Journal: Cancer Gene Therapy

Article Title: Rapamycin rescues APC-mutated colon organoid differentiation

doi: 10.1038/s41417-025-00935-3

Figure Lengend Snippet: Representative images of immunohistochemical staining of the intestinal epithelial markers CDX2, Keratin20 (CK20), Muc2, the proliferation marker Ki67, and the mesenchymal marker VIM, for FAP1 and FAP2 colon organoids at day 45 following rapamycin treatment (R). Non-treated cells served as a negative control, and normal colon tissue served as a positive control. Arrows point for Muc2 staining. Scale bars 200 µM. VIM vimentin, R Rapamycin.

Article Snippet: Confocal microscopy images of organoids and differentiated cell samples were obtained using a Zeiss LSM700 confocal microscope.Organoids were fixed in 4% PFA and stained for the colon markers CDX2 (235R-16, Cell Marque, 1:100), Keratin 20 (320M-16, Cell Marque, 1:100), and MUC2 (PA0155, BOND, 1:100), the stromal marker Vimentin (Dako, M0725, 1:100), KI67 (275R-16, Cell Marque, 1:200) a nuclear marker indicative of cellular proliferation, cMyc (395R-16, Cell Marque, 1:50) as an oncogenic marker and PTEN (31-1147-00, RevMab, 1:500) as mTOR pathway inhibitor.

Techniques: Immunohistochemical staining, Staining, Marker, Negative Control, Positive Control

Journal: Cancer Gene Therapy

Article Title: Rapamycin rescues APC-mutated colon organoid differentiation

doi: 10.1038/s41417-025-00935-3

Figure Lengend Snippet: A The schematic diagram for mTOR signaling pathway. In response to growth factors, PI3K is activated and phosphorylates AKT, which inactivates the tuberous sclerosis (TSC) tumor suppressor protein, the RAS homolog enriched in brain (Rheb) small G protein which activates mTOR. Activated mTOR phosphorylates S6K1 kinase which activates translation, cell growth, and proliferation. In parallel, phosphorylation and inactivation of 4E-BP1 activates eIF4E which leads to protein synthesis, cell growth, and proliferation . B Immunohistochemical staining of phospho-mTOR (p-mTOR) in normal colon tissue, and day 45 organoids derived from isogenic control FAP1, FAP1, and FAP2. Normal colon tissue served as a control for protein expression within the normal environment. This experiment was repeated twice, and representative pictures are shown. p-mTOR expression quantification utilized the open-source image analysis software Ilastik (version 1.4.1rc2), **** P < 0.0001, t test. C Immunohistochemical staining of the tumor suppressor protein PTEN in day 45 organoids derived from isogenic FAP1, FAP1, FAP2, and normal colon tissue served as a control. X10- Scale bars- 100 µm, X20- Scale bars- 200 µm.

Article Snippet: Confocal microscopy images of organoids and differentiated cell samples were obtained using a Zeiss LSM700 confocal microscope.Organoids were fixed in 4% PFA and stained for the colon markers CDX2 (235R-16, Cell Marque, 1:100), Keratin 20 (320M-16, Cell Marque, 1:100), and MUC2 (PA0155, BOND, 1:100), the stromal marker Vimentin (Dako, M0725, 1:100), KI67 (275R-16, Cell Marque, 1:200) a nuclear marker indicative of cellular proliferation, cMyc (395R-16, Cell Marque, 1:50) as an oncogenic marker and PTEN (31-1147-00, RevMab, 1:500) as mTOR pathway inhibitor.

Techniques: Phospho-proteomics, Immunohistochemical staining, Staining, Derivative Assay, Control, Expressing, Software