EMBO/soda-nel huggingface dataset:
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 4

Figure 4. AS-RBM15 enhances RBM15 protein translation.

A Cytoplasmic extracts from MEG-01 cell lines stably expressing AS-RBM15, Exon 1, or shRNA-02 against AS-RBM15 were fractionated through sucrose gradients (15%-60%) (n=4). Global polyribosome profiles of these cell lines were generated by UV absorbance at 254 nm (A254). The arrow indicates increasing polyribosome weights. Low molecular weight, LMW; high molecular weight, HMW.

B The distributions of RBM15 mRNA (top), GAPDH mRNA (middle), and AS-RBM15 (bottom) were measured by real-time PCR and represented as percentages of total RNA in the fractions (n=3).

C MS2 pull-down assays: AS-RBM15 full-length (FL) and exon 1 were fused to MS2 RNA as baits. Plasmids expressing MS2 binding protein (HA-tagged) and fusion baits were co-transfected into 293T cells. HA antibody was used to immunoprecipitate HA-MS2 binding protein as shown by a WB on the bottom panel. RBM15 mRNA and MS2 fusions were assessed by real-time PCR (n=3, mean ± SD). Fold enrichment was calculated as 2 to the power of [(CI.R-Cb.R)-(CI.MS2-Cb.MS2)]. I: input i.e. the RNA in solution before immunoprecipitation, R: RBM15 mRNA, b: bead-bound mRNA, MS2: RNA levels of MS2 fusions, C: number of cycles in real-time PCR. One-way ANONA for p value: p<0.05.

D WB analysis of the endogenous RBM15 protein levels in 293T cells overexpressing two different doses of AS-RBM15-MS2 and MS2 (n=3).

E RBM15 mRNA interacts with AS-RBM15 via RNA-RNA interaction. AS-RBM15-MS2-associated RNAs were purified by phenol extraction and measured by reverse transcription and PCR for detection of RBM15, AS-RBM15 and MS2 under denaturing (heat) and non-denaturing conditions (n =3).

F RNase A protection assays for detecting endogenous association of AS-RBM15 and RBM15 mRNA (n=4). RNA was extracted from MEG-01 cells with phenol and digested with RNase A. cDNA was synthesized after RNase A treatment and detected using AS-RBM15 exon-specific primers as shown in the schematic diagram on top. RT: reverse transcriptase.

G MEG-01 cells expressing AS-RBM15 or vector control were treated with DMSO or rapamycin for 45 minutes. Cells were harvested for WB using antibodies against GAPDH and RBM15 (n=2).

H The RBM15 5’UTR reporter remains sensitive to rapamycin treatment with or without overexpression of antisense AS-RBM15 (n=4, mean ± SD). P values were calculated by one-way ANOVA test (**p<0.01 and ***p<0.001).

Source file is available.
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 5

Figure 5. Upregulation of AS-RBM15 RNA level in the cytoplasm is a response to MK differentiation signal.

The subcellular distribution of RBM15 and AS-RBM15 in CD34+ cells grown in TPO-containing medium for 5 days. Cytoplasmic and nuclear RNA levels of AS-RBM15 and RBM15 were normalized to GAPDH (control for cytoplasmic fraction) and MALAT1 (control for nuclear fraction), respectively, using the ∆∆Ct method (n=4, mean ± SD).
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 6

Figure 6. RUNX1 and RUNX1-ETO regulate the transcription of RBM15 and AS-RBM15.

A RUNX1 expression levels in human CD34+ cells grown in pro-MK medium in a time course were measured by real-time PCR (n=4, mean ± SD).

B CD34+ cells were grown in pro-MK medium for 5 days and stained with antibodies against CD41 and CD42 for flow cytometry analysis. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).

C The mRNA expression levels of RUNX1, RBM15, and AS-RBM15 in RUNX1 knockdown MEG-01 cells were measured by real-time PCR (n=4, mean ± SD).

D Chromatin immunoprecipitation using anti-RUNX1 antibody. Immunoprecipitated DNA fragments were measured by real-time PCR using HEL cells expressing shRUNX1 and control (n=4, means ± SD).

E CD34+ human cord blood cells were transduced with MIG retroviruses co-expressing A/E (AML1-ETO, aka RUNX1-ETO) and GFP. Around 5 days after transduction, the GFP+CD11b- cells were sorted for real-time PCR (n=4). Also see Fig EV6 for RUNX1-ETO binding profile on RBM15 locus.

Data information: Data are presented as mean ± SD. P value was determined using a Student’s t test (E), one-way ANOVA (A, C), and two-way ANOVA (D) (*p<0.05, **p<0.01, and ***p<0.001).
Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling (2020)
Figure 1

Fig 1 INSL5 is elevated in NPC tissue and plasma and is a potential diagnostic biomarker

A, Western blotting showing cellular INSL5 in the immortalized NPEC and NPC cell lines after inhibition of protein secretion by BFA, and β-actin was used as a loading control.

B, ELISA assay showing secreted INSL5 in the supernatants of NPECs and NPC cell lines. For NPECs, n=3; for NPC, n=7.

C, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in training cohort.

D, ROC of the diagnostic prediction model with plasma INSL5 level in the training cohort.

E, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 1(GZ).

F, The concentration of plasma INSL5 in healthy controls (normal EBV (-)), non-tumor individuals with VCA-IgA positive (normal EBV (+)) and NPC patients, in validation cohort 2(ZS).

G, Confusion table of binary results of the diagnostic prediction model in the training cohort, validation cohorts and the whole cohort.

H, ROC of the diagnostic prediction model with plasma INSL5 level in the whole cohort.

I, ROC of the diagnostic prediction model with plasma INSL5 level in individuals with VCA-IgA-negative plasma in all three cohorts.

J, Confusion table of binary results of the diagnostic prediction model in VCA-IgA-negative individuals in the training cohort, validation cohorts and the whole cohort.

Data information: In (B), data are presented as mean ± SD, in (C, E and F), data are presented as mean ± SEM, and P-values were determined by unpaired t test. *P<0.05, **P<0.01, ***P<0.001, ns, no significance. Exact P-values are specified in Appendix Table S4.
Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling (2020)
Figure 3

Fig 3 INSL5 promotes the progression of NPC via accelerating cell proliferation and invasion depending on GPCR142

A. Exogenous expression of INSL5 in NPC cells. Representative immunoblotting (upper panel) and immunofluorescent staining (lower panel) showed stable exogenous expression of INSL5 in both CNE1, CNE2 and HK1 NPC cell lines. Scale bars represent 20μm.B, MTT assay of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines (upper panel) either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2) (lower panel). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1.

C-H, colony formation (C and D), Brdu incorporation (E and F), and migration assays (G and H) of vector control or INSL5 overexpressing CNE1, CNE2 and HK1 NPC cell lines either transfected with control siRNA (NC) or GPCR142 siRNA (#1 and #2). Representative images are shown in (C), (E), and (G) for colony formation, Brdu incorporation and migration assays, respectively. Number of colonies, the percentage of Brdu positive cells and migrated cells per field of view were plotted in D, F and H, respectively. The results are from three different experiments. Scale bars represent 20μm in (E) and 100μm in (G)

I, J, Xenograft tumor growth of INSL5 over expression NPC HK1 stable cell lines in nude mice.. Tumor size (I) and Tumor weight (J) of two groups. n=11 mice per group.

Data information: In (B, I and J), data are presented as mean ± SD, in (D, F and H), data are presented as mean ± SEM, from three different experiments and P-values were determined by unpaired t test. *P<0.05, **P<0.01, ***P<0.001, ns, no significance. Exact P-values are specified in Appendix Table S4.
Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling (2020)
Figure 4

Fig 4 INSL5 induces glucose metabolism to aerobic glycolysis reprogramming in NPC cells.

A, Gene expression profiles from CNE2 cells overexpressed with INSL5 or the vector control. The genes are shaded with green, black or red in the heat map to indicate low, intermediate or high expression, respectively.

B, Functional annotation clustering of genes regulated by INSL5 in CNE2 cells is shown. Significantly enriched groups nominated by the gene ontology term are ranked based on the group enrichment scores. Red: biological process. Blue: signaling pathway.

C, The signal pathway of the subset of genes involved in metabolism was assessed by Gene Set Enrichment Analysis.

D, Analysis of glycolytic gene expression by immunoblot in INSL5 overexpressed or knock-down cell lines.

E, Metabolites were purified from five (CNE1 cell line and HK1 cell line) or seven (CNE2 cell line) independent samples of two vector and INSL5 overexpressed cell lines and analyzed by liquid chromatography-mass spectrometry (LC-MS). Red: glycolytic intermediate metabolites. Green: tricarboxylic acid cycle (TCA cycle) intermediate metabolites. PPP: pentose phosphate pathway. The color scale bar represents Z-score of the metabolites' level.

F, The extracellular acidification rate (ECAR) was measured in cells with or without INSL5 overexpression using a Seahorse XF96 Extracellular Flux analyzer. Glu, glucose; Oligo, oligomycin.

G, Statistical analysis of the effects of INSL5 overexpression on glycolytic activity.

H, CNE1-vector and CNE1-INSL5 cells were grown in the presence of the fluorescent glucose analog 2-NBDG (Invitrogen) for 15min, and glucose uptake was then quantified using flow cytometry (FACS) (left panel). Statistical analysis of the glucose uptake was showed in the right panel.

I-K, glucose consumption and lactate production (I), basic ATP level or the ATP level of cell which was treated by 1uM oligomycin or 50uM 2-DG for 24h (J), HK2 enzyme activity (K) in CNE1-vector and CNE1-INSL5 cells.

L, ATP concentration, HK2 enzyme activity and lactate production in INSL5 wide-type or knock down HNE1-EBV cells.

Data information: In (F, G and L), data are presented as mean ± SEM, in (H-K), data are presented as mean ± SD, from three different experiments and P-values were determined by unpaired t test. (*P<0.05, **P<0.01, ***P<0.001, ns, no significance. ). Exact P-values are specified in Appendix Table S4.
Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling (2020)
Figure 5

Fig 5 Activation of STAT5 by INSL5 facilitates aerobic glycolysis and proliferation

A, Schematic diagram of the mRNA structures of the glycolytic genes and the indicated STAT5 binding site.

B, phosphorylation of STAT5 under INSL5 stimulation. CNE1 cells were starved in FBS-free medium for 24h and then treated with INSL5 peptide (50ng/mL) in the presence of an anti-INSL5 antibody at 100ug/ml, or an isotype-matched IgG control (IgG) for 30min.

C, Localization of STAT5. A confocal analysis of STAT5 staining was performed using CNE1 cells transfected vector or INSL5. Scale bars represent 20μm

D, Cytoplasmic and nuclear proteins from vector or INSL5overexpressing cells were separated and subjected to immunoblot as indicated. The nuclear marker Lamin B1 and the cytoplasmic marker GAPDH were used to demonstrate the purity of fractions.

E, INSL5 overexpression activated AKT, ERK, JAK1 and STAT5 phosphorylation, not STAT3 in HK1 cells. INSL5 overexpressing HK1 cells were treated with MK2206 (50uM), U0126(20uM), MK2206+U0126, or Ruxolitinib (5uM) for 24h. Western blotting was performed to evaluate the effects of those inhibitors on STAT5 and STAT3 phosphorylation level.

F, ChIP-qPCR analyzed the occupancy of potential E-boxes by STAT5 in the genes of HK2, Glut3 and PFK1 in INSL5-overexpressing CNE1 cells using IgG or anti-STAT5 antibodies.

G, Luciferase activity of different glycolytic gene promoter reporters in 293T cells transfected with STAT5 or empty vector.

H, Glucose uptake assessed by 2-NBDG incubation in INSL5 overexpressed CNE1 cells with STAT5 knock down.

I, Glucose uptake was assessed by 2-BNDG incubation in INSL5 overexpressed HK1 cells treated with JAK kinase inhibitor (Ruxolitinib).

J, MTT assay of vector control or INSL5 overexpressing HK1 cells either treated with STAT5 siRNAs or JAK kinase inhibitor.

Data information: In (F-H and J), data are presented as mean ± SEM, in (I), data are presented as mean ± SD, from three different experiments and P-values were determined by unpaired t test. (*P<0.05, **P<0.01, ***P<0.001, ns, no significance).Exact P-values are specified in Appendix Table S4.
Autocrine INSL5 promotes tumor progression and glycolysis via activation of STAT5 signaling (2020)
Figure 6

Fig 6 Overexpression of INSL5 sensitizes NPC to a glycolysis inhibitor and INSL5 neutralized antibody.

A, Brdu assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 20μm.

B, similar to (A), migration and invasion assay for CNE1 cells stimulated with 100ng/ml INSL5 peptide in the presence of an anti-INSL5 antibody at 50 or 100ug/ml, or an isotype-matched IgG control (IgG) for 24h. Scale bars represent 100μm.

C-E Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of glycolysis inhibitor, 2-DG. CNE1 cells (C), CNE2 cells (D) and HK1 cells (E). n=4 biological replicates for CNE1 and CNE2 cell line, n=6 biological replicates for HK1

F, Growth curves of cell lines stably expressing INSL5 or vector 48 h post-treatment with the indicated dose of cis-platinum (DDP) and 2-DG. n=4 biological replicates per group.

G-H, Tumor size (G) and tumor weight (H) of nude mice burdened with NPC PDX tumor and intraperitoneally injected with anti-INSL5, anti-GPCR142 or isotype-matched IgG control (IgG) every two days. n=6 mice per group.

I, Working model for regulation of NPC progression and tumorigenesis by INSL5-GPCR142 axis. INSL5 is highly expressed in NPC tumor cells with poor prognosis in NPC patients. INSL5 can physically bind to the receptor GPCR142 to contribute to oncogenic function by reprogramming glycolysis, which is promoted by activation of the STAT5 signaling pathway. The anti-INSL5 neutralizing antibody, anti-GPCR142 neutralizing antibody and glycolysis inhibitor could be attractive approaches for sensitizing INSL5-overexpressing NPC tumor cells.

Data information: In (A-B and F), data are presented as mean ± SD, in (C-E and G-H), data are presented as mean ± SEM, from three different experiments and P-values were determined by unpaired t test. *P<0.05, **P<0.01, ***P<0.001, ns, no significance. Exact P-values are specified in Appendix Table S4.

FIGURE LEGEND FOR EV FIGURES.
PLETHORA-WOX5 interaction and subnuclear localisation control Arabidopsis root stem cell maintenance (2022)
Figure 1

Figure 1 - WOX5 positively regulates PLT3 expression.

A, Schematic representation of the Arabidopsis root meristem. The QC cells (red) maintain the surrounding stem cells (initials) outlined in black together building the root stem cell niche (SCN). The different cell types are colour coded. QC = quiescent center (red); CSC = columella stem cells (yellow); CC = columella cells (green); LRC = lateral root cap (light purple); ep = epidermis (purple); c = cortex (light turquoise); en = endodermis (dark turquoise); bright turquoise = cortex/endodermis initials; dark purple = epidermis/lateral root cap initals; dark orange = stele initials; stele = light orange; grey dots = starch granules. B, C, Representative images of pPLT3::erCFP (cyan) expressing and PI-stained (red) Arabidopsis roots in Col or wox5 background, respectively. D, Mean fluorescence intensities of the pPLT3::erCFP roots summarized in box and scatter plots. The mean fluorescence intensity of the CFP signal in Col roots was to set to 100 %. E, F, Representative images of pPLT3::PLT3-YFP (yellow) expressing and FM4-64-stained (red) Arabidopsis roots in Col or wox5 mutant background, respectively. g, Mean fluorescence intensities of the pPLT3::PLT3-YFP expressing roots summarized in box and scatter plots. The mean fluorescence intensity of the YFP signal in Col roots was to set to 100%. D, G, Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. The data was statistically analyzed by one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test. Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from two technical replicates. B, C, E, F, Scale bars represent 10 µm. SCN = stem cell niche; PI = propidium iodide; YFP = yellow fluorescent protein; CFP = cyan fluorescent protein.
PLETHORA-WOX5 interaction and subnuclear localisation control Arabidopsis root stem cell maintenance (2022)
Figure 2

Figure 2 - PLTs constrain the WOX5 expression domain.

A-F, Representative FM4-64-stained Arabidopsis roots (grey) expressing pWOX5::mVenus-NLS (green) in Col, plt2, plt3 and plt2, plt3 double mutant background in longitudinal (A-D), or transversal (E-F) optical sections. E', F', Analysis of representative images in (E) and (F) in Imaris to detect and count individual expressing nuclei. E'', F'', Overlay of 10 roots (biological replicates) showing the area of detected fluorescence (high levels in red, low levels in blue) in Col and plt2, plt3 double mutant roots. G, Number of nuclei (biological replicates) expressing pWOX5::mVenus-NLS in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. H, Area of WOX5 expression in µm2 in Col and plt2, plt3 double mutant roots summarized in box and scatter plots. G, H Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, □ = mean value, = minimum/maximum. (G, H) Kruskal-Wallis ANOVA analysis with subsequent Dunns test (G) or one-way ANOVA and post-hoc Holm-Sidak multiple comparisons test was used to test for statistical significance (H). Asterisks indicate statistically significant differences (α = 0.01). Number of analysed roots (n) (biological replicates) is indicated for each genotype and results from three technical replicates per genotype. Scale bars represent 10 µm; NLS = nuclear localisation signal.
PLETHORA-WOX5 interaction and subnuclear localisation control Arabidopsis root stem cell maintenance (2022)
Figure 7

Figure 7 - WOX5 can interact with PLTs.

A-H, Fluorescence Lifetime Imaging (FLIM) results of transiently expressing N. benthamiana leaf epidermal cells. A-H, J, Representative FLIM images of WOX5-mVenus (donor only) plus the indicated acceptors after a pixel-wise mono-exponential fit of the mVenus fluorescence signal. The fluorescence lifetime of WOX5-mVenus in ns is color-coded. Low lifetimes (blue) due to FRET indicate strong interaction of the two proteins and high lifetimes (red) indicate weaker or no interaction. Scale bars represent 5 µm. I, Fluorescence lifetimes in ns are summarized in combined jitter and box plots. The dashed line represents the fluorescence lifetime mean value of the WOX5-mV co-expressed with free mCh as negative control. The one-way ANOVA and Holm-Sidak post-hoc multiple comparisons test was used to test for statistical significance. Samples with identical letters do not show significant differences (α = 0.01). Number of nuclei analyzed (n) (biological replicates) is indicated and results from 2 to 9 technical replicates. Box = 25-75 % of percentile, whisker = 1.5 interquartile range, − = median, ▪ = mean value. K, Individual nuclei showing nuclear bodies during co-expression of WOX5-mV and PLT3-mCh were analysed for WOX5-mV lifetime in the nuclear bodies or nucleoplasm separately (n = 7 (biological replicates), from four technical replicates. mCh = mCherry. mV = mVenus.
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 1

Figure 1. Targeting USP7 triggers mitotic signaling throughout the cell cycle.

(A) HTM of EdU levels per individual nucleus in response to USP7 inhibition in HCT-116 cells treated with 50 μM P22077 for 1-2 hrs or 25 μM BAY 11-7082 for 1-2 hrs, or with DMSO as a control.

(B) Western Blot (WB) showing the levels of USP7, MPM-2, histone H3S10P and histone H3 in whole nuclear extracts of HCT-116 cells treated with 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 8 hrs (CDK1i) or DMSO as a control. Ponceau staining is shown as a loading control.

(C) Flow cytometry profile illustrating the levels of histone H3S10P (left) and MPM-2 (right) in HCT-116 cells in control conditions (Control, black) and after treatment with 50 μM P22077 for 4 hrs (USP7i, red). DNA content was measured with Propidium Iodide (PI).

(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.

(F) Flow cytometry profile representing the levels of histone H3S10P and MPM-2 in HCT-116 cells transfected with two different siRNA against USP7 (red) (si#1 and si#2) or with a non-specific siRNA (Control, black), for 96h. DNA content was measured with Propidium Iodide (PI).

(G) WB illustrating the depletion of USP7 in the HCT-116 cells used in (F). Experiments were repeated 3 times and one representative result is shown.
USP7 limits CDK1 activity throughout the cell cycle (2021)
Figure 2

Figure 2. USP7 suppresses CCNB1/CDK1 activity.

(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.

(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.

(C) Quantification of the nuclear translocation of CCNB1 in RPE cells treated with 25 μM P22077 for 1-2 hrs measured by high throughput microscopy in three different experiments (** p<0.01, ***p<0.005, t-test). Bars represent mean values ± SEM. N>2000 cells were analysed per condition.

(D, E) Flow cytometry profiles representing the levels of MPM-2 (D) and H3S10P (E) in WT and CDK2/4/6 knockout MEF in control conditions (Control, black) and after treatment with 50 μM P22077 for 4 hrs (USP7i, red). The experiment was performed twice using two different wild type and CDK2/4/6 KO MEF and one representative result is shown.

(F, G) Flow cytometry profiles representing the levels of MPM-2 (F) and H3S10P (G) in RPE cells in control conditions (Control, black), after treatment with 25 μM P22077 for 4 hrs (USP7i, red) or after treatment with 10 μM RO3306 for 8 hrs followed by incubation with 25 μM P22077 and 10 μM RO3306 for 4 additional hrs (CDK1i+USP7i, blue). DNA content was measured with Propidium Iodide (PI). One representative experiment is shown, out of five.