EMBO/soda-nel huggingface dataset:
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 2

Figure 2. RBM47 displays antiviral activity.

A The mRNA and protein levels of DENV (left and middle panel) and DENV replicon DGL2 replication (right panel) were tested in 293T cells transfected with RBM47-Flag or empty vector at the indicated time points. The PCR and the luciferase data are represented as the means ± SD of n = 3 biological replicates.

B Knockdown efficiency of three shRNAs against RBM47 was measured using RT-PCR (left panel) and western blot (middle panel). DENV replicon DGL2 replication was tested in 293T cells transfected with scramble shRNA (shNC) or RBM47 shRNAs (right panel). The PCR and the luciferase data are represented as the means ± SD of n = 3 biological replicates.

C, D Levels of viral genome, protein, and progeny of VSV (C) or HSV-1 (D) were quantified in virus-infected 293T cells with or without RBM47 ectopic expression at the indicated time points. The viral protein levels were tested 12 h post VSV infection and 24 h post HSV-1 infection.The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates.

E, F Levels of viral genome, protein, and progeny of VSV (E) or HSV-1 (F) from virus-infected 293T cells transfected with RBM47 shRNA or shNC at the indicated time points. The PCR and viral titer data are represented as the means ± SD of n = 3 biological replicates.

Data information: The data shown are representative of n = 3 independent experiments.**P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 3

Figure 3. RBM47 enhances the expression of ISGs.

A Promoter activity of IFN-β or ISRE in SeV-infected 293T cells with or without RBM47 ectopic expression. Cells were infected with SeV for 12 and 24 h, and the promoter activities were determined using the DLR assay.Data are represented as the means ± SD of n = 3 biological replicates.

B, C Promoter activities of ISRE stimulated by Poly (I: C) (B) or IFN-α (C) in RBM47 overexpressing or knockdown 293T cells. Cells were stimulated with Poly (I: C) (1 μg/mL) or IFN-α (200 U/mL) for the indicated time, and the ISRE promoter activities were determined using the DLR assay. Data are represented as the means ± SD of n = 3 biological replicates.

D, E mRNA expression of IFIT1, ISG15, and Cig5 in SeV-infected 293T cells overexpressing RBM47 (D) or RBM47 knocked down cells (E). Cells were infected with SeV for 24 h, and gene expression was determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates.

F The expression of IFIT1, ISG15, and Cig5 in RBM47-overexpressing or control 293T cells stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates.

G The expression of IFIT1, ISG15, and Cig5 in WT and RBM47+/- peritoneal macrophages stimulated by IFN-α for 12 h. The PCR results are represented as the means ± SD of n = 3 biological replicates.

Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 4

Figure 4. IFNAR1 mRNA is stabilized by RBM47.

A Relative mRNA levels of JAK-STAT signaling adaptors were determined using RIP-PCR analysis. The enrichment folds of each gene relative to the IgG isotype group (which was set to "1") are shown. The PCR results are represented as the means ± SD of n = 3 biological replicates.

B IFNAR1 mRNA stability after DRB treatment. 293T cells were transfected with RBM47-Flag or empty vectors and treated with DRB (50 μg/mL). Total RNA was harvested at 0, 15, 30, 45, and 60 min, and IFNAR1 mRNA levels were measured using qRT-PCR and normalized to β-actin. The PCR results are represented as the means ± SD of n = 3 biological replicates.

C The protein levels of IFNAR1 and TYK2 in RBM47-overexpressing or control 293T cells treated with or without DRB. The relative band density was quantified using the ImageJ software and normalized to GAPDH. The relative density of the control group was set to "1."

D IFNAR1 mRNA expression in WT and RBM47 knockout 293T cells treated with or without RNA synthesis inhibitor Act D. The knockout efficiency of RBM47 is shown in the upper-right panel. The PCR results are represented as the means ± SD of n = 3 biological replicates.

E Locations and sequences of three RBM47-targeted RNA sequences at the 3'UTR of IFNAR1 mRNA. Data were obtained from a genome-wide HITS-CLIP database (POSTAR2, http://lulab.life.tsinghua.edu.cn/postar/index.php). The predicted RBM47 binding sequences were labeled in red.

F RNA pulldown assays using biotin-labeled RNA probes. The cell lysates with RBM47-flag were immunoprecipitated with biotin-conjugated RNA probes, and the elution was blotted with an anti-Flag antibody.

G Interaction between RBM47 and the three RNA fragments of IFNAR1 mRNA confirmed using EMSA assay. RBM47 binds to IFNAR1 RNA probes but not to mutant (1m, 2m and 3m) or negative control (NC) probes.

H The interaction between RBM47 and the three targeted RNA fragments from the 3'UTR of IFNAR1 mRNA was tested using the luciferase reporter assay. The U to A mutants of these three sequences (as shown in Table EV1) were also used as negative controls.Data are represented as the means ± SD of n = 3 biological replicates.

Data information: Data in A-D and F-H are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 5

Figure 5. RBM47 enhances the JAK-STAT pathway.

A mRNA of IFNAR1, but not IFNAR2 or STAT1, was stabilized by RBM47 ectopic expression in 293T cells with or without IFN-α stimulation. Cells were treated with IFN-α or PBS for 6 h, and the relative gene expression levels were determined using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates.

B, C mRNA (B) and protein (C) levels of IFNAR1 were analyzed in WT and RBM47 knockout 293T cells with or without IFN-α stimulation. The relative density of IFNAR1 protein in the control group was set to "1." The PCR results are represented as the means ± SD of n = 3 biological replicates.

D IFNAR1 mRNA expression in PMs from WT and RBM47+/- mice treated with or without IFN-α. The PCR results are represented as the means ± SD of n = 3 biological replicates.

E Flow cytometry analysis of IFNAR1 in the cytoplasmic membrane of BMDMs from WT and RBM47+/- mice. The cells was treated with PBS or IFN-α for 6 h. Data are represented as the means ± SD of n = 3 biological replicates.

F, G Protein levels of IFNAR1 and phosphorylation levels of TYK2, STAT1, and STAT2 in RBM47-overexpressing (F) or knockdown (G) 293T cells. Cells were treated with PBS or IFN-α for 6 h, and the relative band densities were analyzed using ImageJ software and normalized to GAPDH.

H ISRE luciferase was tested to investigate the effects of RBM47 or its variants on ISG activation stimulated by IFN-α.Data are represented as the means ± SD of n = 3 biological replicates.

I, J Effect of overexpression of WT or RBM47 mutants on VSV replication. 293T cells were transfected with RBM47-Flag or two mutants for 24 h and then infected with GFP-expressing VSV (MOI = 0.1) for another 12 h. The replication of VSVG was observed using fluorescence microscopy via GFP signal (I)(Scale bar, 50 μm), and protein levels were determined using western blot (J).The relative band densities were analyzed using ImageJ software and normalized to GAPDH. The relative density of VSVG protein in the control group was set to "1."

Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test). The data shown are representative of three independent experiments.
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 6

Figure 6. RBM47 displays antiviral activity in vivo.

A Immunoblot analysis of RBM47 and IFNAR1 in the lungs, livers, and spleens of RBM47+/+ (WT) and RBM47+/- mice. Each band represents tissue samples from one mouse.

B Analysis of viral mRNA from blood, lung, brain, and spleen samples of RBM47+/+ and RBM47+/- mice (n = 6 per group) infected with VSV (1× 108 pfu), and the results were normalized to those of mouse β-actin. Each dot represents data from one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates.

C Viral titers in serum, lung, brain, and spleen samples of RBM47+/+ and RBM47+/- mice at day 1 post VSV infection.The viral titer data are represented as the means ± SD of n = 6 biological replicates.

D Micrographs of hematoxylin-and-eosin-stained lung sections from mice treated with PBS or VSV (day 1 post infection). Scale bar, 50 μm.

E IFN-β protein levels in the serum of RBM47+/+ and RBM47+/- mice determined using IFN-β ELISA kit.Data are represented as the means ± SD of n = 6 biological replicates.

F qPCR analysis of IFNAR1, IFIT1, and Cig5 mRNA in the blood samples of infected mice. The relative expression levels of genes in WT mice were set to "1.". The PCR results are represented as the means ± SD of n = 6 biological replicates.

G RBM47+/+ and RBM47+/- mice were infected with HSV-1 (1× 107 pfu) via caudal vein injection. Viral genome DNA and progeny titers were tested using blood samples from infected mice. Data are represented as the means ± SD of n = 6 biological replicates.

H qRT-PCR analysis of IFNAR1 and Cig5 mRNA in blood samples from HSV-1-infected mice. The PCR results are represented as the means ± SD of n = 6 biological replicates.

Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; * P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
RNA-binding protein RBM47 stabilizes IFNAR1 mRNA to potentiate host antiviral
activity (2021)
Figure 7

Figure 7. The antiviral activity of RBM47 is IFNAR1 dependent.

A IFNAR1+/+ and IFNAR1-/- MEF cells were stimulated with IFN-β or infected with SeV, and the expression of IFNAR1 and RBM47 was determined using western blot. The gray values of the blots were analyzed using ImageJ software and normalized to GAPDH. The relative density of the control group was set to "1."

B IFNAR1+/+ and IFNAR1-/- MEFs were infected with shmRBM47 or control lentivirus, and the knockdown efficiency of RBM47 was determined using western blot. The relative band densities were analyzed using ImageJ software and normalized to GAPDH.

C RBM47-deficient or control IFNAR1+/+ and IFNAR1-/- MEFs were infected with VSV or HSV-1. The cells were harvested at 12 hpi. (VSV) or 24 hpi. (HSV-1), and viral RNA levels were analyzed using qRT-PCR.The PCR results are represented as the means ± SD of n = 3 biological replicates.

D The mRNA levels of VSV and RBM47 in blood samples of IFNAR1 deficient (IFNAR1-/-) mice (n = 6 per group) infected with VSV. Mice were infected with shmRBM47 or control lentivirus for 7 days and then challenged with VSV for another 24 h. Each dot represents one mouse. The PCR results are represented as the means ± SD of n = 6 biological replicates.

Data information: The data shown are representative of n = 3 independent experiments. NS, non-significant; **P ≤ 0.01, and ***P ≤ 0.001 (Student's t-test).
A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation (2018)
Figure 1

Figure 1. The antitumor activity of olaparib in PDXs identifies a subset of PARPi-sensitive tumors. (A) Waterfall plot showing the percentage of tumor volume change in olaparib-treated tumors compared to the tumor volume on day 1. +20% and -30% are marked by doted lines to indicate the range of PR, SD and PD. The box underneath summarizes different characteristics of each model and the clinical context at the moment of PDX implantation. TNBC, Triple Negative Breast Cancer; ER+BC, Estrogen Receptor positive Breast Cancer; P, primary; M, metastasis. Error bars indicate SEM from independent tumors (n ≥ 3). (B) Graph showing the percentage of tumor volume change during olaparib treatment in PDXs from cohort-1. Olaparib-sensitive models are represented with discontinuous lines. Acquisition of PARPi-resistance in PDX302, STG201 and PDX093 after prolonged exposure to olaparib is shown.
A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation (2018)
Figure 2

Figure 2. HRR-related alterations in PDX cohort-1 and PARPi response. (A) Levels of BRCA1 promoter hypermethylation, levels BRCA1 mRNA and the presence of BRCA1 nuclear foci by immunofluorescence are shown (larger views and separate channels are shown in Appendix Fig S1). T127 and T162 were used as positive controls for hypermethylated BRCA1 promoter. Error bars indicate SEM from independent tumors (n ≥ 2). Dashed line indicates mean of BRCA1 mRNA levels in normal breast. PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Alterations in HRR-related genes in PDX are also indicated. (B) Western blot of PALB2 detected in U2OS cells and PDXs. Three biological replicates of PDX093 are shown; PDX302 is used as PALB2 wild type PDX control. (C) YFP-PALB2 recruitment to laser-induced DSBs is impaired in HeLa cells expressing PALB2 p.M296Nfs (n=4, unpaired t-test at 16min). Gene-targeting efficiency using Cas9/mClover-LMNA1 homologous recombination assay of (D) siRNA PALB2 cells (n=4, one-way ANOVA) or (E) cells with no PALB2 depletion complemented with wild type and p.M296Nfs siRNA-resistant constructs (n=7, one-way ANOVA). Western blots of PALB2 wild type and PALB2 p.M296Nfs for each condition are shown. Error bars indicate SEM from independent experiments.

Source data are available online for this figure.
A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation (2018)
Figure 3

Figure 3. Lack of RAD51 nuclear foci identifies PARPi-sensitive PDX tumors. (A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi (larger views and separate channels are shown in Appendix Fig S2). Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Immunofluorescence staining of RAD51 foci in PARPi- and vehicle-treated PDX tumors is shown. Alterations in HRR-related genes are also summarized: hBRCA1: BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation. (B) Restoration of RAD51 foci formation in PARPi acquired-resistant PDXs. Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi-treated tumors from STG201, PDX302 and the corresponding PARPi acquired-resistant models (STG201OR and PDX302OR). Scale bars: 10µm. (C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.
A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation (2018)
Figure 4

Figure 4. RAD51 score predicts PDX response to PARPi in an independent PDX panel (cohort-2). (A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization. (B) ROC curves of the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test. (C) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [with an antibody towards the N-terminus (B1-NT) or C-terminus (B1-CT) of BRCA1] in three BRCA1-mutant, PARPi-resistant models from PDX cohort-2. The location of the mutation within the gene is indicated. Scale bars: 10µm.
A RAD51 assay feasible in routine tumor samples calls PARP inhibitor response beyond BRCA mutation (2018)
Figure 5

Figure 5. RAD51 score in tumors from patients with HBOC syndrome, including germline PALB2-mutation carriers. (A) Consort diagram of the collected and analyzed FFPE tumor samples from patients with HBOC syndrome following the scoring criteria for the RAD51 assay. (B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples. (C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.

Expanded View Figure legends
The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel (2008)
figf2

a, Current densities (mean ± s.e.m., n = 4–10) of IFe (30 mM Fe2+, pH 4.6) for TRPML1Va and ML4 mutant TRPML1Va channels. Asterisk indicates statistical difference (P  0.01) compared to TRPML1Va. b, 55Fe2+ uptake (normalized) in HEK293T cells transfected with vector control, with TRPML1Va, with F408Dgr;-TRPML1Va and with T232P-TRPML1Va constructs. Error bars indicate the standard deviation on the basis of two independent triplicate experiments. c, [Fe2+]o-dependent quenching of Fura-2 fluorescence in TRPML1Va-transfected cells (arrows), but not in non-transfected control cells (arrowheads) or T232P-TRPML1Va-transfected cells (bottom row). The fluorescence intensity was measured at an excitation wavelength of 360 nm (F360). The original magnification used for all micrographs was ×200. d, Average normalized responses of EGFP-positive TRPML1Va-transfected cells (typically n = 20–40 cells) to 1 or 10 mM Fe2+ (pH 4.6). e, No significant quenching was seen in T232P-TRPML1Va-transfected cells. f, Slightly less quenching was observed for the F408Dgr;-TRPML1Va-expressing cells. g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.
The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel (2008)
figf3

a, Co-localization of mCherry–TRPML1 and EGFP–LAMP1 at the membrane of an isolated enlarged LEL (see Methods). The patch pipette was filled with red rhodamine B dye (shown in blue for the purpose of illustration). b, Cartoons of three distinct patch-clamp configurations of lysosomal recordings: lysosome-attached, lysosome luminal-side-out and whole-lysosome. In each configuration, the pink arrow indicates the direction of the inward (at negative potentials; flow out of the lysosomes) current mediated by TRPML1 (as shown in c–i). c, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode’s solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations. d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+. g, Whole-lysosome current in an enlarged lysosome expressing wild-type TRPML1. The pipette (lumen) solution contained nominal divalent-free Tyrode solution. A Cs+-based bath solution (147 mM Cs-MSA) was used. h, Whole-lysosome ITRPML1Va. i, Whole-lysosome IFe/TRPML1. The pipette (lumen) solution contained 105 mM Fe2+ (pH 4.6).
The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel (2008)
figf4

a, Cultured TRPML1-/- (ML1-/-) skin fibroblasts showed less de-quenching of the iron-sensitive fluorescence than ML1+/- cells did. De-quenching was achieved by preloading the fibroblasts with an iron-sensitive dye, Phen Green SK (PG SK), and then adding the membrane-permeable transition metal chelator, 2,2′-bipyridyl (BPD). 2,2′-BPD is predicted to chelate free cellular iron (also referred to as chelatable or labile iron), which subsequently increases PG SK fluorescence. The original magnification used was ×200. b, The average 2,2′-BPD induced normalized change of fluorescence (Dgr;F/F0) for ML4 cells (ML1-/-; n = 6 experiments) is significantly (asterisk, P  0.01) lower than for the parental ML1+/- cells. Fibroblast cells (n = 10–20) were analysed for each individual experiment. 4,4′-BPD, a 2,2′-BPD analogue that cannot bind Fe2+, did not induce any significant restoration of PG SK fluorescence. Error bars, s.e.m. c, An ML1-/- skin fibroblast cell showed autofluorescence in LAMP1-positive compartments. Autofluorescence (green) was detected within a range of excitation wavelengths (shown with excitation at 480 nm). No significant autofluorescence was observed for a ML1+/+ cell. Lysosomes were stained with a LAMP1 antibody (red). Differential interference contrast (DIC) images are shown for comparison.
An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury (2021)
Figure 1

Figure 1. Injured epithelial secretions initiate a fibrotic cascade with mesenchymal cells.

A. Schematic workflow of the co-culture experiment in which AECs were subjected to apical TGFβ1 stimulation for 72 h in the co-culture system. Mono-cultures were employed as negative controls.

B. Representative E-cadherin (E-cad) and DAPI immunofluorescence images of AECs treated apically with TGFβ1 or vehicle control after 72 h in the co-culture system (scale bars 25 µm).

C. ELISA analysis shows decreased E-cad levels in TGFβ1-treated AECs after 72 h in the mono- and co-culture systems (n = 6 biological replicates, mean + s.d., ***p < 0.001, ANOVA/Tukey´s).

D. Representative αSMA and DAPI staining of healthy and IPF-derived MCs from the mono- and co-culture systems after 72 h apical treatment with TGFβ1. Note an increase in αSMA levels only in the presence of AECs and further enhanced in IPF-MCs (scale bars 100 µm).

E. ELISA analysis shows increased αSMA levels in a TGFβ1 dose-dependent manner from MCs and IPF-MCs in the co-culture system. Note a stronger αSMA response in IPF-MCs (mean + s.d., n = 3 biological replicates from 6 MCs and 6 IPF-MCs donors, **p = 0.0014, ****p < 0.0001, Nonlinear Regression followed by ANOVA/Sidak's).

F. Schematic workflow of the substitution system in which AECs were subjected to TGFβ1 stimulation for 3 h in co-culture with MCs, after which (1) MCs or (2) AECs were replaced with untreated counterparts or (3) both fresh media and untreated MCs for a further 72 h.

G, H. 72 h after substitutions in the co-culture, αSMA and E-cad levels of MCs and AECs, respectively, were measured with ELISA and normalised to the control. Note replacement of injured AECs with untreated AECs or replacement of media and MCs attenuates αSMA expression and increases E-cad levels (mean + s.d., ns = non-significant, ***p < 0.001, **p < 0.01, n = 3 biological replicates with 6 MCs donors, repeated measurement ANOVA for MCs or ordinary ANOVA for AECs with Dunnett´s posthoc test).

See also Appendix Figure S1.