Journal: eLife

Article Title: Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells

doi: 10.7554/eLife.61630

Figure Lengend Snippet: ( A ) Heat map showing k-means Pearson correlation clustering of TMM-normalized RNAseq data of FLCN pos versus FLCN neg RPTECs. We analyzed published TFEB/TFE3 target genes. Yellow boxed cluster three shows the subset (n = 115) of TFEB/TFE3 targets upregulated in all three FLCN NEG clones. ( B ) Upregulation of TFE target genes FNIP2, GPNMB, RRAGD, SQSTM1, RRAGC, GABARAP, ARHGAP12, AMDHD2, and WIPI1 in FLCN NEG RPTECs. Results of three independent experiments with three technical replicates. To determine quantitative gene expression levels, data were normalized to the geometric mean of two housekeeping genes. See for raw qRT-PCR values and fold change calculations. ( C ) Western blots of RPTEC/TERT1 tet-on Cas9 cell lines. All FLCN NEG clones show strong induction of protein expression of TFE targets GPNMB, RRAGD, SQSTM1, and FNIP2. GAPDH and Actin were used as loading controls. Western blots were performed three times. ( D ) Knock down of TFE3/TFEB (10 nM siRNA, 72 hr) ameliorates the TFE expression gene signature induced by FLCN loss in three FLCN NEG clones. Expression levels were determined by qRT-PCR, normalized to siNT-treated clones and are representative of three independent experiments. To determine quantitative gene expression data levels were normalized to the geometric mean of two housekeeping genes. Also see . Effects of siTFE3 alone are shown in . See for raw qRT-PCR values and fold change calculations. Figure 3—source data 1. Raw qRT-PCR values and fold change calculations belonging to and .

Article Snippet: For western blot experiments following antibodies were used according to individual datasheets: Vinculin (H-10, sc-25336), FLCN (D14G9, CST 3697S), Cas9 (epigentek A-9000–050), AQP1 (sc-25287), GPNMB (AF2550-SP), SQSTM1 (CST 88588), RRAGD (CST_ 4470S), FNIP1 (ab134969) FNIP2 (HPA042779), STAT2 (GTX103117), pSTAT1 Y701(7649S), TFE3 (HPA023881), H3 (9715S), Tubulin (B-5-1-2, SC-23948), p70S6Kinase T389 (CST 9205), pAKT S473 D9E (CST 4060), total p70S6K (49D7 CST 2708), panAKT 40D4 (CST 2920), 4E-BP1 53H11 (CST 9644), GAPDH (sc-47724) and (MAB374; Merck Millipore).

Techniques: Clone Assay, Gene Expression, Quantitative RT-PCR, Western Blot, Expressing, Knockdown

Journal: eLife

Article Title: Loss of FLCN-FNIP1/2 induces a non-canonical interferon response in human renal tubular epithelial cells

doi: 10.7554/eLife.61630

Figure Lengend Snippet:

Article Snippet: For western blot experiments following antibodies were used according to individual datasheets: Vinculin (H-10, sc-25336), FLCN (D14G9, CST 3697S), Cas9 (epigentek A-9000–050), AQP1 (sc-25287), GPNMB (AF2550-SP), SQSTM1 (CST 88588), RRAGD (CST_ 4470S), FNIP1 (ab134969) FNIP2 (HPA042779), STAT2 (GTX103117), pSTAT1 Y701(7649S), TFE3 (HPA023881), H3 (9715S), Tubulin (B-5-1-2, SC-23948), p70S6Kinase T389 (CST 9205), pAKT S473 D9E (CST 4060), total p70S6K (49D7 CST 2708), panAKT 40D4 (CST 2920), 4E-BP1 53H11 (CST 9644), GAPDH (sc-47724) and (MAB374; Merck Millipore).

Techniques: Derivative Assay, Knock-Out, CRISPR, Mutagenesis, Knockdown, Control, Transfection, Construct, Over Expression, Plasmid Preparation, Sequencing, Modification, Mass Spectrometry, Immunofluorescence, Expressing, Isolation, cDNA Synthesis, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Software, Microscopy

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 5 IL4 induces RRAGD expression and combined IL4 treatment and BCR crosslinking hyperactivates mTOR signaling. A–C Lymphoma cell line pools transduced with lentiviral CRISPR-Cas9 guide pools targeting AAVS (control) or RRAGD, were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. D Non-malignant normal human LN derived B cells were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were treated ex vivo with IL4 for 0–6 h (time course). Cell lysates were made and prepared for immunoblotting using the indicated epitopes. E, F Non-malignant normal human LN derived B cells (N = 4) were purified via Miltenyi columns and depletion of CD3 T cells. Purified B cells were left untreated or treated ex vivo with IL4, anti-IgM or both as indicated. Cell lysates were prepared for immunoblotting using the indicated epitopes. G densitometry results for p-p70- S6K and RRAGD normalized to actin for all conditions for four primary human LN-derived B lymphocyte samples (# 111, 89, 94, and 95) combined. Left panel: One-way Anova with post hoc Tukey’s test. Right panel: paired nonparametric Wilcoxon test. *p < 0.05, ***p < 0.001. IL4 interleukin 4, BCR B cell receptor, AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, CRISPR, Control, Ex Vivo, Western Blot, Derivative Assay, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 6 RRAGD is required for S6 Kinase phosphorylation in lymphoma. A The RRAGD gene or AAVS (control) was targeted with lentivirus carrying pooled CRISPR-Cas9 guides in four lymphoma cell lines and following puromycin selection the pools were analyzed for RRAGD protein expression by immunoblotting. B, C RRAGD −/−or AAVS targeted lymphoma cell line pools were treated with anti-IgM or anti-IgG for 10’, cell lysates were made, and protein prepared for immunoblotting using the indicated epitopes. Densitometry data for mean p-p70- S6K:HSP90 for AAVS control cells are shown, while signals for RRAGD −/−cells were close to background. One-Way ANOVA with post hoc Tukey’s test. Densitometry based on three cell lines and N = 2 repeats *p < 0.05, **p < 0.01. D–F RRAGD −/−or AAVS targeted lymphoma cell line pools were treated +/−IL4 for 6 h and +/−anti-IgM for 10’, cell lysates were made and protein prepared for immunoblotting using the indicated epitopes. D A representative blot for OCI-LY7. E Densitometry of mean p-4E-BP1:total 4E-BP1 for AAVS control cells +/−IL4 for 6 h and +/−anti-IgM/G. One-Way ANOVA with post hoc Dunn’s test. F Densitometry of comparative data for AAVS and RRAGD −/−cells; Mann–Whitney test. Densitometry based on three cell lines and N = 2 repeats. ns, not significant, *p < 0.05, **p < 0.01. AAVS adeno-associated virus integration site, CRISPR-Cas9 clustered regularly interspaced short palindromic repeats CRISPR associated protein 9, IgM immunoglobulin M, IgG immunoglobulin G, IL4 interleukin 4, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Phospho-proteomics, Control, CRISPR, Selection, Expressing, Western Blot, MANN-WHITNEY, Virus

Journal: Leukemia

Article Title: STAT6 mutations compensate for CREBBP mutations and hyperactivate IL4/STAT6/RRAGD/mTOR signaling in follicular lymphoma.

doi: 10.1038/s41375-025-02525-6

Figure Lengend Snippet: Fig. 7 STAT6 mutations hyperinduce RRAGD expression and IL4 and BCR induced mTOR signaling. Three lymphoma cell lines were lentivirally transduced with cDNA/ORFs for WT or MUT STAT6 and cells sorted via GFP fluorescence. Cells were stimulated with IL4 and BCR crosslinking as indicated or left untreated and detergent lysates prepared for immunoblotting using the indicated epitopes. A–C Representative immunoblotting results for N = 2 independent experiments per cell pool. D Results from densitometry for phospho-S6K normalized to HSP90 for indicated conditions across three cell lines (N = 6). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05. E Results from densitometry for RRAGD normalized to HSP90 for indicated conditions for N = 2 independent experiments across three cell lines (N = 6 measurements). Two-way ANOVA with post hoc Holm Šidák test *p < 0.05; **p < 0.01. STAT6 Signal Transducer and Activator of Transcription 6, IL4 interleukin 4, WT wild type, MUT mutated, GFP green fluorescence protein, cDNA complementary DNA, ORFs open reading frames, IL4 interleukin 4, BCR B cell receptor, ANOVA analysis of variance.

Article Snippet: Cell lysates from parental 293 T cells or transfected with a RRAGD cDNA (Addgene, # 19316) and Hela cells treated with and without insulin were used as blotting and epitope controls.

Techniques: Expressing, Transduction, Western Blot