS-atg8GFP, UAS-foxoGFP, UAS-pirkRNAi or UAS-toll10B in the progenitors as compared to wildtype control. For autophagic flux estimation by p62 staining, tepIVGal4 x UAS-pirkRNAi (N = 11, n = 21) and tepIVGal4 x UAS-atg8GFP (N = 11, n = 21) were analyzed as compared to wildtype control, tepIVGal4 x wt (N = 11, n = 21). For estimation of ROS levels by Cell-ROX deep red staining, tepIVGal4 x UAS-atg8GFP (N = 5, n = 10), tepIVGal4 x UAS-foxoGFP (N = 9, n = 18) and tepIVGal4 x UAS-toll10B (N = 10, n = 20) were analyzed as compared to wildtype control, tepIVGal4 x wt (N = 10, n = 20). For estimation of protein turnover by Proteostat detection reagent staining, tepIVGal4 x UAS-atg8GFP (N = 10, n = 20), tepIVGal4 x UAS-foxoGFP (N = 10, n = 20), tepIVGal4 x UAS-pirkRNAi (N = 10, n = 20) and tepIVGal4 x UAS-toll10B (N = 10, n = 20) were analyzed as compared to tepIVGal4 x wt (N = 10, n = 20) as wildtype control . N denotes the number of larvae & n denotes the number of individual primary lymph gland lobes analyzed per genotype. Individual data points in the graphs represent individual primary lobes of the Lymph gland. GFP expression (green) is driven by tepIVGal4 ( - , - , - ). Nuclei are stained with DAPI (Blue). Values are mean ± SD, and asterisks denote statistically significant differences (ns denotes not significant, *** p < 0.001, ****p < 0.0001). Student’s t-test with Welch’s correction was performed for the statistical analysis. Scale Bar: 30 µm (A-C, J-N), 50 µm (E-H).

Article Snippet: Antibodies used were mouse-raised anti-P1 (1:100, kind gift from Dr. Istvan Ando), mouse- raised anti-Hindsight (1:25, 1G9 – DSHB; RRID:Ab_528278), mouse-raised anti-Antp (1:25, 8C11- DSHB; RRID:Ab_528083), mouse-raised anti-γ2AX (1:200, UNC93-5.2.1 - DSHB; RRID:Ab_2618077), mouse-raised anti-Myospheroid (1:25, 6G11 - DSHB; RRID: Ab_528310), rabbit-raised anti-p-eIF2α (1:100, Cell Signaling technologies, RRID:Ab_119A11), CellROX Deep Red Reagent (2.5mM in DMSO, Invitrogen, C10422), PROTEOSTAT Protein detection assay (1:100, Enzo life sciences, CAT No.: ENZ-51023- KP050), rabbit-raised anti-P62/SQSTM1 (1:250, Proteintech, RRID:AB_10694431) and rabbit-raised anti-ATG8 (1:200, Sigma Aldrich, RRID:Ab_2939040) for immunofluorescence based experiments.

Techniques: Knockdown, Over Expression, Control, Fluorescence, Expressing, Staining

Journal: bioRxiv

Article Title: Modulation of cellular ageing regulates Integrated Stress Response signalling to control blood cell homeostasis

doi: 10.1101/2025.01.09.632127

Figure Lengend Snippet: Plasmatocyte differentiation marked by P1 (magenta) or crystal cell differentiation marked by Hindsight (Hnt, magenta) or lamellocyte differentiation marked by β-integrin-specific Myospheroid (Mys, magenta) or evaluation of p62 or Atg8 positive punctae per cell marked by p62 or Atg8 antibody (magenta) upon distal progenitor-specific (using dome-mesoGal4 ) hyperactivation of components of ISR pathway viz. gcn2 act & crcRA in an IMD upregulated background (via pirkRNAi ) as compared to wildtype control . Graphical representation of plasmatocyte differentiation index or number of crystal cells or percentage of lamellocyte positive lymph glands or ratio of p62 or Atg8 positive punctae per cell upon dome-mesoGal4 mediated over-expression of gcn2 act & crcRA in a pirk knockdown background as compared to wildtype control . UAS-gcn2 act /UAS- pirkRNAi; dome-mesoGal4 & UAS-crcRA/UAS-pirkRNAi; dome-mesoGal4 were analyzed for plasmatocyte differentiation ( gcn2 act : N = 15, n = 30; crcRA: N = 19, n = 37) or crystal cell numbers ( gcn2 act : N = 17, n = 33; crcRA: N = 14, n = 28) or lamellocyte differentiation ( gcn2 act : N = 16, n = 32; crcRA: N = 15, n = 30) or p62 levels ( gcn2 act : N = 11; crcRA: N = 10) or Atg8 levels per cell ( gcn2 act : N = 10; crcRA: N = 12) as compared to wildtype control (plasmatocytes: N = 19, n = 38; crystal cells: N = 14, n = 28; lamellocytes: N = 15, n = 30; p62: N = 12; Atg8: N = 10). N denotes the number of larvae & n denotes the number of individual primary lymph gland lobes analyzed per genotype. Individual data points in the graphs represent individual primary lobes of the Lymph gland. GFP expression (green) is driven by dome-mesoGal4 . Nuclei are stained with DAPI (Blue). Values are mean ± SD, and asterisks denote statistically significant differences (ns denotes not significant, **p<0.01, *** p<0.001, ****p<0.0001). Student’s t-test with Welch’s correction was performed for the statistical analysis. Our working model summarizing how restoration of blood cell homeostasis in the lymph gland occurs upon ectopic ISR hyperactivation in the hematopoietic progenitors in an accelerated ageing genetic background (NF-κB (Imd) upregulated) . Scale Bar: 50 µm (A-K’), 30 µm (M-R’).

Article Snippet: Antibodies used were mouse-raised anti-P1 (1:100, kind gift from Dr. Istvan Ando), mouse- raised anti-Hindsight (1:25, 1G9 – DSHB; RRID:Ab_528278), mouse-raised anti-Antp (1:25, 8C11- DSHB; RRID:Ab_528083), mouse-raised anti-γ2AX (1:200, UNC93-5.2.1 - DSHB; RRID:Ab_2618077), mouse-raised anti-Myospheroid (1:25, 6G11 - DSHB; RRID: Ab_528310), rabbit-raised anti-p-eIF2α (1:100, Cell Signaling technologies, RRID:Ab_119A11), CellROX Deep Red Reagent (2.5mM in DMSO, Invitrogen, C10422), PROTEOSTAT Protein detection assay (1:100, Enzo life sciences, CAT No.: ENZ-51023- KP050), rabbit-raised anti-P62/SQSTM1 (1:250, Proteintech, RRID:AB_10694431) and rabbit-raised anti-ATG8 (1:200, Sigma Aldrich, RRID:Ab_2939040) for immunofluorescence based experiments.

Techniques: Cell Differentiation, Control, Over Expression, Knockdown, Expressing, Staining

Journal: Development (Cambridge, England)

Article Title: The role of integrins in Drosophila egg chamber morphogenesis

doi: 10.1242/dev.182774

Figure Lengend Snippet: Mys is required for interfollicular stalk formation. (A) A wild-type ovariole expressing Mys-GFP (green) from a genomic BAC transgene. Mys is enriched on the basal side of all pre-follicle cells in the germarium (bracket) and in the forming and mature interfollicular stalks (yellow arrowheads and white arrowhead, respectively) ( n =4). (B) A section of ovariole stained for Lgl (red), DNA (blue) and β-galactosidase (green and B′) showing the pattern of UAS-LacZ expression under the control of the 24B-Gal4 driver. 24B-Gal4 drives expression in the interfollicular stalk cells only (arrowhead and arrows) ( n =6). (C) A stage 4 egg chamber stained for Dlg (white) and DNA (blue) containing mys XG43 clones marked by the loss of RFP (red). 24B-Gal4 was used to drive expression of UAS-FLP to generate mys XG43 clones specifically in the interfollicular stalks. The presence of mys mutant cells disrupts the organisation of the interfollicular stalks ( n =14/15). (D) A wild-type stage 4 egg chamber stained for F-actin (white) and DNA (blue) showing the fully formed stalks that separate it from the adjacent younger and older egg chambers ( n =5). (E-J′) For these experiments, mys XG43 clones [marked by the absence of RFP (red)] were induced in interfollicular stalks by the expression UAS-FLP under the control of 24B-Gal4. (E,E′) A disorganised stalk containing mys clones stained for F-actin (green), DNA (blue) and Lamin C (white in E′). Lamin C is expressed in both mutant and wild-type cells in disrupted interfollicular stalks (E) ( n =8). (F,F′) A disorganised stalk containing mys clones stained for Eya (green) and DNA (blue). Eya is turned off normally in both the mutant and wild-type stalk cells ( n =7). (G-I′) Mutant interfollicular stalk cells are round in appearance ( n =33) compared with the stalk made from all wild-type cells (I) ( n =4). Wild-type cells in disrupted interfollicular stalks range from rounded (white asterisks in G,G′) ( n =18/51) to wild type (white asterisks in H,H′) ( n =33/51). Dlg only localises to the regions where neighbouring interfollicular stalk cells contact each other in disrupted interfollicular stalks (arrowheads G′ and H′) ( n =18/18) as in interfollicular stalks containing only wild-type cells (I′) ( n =8/8). (J) A disorganised interfollicular stalk stained for aPKC (green) and Dlg (white). (J′) A z -projection of 16 planes taken 1 μm apart of the stalk in J. This shows that aPKC is not present in mature stalks containing mys XG43 mutant cells ( n =11). (K-L′) Regions of ovarioles stained for DNA (blue) and β-galactosidase (green) containing hs-FLP-induced mys XG43 clones marked by the loss of RFP (red). The interfollicular stalk cells are marked by β-galactosidase (green) expressed from UAS-LacZ under the control of 24B-Gal4. The disorganised region caused by mys XG43 clones at the terminus of the stage 9 egg chamber in the boxed area in K (enlarged in K′) does not contain cells expressing the β-galactosidase stalk marker, which lie only in a malformed stalk posteriorly (K′) ( n =4). (L,L′) The stalk cells lie at the posterior of the region between the two younger egg chambers ( lacZ -positive cells, green). The black dashed line identifies cells that could potentially contribute to the disorganised region at the terminus of the egg chamber later in development, like that seen in K ( n =4). Scale bars: 10 μm.

Article Snippet: The following antibodies were used in this study: mouse anti-Fasciclin II (1D4), mouse anti-Discs large (4F3), rabbit anti-Myospheroid (CF.6G11), mouse anti-Lamin C (LC28.26), mouse anti-Eyes absent (eya10HB), mouse anti-Armadillo (N2 7A1) (all Developmental Studies Hybridoma Bank), rabbit anti-aPKC (Sigma-Aldrich, P0713), rabbit anti-β-galactosidase (MP Biomedicals, 0855976), rabbit anti-Lethal giant larvae (Santa Cruz Biotechnology, sc-98260) and mouse anti-Fasciclin III (gift from C. Goodman, UC Berkeley, USA).

Techniques: Expressing, Staining, Clone Assay, Mutagenesis, Marker