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: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: FL-associated RRAGC mutations cluster on the protein surface surrounding the GTP/GDP binding site. Upper: Location of identified RRAGC missense mutations in a linear schema of RRAGC. Lower: Location of identified RRAGC missense mutations in the crystal complex (PDB:3LLU) of the RRAGC nucleotide binding domain (G-domain) bound with the nucleotide analog phosphoaminophosphonic acid guanylate ester (GNP), shown in stick representation. Amino acid residues undergoing mutation are labeled and shown in stick representation, while other residues within 4 angstroms of GNP are shown in line representation. A magnesium ion is shown as a sphere. The figure was generated with PyMOL.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Binding Assay, Mutagenesis, Labeling, Generated

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Details of gene mutations in FL cases carrying RRAGC mutations.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: FL-associated RRAGC mutations are intrinsically mTOR activating. Upper: HEK293T cells stably retrovirally infected expressing HA-tagged RRAGC wild type or various RRAGC mutants were grown in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS; +) or alternatively for the last 1 h of culture in RPMI1640 medium that was free of the amino acid leucine and supplemented with dialyzed 10% FBS (−). Cells were harvested and prepared for immunoblotting with various antibodies as indicated. Short and long exposures for RPS6KB/S6K (S6K) and p-Thr389-RPS6KB/S6K (p-S6K) are shown. CHO-IR + INS: Insulin receptor transfected CHO cells stimulated with insulin (immunoblot controls). Measurements of HA-RRAGC expression levels were aided by the slower migration of the HA-tagged RRAGC WT and mutant proteins in SDS-PAGE resulting in a doublet band. Lower: Displayed are combined quantitation results (p-S6K/total S6K) from 3 independent experiments using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Stable Transfection, Infection, Expressing, Western Blot, Transfection, Migration, Mutagenesis, SDS Page, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Measurements of p-Thr389-RPS6KB/S6K levels in RRAGC WT or mutant stably lentivirally transduced lymphoma cell lines. Upper: Lymphoma cell lines stably lentivirally infected with constructs expressing HA-tagged RRAGC wild type or various RRAGC mutants as indicated were grown in RPMI or DMEM medium supplemented with 10% FBS (+) or alternatively for the last 1 h of culture in medium that was free of the amino acid leucine and supplemented with dialyzed 10% FBS (−). Cells were harvested and prepared for immunoblotting with various antibodies as indicated. Short and long exposures for p-Thr389-RPS6KB/S6K are shown. CHO-IR + INS: Insulin receptor transfected CHO cells stimulated with insulin (immunoblot controls). Measurements of HA-RRAGC expression levels were aided by the slower migration of the HA-tagged RRAGC WT and mutant proteins in SDS-PAGE resulting in a doublet band. Lower: Displayed are combined quantitation results (p-S6K/total S6K) from 3 independent experiments per cell line using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Mutagenesis, Stable Transfection, Infection, Construct, Expressing, Western Blot, Transfection, Migration, SDS Page, Quantitation Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: Analysis of TOR activation resulting from Gtr2 mutations in yeast. (A) Alignment of human and yeast RRAGC and Gtr2 proteins, respectively. Mutated residues are boxed. (B) Yeast cells with the PHO8 locus replaced with pho8∆60 were chromosomally-tagged with Gtr2-3HA WT and the indicated mutants. Protein extracts were prepared from cells in growing conditions (0 h) or after 2 h of nitrogen starvation, and assayed for Pho8∆60-dependent phosphatase activity.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Activation Assay, Activity Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Recurrent mutations in the MTOR regulator RRAGC in follicular lymphoma

doi: 10.1158/1078-0432.CCR-16-0609

Figure Lengend Snippet: A RRAGC mutants demonstrate increased HA-RPTOR binding in co-immunoprecipitation studies in transiently transfected HEK293T cells. Upper: HEK293T cell were transiently co-transfected as indicated with expression plasmids coding for HA-RPTOR, RRAGB, RRAGC WT or the indicated RRAGC mutants. CHAPS detergent lysates were prepared and subjected to anti-HA-bead conjugate-mediated immunoprecipitations. Bound protein was eluted and fractioned by SDS-PAGE and prepared for immunoblotting with the indicated antibodies. Leucine starvation (Leu −) was performed in parallel transfections. Lysate: aliquots of lysates prior to immunoprecipitation. Lower: Displayed are combined quantitation results (RRAGC IP band intensities divided by RRAGC detergent cell lysates band intensities normalized to raptor IP band densities) from 3 independent experiments using ImageJ densitometry results indexed to the measurements for RRAGC wt.

Article Snippet: RRAGC cDNA mutagenesis, retroviral and lentiviral vector generation, cell transfection and cell transduction to generate stable cell lines Reagents and mutagenesis A pCMV-SPORT plasmid containing the RRAGC cDNA (cat#: MHS6278-202757712; accession: {"type":"entrez-nucleotide","attrs":{"text":"BC016668","term_id":"16741747","term_text":"BC016668"}} BC016668 ) was purchased from ThermoScientific, and used as a template to generate mutant RRAGC cDNAs using the QuikChange® Lightning Site-Directed Mutagenesis Kit (Stratagene/Agilent, La Jolla, CA).

Techniques: Binding Assay, Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot, Quantitation Assay

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