Supplementary MaterialsS1 Fig: is definitely ubiquitously expressed in planarians and RNAi produces overgrowths. for 3 weeks ( 5). Images correspond to confocal Z-projections. (B) Quantification of caspase-3 activity after 1, 2, and 3 weeks of inhibition. Results are presented as units of caspase-3 activity per g of protein. Bars correspond to the mean of 3 biological replicates. Error bars represent standard deviation. (C) Immunostaining with anti-H3P antibody in planarians subjected to RNAi for 3 weeks ( 10). (D) Graph showing the total cell number in planarians subjected to RNAi for 3 weeks, as determined using a Neubauer chamber. Bars correspond to the mean of 3 biological replicates. Error bars represent standard deviation. Data were analyzed by Student test. ** 0.01; *** 0.001. Data used in the generation of this physique can be found in S1 Data. Scale bars: 250 m (A); 1 mm (B). n.s., not significant; RNAi, RNA interference.(TIF) pbio.2002399.s002.tif (557K) GUID:?D1E05785-FA3A-439A-A025-C3001CEE11D2 S3 Fig: is essential for G2/M transition and M exit in planarians. (A) Cartoon illustrating the EdU pulse procedure. Animals were starved for 1 week, injected with dsRNA for 3 weeks, and then injected with EdU and fixed 16 h later. (B) EdU labeling in transverse sections combined with immunostaining with anti-H3P antibody in the pharynx region IL1RA in controls and in planarians subjected to RNAi for 3 weeks. Scale bars: 50 m. dsRNA, double-stranded RNA; EdU, 5-ethynyl-2-deoxyuridine; H3P, phospho-histone-H3-Ser10; RNAi, RNA interference.(TIF) pbio.2002399.s003.tif (500K) GUID:?F4949D63-ED69-4B7B-9254-7B71389521B5 S4 Ciwujianoside-B Fig: Cellular and molecular analysis of overgrowths and unpigmented regions in animals. (A) Analysis of overgrowths. FISH combined with immunostaining showing the localization of mRNA and SMEDWI-1 protein. Colocalization of both signals appears to be concentrated in the overgrowths, indicating that they consist of undifferentiated cells. Arrowhead indicates an epidermal cell of an overgrowth stained with SMEDWI-1. (B) Analysis of unpigmented regions. Immunostaining using different markers. From left to right: staining of the epithelia with anti-anti-Bcat2 antibody; digestive system labeled with anti-Bcat2 antibody (white arrows indicate gut branches); pharynx labeled with anti-Bcat2 antibody; head region stained with anti-synapsin, anti-H3P, and anti-Bcat2 antibodies (arrowheads indicate mitotic cells); sagittal section showing a head region stained with anti-H3P (arrowheads indicate mitotic cells; discontinuous line delimits the brain); visual system stained with anti-arrestin (VC-1). Blue corresponds to nuclei stained with DAPI. All experiments were performed in planarians subjected to RNAi for 3 weeks. All images correspond to confocal Z-projections. Scale bars: 100 m; 200 m (A); 100 m; 250 m; 150 m; 250 m; 150 m; 100 m (B). Bcat2, -catenin-2; Br, brain; FISH, fluorescent in situ hybridization; H3P, phospho-histone-H3-Ser10; RNAi, RNA interference.(TIF) pbio.2002399.s004.tif (2.5M) GUID:?B266CE07-319E-43C9-A9F2-67972C970A1D S5 Fig: Inhibition of increases in vivo PI incorporation. Staining of dead cells using PI in live control and animals. Nuclei are stained with Hoechst. Magnifications of the indicated area are shown below. Arrowhead indicates some cells positive for PI. A stereomicroscopic view of live control and animals used in the experiment is usually shown. Quantification of the PI+ cells Ciwujianoside-B per nuclei area in the head region is usually shown. Data were analyzed by Student test (= 4). *** 0.001. Data used in the generation of this physique are available in S1 Data. Size pubs: 100 m (best pictures); 25 m (bottom level pictures). PI, propidium iodide; RNAi, RNA disturbance.(TIF) pbio.2002399.s005.tif Ciwujianoside-B (1.2M) GUID:?DDCB3E12-C998-4374-8328-7756CBEC85E1 S6 Fig: A sign regulates cell differentiation during planarian regeneration. (A) Cartoon illustrating the RNAi treatment in regenerating circumstances. Pets had been starved for a week prior to the test and injected on 3 consecutive times. The following week, animals were injected again on 3 consecutive days, cut the next day, and fixed at different time Ciwujianoside-B points. (B) Anti-synapsin immunostaining of Ciwujianoside-B the brain region of control and animals. Nuclei are stained with DAPI. Images correspond to planarians after 12 dR. (C) In situ hybridization with (digestive system) in and control animals. Images correspond to planarians after 10 dR. (D) Fluorescent in.

Supplementary MaterialsS1 Fig: is definitely ubiquitously expressed in planarians and RNAi produces overgrowths