Journal: bioRxiv

Article Title: Lipid metabolism of hepatocyte-like cells supports intestinal tumor growth by promoting tracheogenesis

doi: 10.1101/2025.04.04.647255

Figure Lengend Snippet: (A-B) Heatmap of the normalized total ion intensity of major lipid classes identified in whole bodies from control flies ( Esg-LexA ) or Yki flies ( Esg-LexA/LexAop-yki S3A ; PromE-GAL4, Tub-GAL80 TS /+ ), with four biological replicates per condition. Scaled colors are presented as the values relative to the average of each row. (B) Untargeted lipidomic profiling using LC-MS/MS of whole-body extracts revealed increased wax ester (WE) levels in Yki flies. (C) WE level in oenocyte-specific knockdown of Hnf4 using the RU486-inducible PromE gs driver (+RU) compared to controls (-RU). (D) WE level in control flies, Yki flies, and Hnf4 knockdown in oenocytes of Yki flies, N = 4 biological replicates. (E-F) Immunostaining of Nb127D01 for nuclear-localized, endogenously tagged Hnf4 (Hnf4-127D01) in oenocytes in controls and in Yki flies. DAPI staining marks nuclei, with dashed lines enclosing oenocytes. N = 8 independent experiments. (G) Quantification of intestinal stem cell (ISC) proliferation, as indicated by phospho-histone H3 (pH3+) cells in control. (H-I) Ovary sizes and quantification. (J) Abdominal bloating. (K) UMAP from whole body (without head) snRNAseq, mElo expression is enriched in adult oenocytes. (L) mElo expression in oenocytes, normalized to housekeeping gene, in Yki flies and in Yki flies with Hnf4 knockdown. N = 2 biological replicates from 2 independent experiments. (M) Quantification of pH3+ cell number, N =8 flies. (N) Oil Red O staining in oenocytes of Hnf4 knockdown or mElo knockdown. N = 8 flies. (O-P) Lifespan analysis showing that oenocyte-specific knockdown of Hnf4 (O) or mElo (P) in Yki flies, compared to only Yki or control flies, N = 40. Scale bar shows 20μm. Data are presented as mean ± s.e.m. Statistical significance was determined using Student’s t-test or one-way ANOVA with appropriate post hoc tests, or Log-rank (Mantel-Cox) test. *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: The following primary antibodies were used: rabbit-anti-pH3 (1:500, Cell Signaling, 9701), rabbit-anti-p4E-BP (1:800, Cell Signaling, 2855S).

Techniques: Control, Liquid Chromatography with Mass Spectroscopy, Knockdown, Immunostaining, Staining, Expressing

Journal: bioRxiv

Article Title: Lipid metabolism of hepatocyte-like cells supports intestinal tumor growth by promoting tracheogenesis

doi: 10.1101/2025.04.04.647255

Figure Lengend Snippet: (A) snRNAseq analysis showing Hnf4 expression in fat body, muscle and oenocytes upon overexpression of TSC1,2 in oenocytes. (B-C) Immunostaining of Hnf4-127D01 in oenocytes when TSC1,2 is overexpressed in oenocytes compared to control. DAPI marks nuclei, with dashed lines enclosing oenocytes, quantification of Hnf4-127D01 signal intensity in the nucleus, N = 8-10 flies. (D) ModuleScore from snRNAseq analysis visualizes VLCFA synthesis gene expression across muscle, fat body and oenocytes, in control and upon oenocyte TSC1,2 overexpression. (E) ORO staining indicates steatosis in oenocytes induced by TSC1,2 overexpression, and in TSC1,2 with Hnf4 overexpression, N = 8. (F-G) Expression of Hnf4 targets mElo and FASN2 in TSC1,2 overexpression and in TSC1,2 with Hnf4 overexpression. N = 2 biological replicates from 2 independent experiments. (H) Whole body WE level in ISC Yki flies and Yki with oenocyte TSC1,2 overexpression. (I-J) P-4EBP immunostaining indicates TORC1 activity in oenocytes of Yki flies, quantification of P-4EBP intensity is shown on the right (J), N = 8-10 flies. (K) mElo expression in whole body mRNA extracts in Yki flies and Yki flies with TSC1,2 overexpression in oenocytes, N = 6 biological samples. (L) Quantification of pH3+ cell number, N = 8 – 10 flies. (M) Ovary size measurements are rescued by TSC1,2 overexpression in Yki flies, N = 15-30 flies.

Article Snippet: The following primary antibodies were used: rabbit-anti-pH3 (1:500, Cell Signaling, 9701), rabbit-anti-p4E-BP (1:800, Cell Signaling, 2855S).

Techniques: Expressing, Over Expression, Immunostaining, Control, Gene Expression, Staining, Activity Assay

Journal: bioRxiv

Article Title: Lipid metabolism of hepatocyte-like cells supports intestinal tumor growth by promoting tracheogenesis

doi: 10.1101/2025.04.04.647255

Figure Lengend Snippet: (A-B) Immunostaining of Hnf4-127D01 nuclear localization in oenocytes upon Pvf1 overexpression in ISCs ( Esg>Pvf1 ). Dashed lines enclose oenocytes. N = 10 biological replicates. (C) mElo expression in oenocytes following ISC-specific Pvf1 overexpression. N = 4 biological replicates from 2 independent experiments. (D-F) Immunostaining and qPCR analysis reveal Hnf4-127D01 nuclear localization and mElo expression in oenocytes overexpressing active PvR . (G-H) Immunostaining of Hnf4-127D01 in oenocytes of Yki flies ( Esg>yki 3SA ;Luc-i ) and flies with knockdown of Pvf1 in Yki flies ( Esg> yki 3SA ;Pvf1-i ). N = 10 biological replicates. (I) mElo expression in oenocytes of Esg>yki 3SA ;Pvf1-i flies, N = 4 biological replicates from 2 independent experiments. (J-K) Inhibition of Pvf1 in the gut ajects bloating and ovary size in Yki flies, n = 15-20 flies. (L) ISC mitosis (pH3+ cells) in Esg>yki 3SA ;Pvf1-i flies, n = 8-10 biological replicates. (M) Yki-induced mElo expression. Dual LexA and Gal4 systems to express yki 3SA in ISCs and mediate knockdown of PvR in oenocytes ( Esg-LexA> yki 3SA ; PromE>PvR-i ). N = 2 biological replicates from 2 independent experiments. (N-P) PvR knockdown in oenocytes and its eject on tumor-associated bloating (N), ovary wasting (O), and pH3+ cell number (P), N = 10-20 flies. (Q) Lifespan extension in Yki flies with oenocyte-specific PvR knockdown, N = 40 flies. Dashed lines enclose oenocytes in all immunostaining images. Data are presented as mean ± s.e.m.; statistical significance was assessed using Student’s t-test or ANOVA.

Article Snippet: The following primary antibodies were used: rabbit-anti-pH3 (1:500, Cell Signaling, 9701), rabbit-anti-p4E-BP (1:800, Cell Signaling, 2855S).

Techniques: Immunostaining, Over Expression, Expressing, Knockdown, Inhibition

Journal: bioRxiv

Article Title: Lipid metabolism of hepatocyte-like cells supports intestinal tumor growth by promoting tracheogenesis

doi: 10.1101/2025.04.04.647255

Figure Lengend Snippet: Untargeted lipidomic analysis of fly hemolymph showing lipid species in control, Yki flies, and Yki flies with oenocyte-specific Hnf4 knockdown ( Esg>yki 3SA ;PromE>Hnf4-i ). N = 4 biological replicates. (B-C) Levels of very-long-acylcarnitines (AcCa 24:1 and AcCa 24:2) in Yki flies with or without Hnf4 knockdown in oenocytes. (D-E) Levels of circulating phospholipids, including PC 36:4 PE 32:5, across experimental conditions. (F-G) Levels of circulating TG 18:3_16:0_16:1 and TG 18:2_16:0_18:0 in the fly hemolymph. (H) Counts of pH3+ cells in the midgut of flies with oenocyte-specific TSC1 ,2 overexpression or Hnf4 knockdown or overexpression. N = 8-20 flies. (I-K) Number of tracheal branches, tube area, and skeleton length in Yki flies with ISC-specific knockdown of LpR1 and LpR2 . (L) pH3+ cell counts in ISCs of Yki flies with ISC-specific knockdown of LpR1 and LpR2 . (M-O) Ejects of trachea-specific knockdown of LpR1 and LpR2 using the Btl-Gal4 driver on trachea parameters, including number of branches, tube area, and skeleton length, compared to control flies ( Btl>ctrl ). N = 8-12 flies. Dashed lines enclose oenocytes in all relevant images. Data are presented as mean ± s.e.m.; statistical analysis was performed using Student’s t-test or ANOVA as appropriate.

Article Snippet: The following primary antibodies were used: rabbit-anti-pH3 (1:500, Cell Signaling, 9701), rabbit-anti-p4E-BP (1:800, Cell Signaling, 2855S).

Techniques: Control, Knockdown, Over Expression

Journal: bioRxiv

Article Title: Lipid metabolism of hepatocyte-like cells supports intestinal tumor growth by promoting tracheogenesis

doi: 10.1101/2025.04.04.647255

Figure Lengend Snippet: (A-C) Trachea parameters, including number of branches, tube area, and skeleton length, in flies with ISC-specific knockdown of LpR1 and LpR2 using Esg driver, compared to control ( Esg>ctrl ). N = 7-11 flies. (D) Counts of pH3+ cells in the midgut of flies with ISC-specific knockdown of LpR1 and LpR2 , compared to controls. (E) Counts of pH3+ cells in the midgut of flies with trachea-specific knockdown of LpR1 and LpR2 , compared to controls. N=8-10 flies. Data are presented as mean ± s.e.m.; statistical analysis was performed using Student’s t-test or ANOVA as appropriate.

Article Snippet: The following primary antibodies were used: rabbit-anti-pH3 (1:500, Cell Signaling, 9701), rabbit-anti-p4E-BP (1:800, Cell Signaling, 2855S).

Techniques: Knockdown, Control