Journal: The American Journal of Pathology
Article Title: Folliculin Deletion in the Mouse Kidney Results in Cystogenesis of the Loops of Henle via Aberrant TFEB Activation
doi: 10.1016/j.ajpath.2025.05.010
Figure Lengend Snippet: Transcription factor E3 (TFE3) and mammalian target of rapamycin complex 1 (mTORC1) are activated in Flcn knockout (KO) nephrons. A–D: In situ hybridization of Atp6v0d2 and Rab27a in Flcn flox/flox and KO kidneys. Slc12a1 is used as a marker for loop of Henle (LoH). The right panels are higher magnification of the boxed regions in the left panels . E–H: Immunofluorescence staining for TFE3, SLC12A1 (LoH), and LTL (proximal tubule) showing TFE3 nuclear reactivity in the cortex ( E and F ) but not in the medulla ( G and H ). Nuclear localization of TFE3 is detected in glomerular podocytes, Bowman's capsule ( white arrowheads ), and proximal tubules ( yellow arrowheads ) in Flcn KOs but not in LoH. The right panels are higher magnification of the boxed regions in the left panels . I–L: Immunofluorescence staining of phosphorylated S6 ribosomal protein (pS6), SLC12A1 (LoH), and LTL (proximal tubule) showing the increased pS6 in the LoH ( yellow arrows ) of Flcn KO kidneys. The right panels are higher magnification of the boxed regions in the left panels . Scale bars: 500 μm ( A–D ); 100 μm ( E–L ).
Article Snippet: The following primary antibodies were used: rabbit anti-SIX2 (#11562-1-AP; 1:400 dilution; Proteintech, Rosemont, IL), rat anti-KRT8 (1:10; Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti–E-cadherin (CDH1) (#610181; 1:100; BD, Franklin Lakes, NJ); rabbit anti-NKCC2 (SLC12A1) (#SPC-401D; 1:100; StressMarq Biosciences, Victoria, BC, Canada), goat anti-NKCC2 (SLC12A1) (#PAS-142445; 1:100; Invitrogen, Carlsbad, CA), rabbit anti-phospho-S6 ribosomal protein (Ser 240/244) (#5364; 1:100; Cell Signaling Technology, Danvers, MA), rabbit anti-WT1 (#GR 3270281-4; 1:100; Abcam, Cambridge, UK,), LTL (#B-132; 1:200; Vector Laboratories, Newark, CA), guinea pig anti-NEPHRIN (#GP-N2, 1:200; PROGEN, Heidelberg, Germany), rabbit anti-TFE3 (#HPA023881; 1:100; Sigma-Aldrich, St. Louis, MN), and rabbit anti-TFEB (#A303-673A, lot numbers 8 and 9; Bethyl Laboratories, Montgomery, TX).
Techniques: Knock-Out, In Situ Hybridization, Marker, Immunofluorescence, Staining