Journal: bioRxiv

Article Title: Axial Nephron Fate Switching Demonstrates a Plastic System Tunable on Demand

doi: 10.1101/2025.03.29.646044

Figure Lengend Snippet: (a) Annotated unsupervised clustering of PAX2 + nephron-like DD14 control and DE organoid cells represented in a UMAP. (Inset) Sample contribution of each condition. (b) Violin plot with annotations of select gene markers (top x axis) showing expression levels (bottom x axis) in the DD14 organoid dataset. The y axis shows cluster numbers and sample origin (gray square – control organoids, pink square – DE organoids). (c) Volcano plot of DESeq analysis comparing DD14 DMSO control and DE organoid nephron scRNA sequencing data. Dashed lines show 0.05 p-value cutoff (horizontal) and ±1.5-fold change cutoff (vertical). (d) Bar plot showing normalized percent contribution of cells expressing select nephron markers (log 2 FC > 0) separated by sample origin. Numbers in bars show raw cell counts. (e) Feature plots of select genes in the DD14 DE organoid dataset. (f) Immunofluorescence stain of week 16 human kidney from SSB-CLSN stage marking DCT/CNT precursor and loop of Henle/macula densa precursor segments. (g) Immunofluorescence stains of DD14 control and DE organoids showing expression of DCT/CNT precursor and loop of Henle/macula densa precursor markers. Yellow arrow – autofluorescence. Scale bar: 50 microns.

Article Snippet: The primary antibodies used were as follows: Laminin β1 (Santa Cruz Biotechnology, sc-33709, 1:250), HNF4A (R&D Systems, MAB4605, 1:200), POU3F3 (Thermo Scientific, PA564311, 1:100), Mafb (Novus (R&D), MAB3810, 1:500), GATA3 (Novus (R&D), AF2605, 1:200), Jag1 (Novus (R&D), AF599, 1:100), SLC12A1 (Sigma-Aldrich, HPA018107, 1:200), PAX8 (abcam, ab189249, 1:100), WT1 (abcam, ab89901, 1:500), LEF1 (Cell signaling, 2286S, 1:200), CDH1 (BD Laboratories, 610181, 1:200), PAPPA2 (R&D Systems, AF1668, 1:100), HNF1B (Thermo Scientific, MA5-24605, 1:500), Pax2 (Thermo Scientific, 716000, 1:100), TFAP2A (Santa Cruz Biotechnology, sc-12726, 1:200), IRX1 (Sigma-Aldrich, HPA043160, 1:200), SIX1 (Cell signaling, 12891S, 1:300), Pax2 (R&D Systems, AF3364, 1:50), TMEM52B (Novus (R&D), NBP2-49272, 1:100), MEIS1/2/3 (Active Motif, 39096, 1:1000), ZO-1 (Thermo Scientific, 33-9100, 1:200), PODXL (R&D Systems, AF1658, 1:300), Renin (R&D Systems, AF4090, 1:100), MECOM (R&D Systems, MAB75061, 1:100), hErbB4 (R&D Systems, MAB1131, 1:100), LEF1 (Santa Cruz Biotechnology, sc-374412, 1:200), CD31 (abcam, ab9498, 1:250), ESRRγ (Sigma-Aldrich, HPA044678, 1:100), GATA3 (Cell Signaling Technology, 5852T, 1:100), and acetyl-alpha tubulin (Sigma-Aldrich, MABT868, 1:500).

Techniques: Control, Expressing, Sequencing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

doi: 10.1101/2024.11.21.624702

Figure Lengend Snippet: (A) Dual IHC for PAX8 (brown) and GPNMB (teal) in tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 2 weeks following injection of tamoxifen. Scale bar = 100 µm. (B) H&E (top row) and dual PAX8/GPNMB IHC staining (bottom row) of kidneys from representative tamoxifen-injected, SFPQ-TFE3 LSL ; Pax8-CreERT transgenic mice and age-matched, littermate controls, at 3.5 months following injection of tamoxifen. Scale bar= 100 µm. (C) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and SFPQ-TFE3 LSL ; Ksp-Cre transgenic mice at post-natal day 15. Scale bar = 100 µm. (D) H&E (top row), dual PAX8/GPNMB IHC staining (middle row) and PAX2 IHC staining (bottom row) of kidneys from representative age-matched, control and PRCC-TFE3 LSL ; Ksp-Cre transgenic mice at 7 to 9 months. (E) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at day 1 and day 15 (Fix Figure), from experiments in C , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=11 (day 1) and n=9 (day 15). Statistical analyses were performed using two-tailed Mann-Whitney test. (F) Digital quantification of mean nuclear PAX8 H-scores in GPNMB-cells (blue) and GPNMB+ cells (red) at 4 months and 7 months, from experiments in D , as depicted by scatter plots, with the central line denoting the median. The number of biological replicates analyzed for both, GPNMB – and + cells were n=9 at 4 and 7 months. Statistical analyses were performed using two-tailed Mann-Whitney test. (G) Gene Set Enrichment Analysis (GSEA) comparing 15-day STK (top panels), 3.5-month tamoxifen-treated, STP (middle panels) and 7-month PTK (bottom panels), transgenic kidneys and their controls, for genes differentially expressed in renal TSC-related PEComas from . (H) Immunoblotting of lysates from primary renal tubular epithelial cells from SFPQ-TFE3 LSL transgenic mice treated with control or Cre-recombinase expressing adenovirus in vitro for the indicated markers. Source data are provided as a Source data file.

Article Snippet: Primary antibodies: 4E-BP1 (#9644 CST; 1:2000), CTSK (#ab19027 Abcam; 1:1000), FLCN (#3697 CST; 1:1000), GAPDH (#5174 CST; 1:1000), GATA3(#5852 CST; 1:500), GPNMB (#38313 CST; 1:1000), GPNMB (Mouse specific) (#90205 CST; 1:1000), Histone-3 (#4499 CST; 1:4000), HNF4A (#3113 CST; 1:500), Keratin 8 (#ab53280 Abcam; 1:1000), LC3 A, B (#12741 CST; 1:2000), MelanA (#ab210546 Abcam; 1:1000), PAX2 (#9666 CST; 1:500), PAX8 (#59019 CST; 1:500), PMEL (#ab137078 Abcam; 1:1000), Phospho-p70 S6 Kinase (Thr389) (#9205 CST; 1:1000), p70 S6 Kinase (#9202 CST; 1:1000), Phospho-S6 Ribosomal Protein (Ser235/236) (#4858 CST; 1:2000), Phospho-4E BP1 (Ser65) (#9451 CST; 1:2000), Phospho-4E BP1 (Thr37/46) (#2855 CST; 1:1000), p-TFEB(S122) (#86843 CST; 1:2000), p-TFEB(S211) (#37681 CST; 1:2000), Pan-Keratin (Type1) (#83957 CST; 1:1000), RAB7 (#9367 CST; 1:1000), RHEB (#13879 CST; 1:1000), RRAGC (#9480 CST; 1:1000), RRAGD (#4470 CST; 1:1000), Synaptophysin (#5461 CST; 1:1000), S6 Ribosomal Protein (#2317 CST; 1:2000), TFE3 (#14779 CST; 1:4000), TFE3 (#ABE1400 Sigma; 1:4000), TFEB (#4240 CST; 1:2000), WT1 (#83535 CST; 1:1000).

Techniques: Injection, Transgenic Assay, Immunohistochemistry, Control, Two Tailed Test, MANN-WHITNEY, Western Blot, Expressing, In Vitro

Journal: bioRxiv

Article Title: SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

doi: 10.1101/2024.11.21.624702

Figure Lengend Snippet: (A) Immunoblotting of HK2 proximal tubular epithelial cells with doxycycline-inducible expression of WT-TFE3 and TFE3 fusion-proteins, for the indicated markers. Cells were treated with the indicated doses of doxycycline for 72 hrs, prior to lysis and immunoblotting. (B) Immunoblotting of cytosolic and nuclear fractions of HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 for the indicated markers. (C) Indirect immunofluorescence for PAX8 in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3. (D) Quantitative real time PCR (qRT-PCR) for PMEL and GPNMB in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 (n>3, error bars represent SEM; p values by Student’s T-test). (E) Quantitative real time PCR (qRT-PCR) for PAX2, PAX8, HNF1B, LHX1 and GATA3 in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 (n>3, error bars represent SEM; p values by Student’s T-test). (F) Immunoblotting of HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 with CRISPR-Cas9-mediated genomic inactivation of TFE3, for TFE3 and PAX8. Two clones representing two unique guide RNAs targeting TFE3 are shown. (G) Immunoblotting of lysates from TFE3 -fusion RCC cell lines [UOK145(SFPQ-TFE3), UOK120,124,146 (PRCC-TFE3) and UOK109(NONO-TFE3)], and ccRCC controls (UOK111, UOK140 and UOK150) for PAX8 and PAX2. (H) Normalized densitometric quantifications for PAX8 and PAX2 immunoblots, from experiments in G (n=4, error bars represent SEM; p values by one-way ANOVA). (I) Comparison of PAX8 and PAX2 gene expression in TFE3 fusion-RCC (n=8) and TFEB -amplified RCC (n=4) to the remainder of papillary RCC cases without underlying TFE3 fusions or TFEB amplifications (n=273) in the papillary RCC (KIRP) cohort from TCGA. Data are represented as a box-and-whisker plot. ( P -values indicated are by Wilcoxon rank sum test adjusted with multiple comparisons using the false discovery rate (FDR) method). Source data are provided as a Source data file.

Article Snippet: Primary antibodies: 4E-BP1 (#9644 CST; 1:2000), CTSK (#ab19027 Abcam; 1:1000), FLCN (#3697 CST; 1:1000), GAPDH (#5174 CST; 1:1000), GATA3(#5852 CST; 1:500), GPNMB (#38313 CST; 1:1000), GPNMB (Mouse specific) (#90205 CST; 1:1000), Histone-3 (#4499 CST; 1:4000), HNF4A (#3113 CST; 1:500), Keratin 8 (#ab53280 Abcam; 1:1000), LC3 A, B (#12741 CST; 1:2000), MelanA (#ab210546 Abcam; 1:1000), PAX2 (#9666 CST; 1:500), PAX8 (#59019 CST; 1:500), PMEL (#ab137078 Abcam; 1:1000), Phospho-p70 S6 Kinase (Thr389) (#9205 CST; 1:1000), p70 S6 Kinase (#9202 CST; 1:1000), Phospho-S6 Ribosomal Protein (Ser235/236) (#4858 CST; 1:2000), Phospho-4E BP1 (Ser65) (#9451 CST; 1:2000), Phospho-4E BP1 (Thr37/46) (#2855 CST; 1:1000), p-TFEB(S122) (#86843 CST; 1:2000), p-TFEB(S211) (#37681 CST; 1:2000), Pan-Keratin (Type1) (#83957 CST; 1:1000), RAB7 (#9367 CST; 1:1000), RHEB (#13879 CST; 1:1000), RRAGC (#9480 CST; 1:1000), RRAGD (#4470 CST; 1:1000), Synaptophysin (#5461 CST; 1:1000), S6 Ribosomal Protein (#2317 CST; 1:2000), TFE3 (#14779 CST; 1:4000), TFE3 (#ABE1400 Sigma; 1:4000), TFEB (#4240 CST; 1:2000), WT1 (#83535 CST; 1:1000).

Techniques: Western Blot, Expressing, Lysis, Immunofluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CRISPR, Clone Assay, Comparison, Amplification, Whisker Assay

Journal: bioRxiv

Article Title: SFPQ-TFE3 gene fusion reciprocally regulates mTORC1 activity and induces lineage plasticity in a novel mouse model of renal tumorigenesis

doi: 10.1101/2024.11.21.624702

Figure Lengend Snippet: (A) Immunoblotting of HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with doses of the mTOR kinase inhibitor torin1, for the indicated antibodies. (B) Densitometry quantification of normalized SFPQ-TFE3 and PAX8 expression, in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 following treatment with torin1, from experiments in (A) (n>3, error bars represent SEM; p values by one-way ANOVA with Dunnett’s test for multiple comparisons). (C) Immunoblotting of HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with Rheb siRNA, for the indicated antibodies. (D) Densitometry quantification of normalized SFPQ-TFE3 and PAX8 expression, in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3 following treatment with Rheb siRNA from experiments in ( C) (n>3, error bars represent SEM; p values by one-way ANOVA with Dunnett’s test for multiple comparisons). (E) Immunoblotting of cytosolic and nuclear fractions of HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with the mTOR kinase inhibitor torin1 for the indicated markers. (F) Indirect immunofluorescence for TFE3 and PAX8 in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with the mTOR kinase inhibitor torin1. (G) Quantitative real time PCR (qRT-PCR) for PMEL and GPNMB in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with the mTOR kinase inhibitor torin1 (n=3, error bars represent SEM; p values by one-way ANOVA with Dunnett’s test for multiple comparisons). (H) Quantitative real time PCR (qRT-PCR) for PAX2, PAX8, HNF1B, LHX1 and GATA3 in HK2 cells with doxycycline-inducible expression of SFPQ-TFE3, following treatment with the mTOR kinase inhibitor torin1 (n=3, error bars represent SEM; p values by one-way ANOVA with Dunnett’s test for multiple comparisons). Source data are provided as a Source data file.

Article Snippet: Primary antibodies: 4E-BP1 (#9644 CST; 1:2000), CTSK (#ab19027 Abcam; 1:1000), FLCN (#3697 CST; 1:1000), GAPDH (#5174 CST; 1:1000), GATA3(#5852 CST; 1:500), GPNMB (#38313 CST; 1:1000), GPNMB (Mouse specific) (#90205 CST; 1:1000), Histone-3 (#4499 CST; 1:4000), HNF4A (#3113 CST; 1:500), Keratin 8 (#ab53280 Abcam; 1:1000), LC3 A, B (#12741 CST; 1:2000), MelanA (#ab210546 Abcam; 1:1000), PAX2 (#9666 CST; 1:500), PAX8 (#59019 CST; 1:500), PMEL (#ab137078 Abcam; 1:1000), Phospho-p70 S6 Kinase (Thr389) (#9205 CST; 1:1000), p70 S6 Kinase (#9202 CST; 1:1000), Phospho-S6 Ribosomal Protein (Ser235/236) (#4858 CST; 1:2000), Phospho-4E BP1 (Ser65) (#9451 CST; 1:2000), Phospho-4E BP1 (Thr37/46) (#2855 CST; 1:1000), p-TFEB(S122) (#86843 CST; 1:2000), p-TFEB(S211) (#37681 CST; 1:2000), Pan-Keratin (Type1) (#83957 CST; 1:1000), RAB7 (#9367 CST; 1:1000), RHEB (#13879 CST; 1:1000), RRAGC (#9480 CST; 1:1000), RRAGD (#4470 CST; 1:1000), Synaptophysin (#5461 CST; 1:1000), S6 Ribosomal Protein (#2317 CST; 1:2000), TFE3 (#14779 CST; 1:4000), TFE3 (#ABE1400 Sigma; 1:4000), TFEB (#4240 CST; 1:2000), WT1 (#83535 CST; 1:1000).

Techniques: Western Blot, Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Quantitative RT-PCR