Fig 1: Salr inhibits cell growth and lipid storage cell autonomously in the fat body.(A) Representative image of early third instar larvae overexpressing Salr in the fat body. (B) Fat body-specific Salr overexpression leads to a delayed pupariation, dAEL: days after egg laying. (C) Salr inhibits cell growth autonomously. Representative image of Salr overexpressing clones (Tubulin flipout) marked with GFP and YFP. Quantification of relative nuclear and nucleolar sizes. (D) SALL1 overexpression in HEPG2 cells reduces cell proliferation. Quantification of sulforhodamine B (SRB) assay on days 3 and 5 after seeding. (E) Fat body-specific CRISPR/Cas12a knockout of Salr leads to increased pupal volume. (F) Fat body-specific CRISPR/Cas12a knockout of Salr leads to increased nuclear and nucleolar sizes. Representative image of Salr knockout and control nuclei stained with DAPI. Quantification of nuclear and nucleolar sizes. (G) Salr overexpression decreases protein synthesis. Representative image of GFP and YFP marked Salr overexpressing clones (Tubulin flipout) stained with DAPI and OPP. Quantification of OPP intensity in Salr overexpressing and control cells. (H) Salr CRISPR/Cas12a knockout in the fat body increases protein synthesis. Representative image of Salr knockout cells stained with DAPI and OPP. Quantification of OPP intensity of Salr knockout and control cells. (I) Salr overexpression decreases lipid droplet volume in fat body cells. Representative image of GFP and YFP marked Salr overexpressing clone (Tubulin flipout) stained with DAPI and LipidTox (above). 3D model of lipid droplets inside the white line (below). Lipid droplets of Salr overexpressing cell highlighted (orange). Quantification of the total lipid volume to cell volume ratio in Salr overexpressing cells. (J) Salr CRISPR/Cas12a knockout increases lipid droplet volume in fat body cells. Representative image of Salr knockout cells stained with DAPI, LipidTox, and Phalloidin. Quantification of lipid droplet volume to cell volume ratio. All experiments were replicated at least twice. P value and chi-square in (B) were calculated by log-rank test for trend. P values in (C–J) were calculated with unpaired t test with Welch correction. N = biological replicates, n = technical replicates. In box plots, the center line represents the median, the box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minimum and maximum observed values. Error bars display standard deviations (SD).
Fig 2: Salr shares common targets with FoxO, related to Fig. 4.(A) Called peaks and track coverage of Salr and FoxO ChIP-Seq on bmm. (B) Called peaks and track coverage of Salr and FoxO ChIP-Seq on Atg17. (C) FoxO positively regulates Sesn. qPCR of Sesn mRNA in FoxO gain- and loss-of-function fat bodies. Foxo-i 097: VDRC 106097, Foxo-i 427: BDSC 32427. (D) Called peaks and track coverage of SALL1 and FOXO1 ChIP-Seq on SESN3 (one of the human homologs of Drosophila Sesn). (E) Called peaks and track coverage of SALL1 and FOXO1 ChIP-Seq on RB1CC1 (human ortholog of Atg17). (F) InR knockdown does not activate Salr. Quantification of Salr-GFP intensity of fat body-specific InR knockdown and control. P values in (C) (left panel) and (F) were calculated with unpaired t test with Welch correction. P values in (C) (right panel) were obtained by one-way ANOVA followed by Tukey’s test with multiple comparison correction. N = biological replicates, n = technical replicates. In box plots, the center line represents the median, the box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minimum and maximum observed values. Error bars display standard deviations (SD).
Fig 3: Salr inhibits cell growth in the fat body, related to Fig. 1.(A) Salr regulates cell size in the fat body. Representative images of volume quantification of Salr overexpressing clones (Tubulin flipout) and control cells. Quantification of cell volume upon gain- and loss-of-function of Salr in the fat body. (B) Representative images of anti-fibrillarin staining of Salr CRISPR/Cas12a knockout and control fat body cells. Fibrillarin localizes to the nucleolus and colocalizes with the DAPI-deficient area. (C) qPCR analysis of SALL1 mRNA levels from SALL1 stably transfected HepG2 cells. (D) Salr CRISPR/Cas12a target sites on wild-type Salr protein. (E) Confirmation of Salr CRISPR/Cas12a knockout using GFP-tagged Salr reporter. (F) Fat body-specific Salr CRISPR/Cas12a knockout does not lead to accelerated pupariation. (G) Salr knockdown with Dicer2 increases nucleus size. Representative image of fat body cells that are Salr knockdown with Dicer2, control, and Dicer2, stained with DAPI. Quantification of nucleus size. P values in (A–C, G) were calculated with unpaired t test with Welch correction. P values in (E) were obtained by one-way ANOVA followed by Tukey’s test with multiple comparison correction. P value and chi-square in (F) were calculated by the log-rank test for trend. N = biological replicates, n = technical replicates. In box plots, the center line represents the median, the box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minimum and maximum observed values. Error bars display standard deviations (SD).
Fig 4: Salr shares common targets with and antagonizes FoxO.(A–D) Salr and FoxO share common direct targets in Drosophila and humans. (A) Venn diagram of direct targets of FoxO and Salr. (B) Called peaks and coverage tracks of Salr and FoxO ChIP-Seq on Atg2, Sesn, and PRAS40 loci (highlighted in red). (C) Venn diagram of direct targets of human SALL1 and FOXO1. (D) Called peaks and coverage tracks of human SALL1 and FOXO1 on ATG2A, SESN1, and AKT1S1 (PRAS40 ortholog) loci (highlighted in red). (E, F) Salr antagonizes the nuclear localization of FoxO. (E) Representative image of fat body-specific Salr CRISPR/Cas12a knockout and control fat body cells stained with anti-FoxO antibodies. Quantification of the nuclear versus cytoplasmic FoxO ratio. (F) Representative image of Salr overexpressing clone stained with anti-FoxO antibodies. Quantification of the nuclear versus cytoplasmic FoxO ratio. All experiments were replicated at least twice. P values in (E, F) were calculated with unpaired t test with Welch correction. N = biological replicates, n = technical replicates. In box plots, the center line represents the median, the box boundaries represent the 25th and 75th percentiles, and the whiskers represent the minimum and maximum observed values. Error bars display standard deviations (SD).
Supplier Page from Sino Biological, Inc. for Human SALL1 Gene ORF cDNA clone expression plasmid