EMBO/soda-nel huggingface dataset:
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 5

Figure 5 ZNF596 is important for LINC00115-driven GSC tumorigenicity

Schematics of the 5'-terminus of LINC00115 that is homologous to the 5'-UTR of ZNF596 mRNA.

Sequence alignment of the 5'-terminus of LINC00115 and the 5'-UTR of ZNF596 mRNA.

C and D. LINC00115 knockdown reduced ZNF596 mRNA (C) and ZNF596 protein (D) expression.

E. Effects of exogenous ZNF596 expression on LINC00115 knockdown-inhibited ZNF596 protein expression.

F. ZNF596 overexpression had no effect on LINC00115 expression.

G. Expression of exogenous ZNF596 rescued LINC00115 knockdown-inhibited cell proliferation.

H. Representative H&E staining images of mouse brain sections with 1123/shC and 1123/shL115 GSC tumor xenografts with empty vector (EV) or ectopic ZNF596 expression. Images represent results of five mice per group of two independent experiments. Scale bars: 1 mm.

I. Quantification of tumor size in H.

J and K. Correlation of expression between LINC00115 and ZEB1 from TCGA GBM RNA-Seq dataset (J) and REMBRANDT GBM array dataset (K). **, p < 0.01, by two-tailed t-test.

Data information: In C to G, data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA in C and F, by two-tailed t-test in G.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 6

Figure 6 LINC00115 promotes ZNF596 transcription by sequestrating miR-200s from binding to the 5'-UTR of the ZNF596 gene

A and B. Expression of shRNA-resistant LINC00115 WT but not mutant or empty vector (EV) rescued LINC00115 shRNA-inhibited ZNF596 mRNA (A) and ZNF596 protein (B) expression in 1123 GSCs.

C. Schematics of the predicted binding sites of miR-200s on ZNF596 sequence. ZNF596-5'-UTR-mut contains a mutated sequence in the miR-200 binding site.

D. MS2-RIP followed by qRT-PCR for endogenous microRNAs association with ZNF596. 12XMS2 empty vector, ZNF596-5'-UTR-WT-12XMS2, or ZNF596-5'-UTR-mut -12XMS2 with Flag-tagged MS2 were co-transfected into 1123 GSCs.

E. Schematics of ZNF596 wild type (ZNF596-WT) and miR-200 binding site mutant (ZNF596-mut) promoter.

F. Luciferase activity in 1123 GSCs co-transfected with miR-200s and luciferase reporters containing ZNF596 5'-UTR WT, mutant, or empty vector (EV).

Data information: In B, D and F, data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, by One-way ANOVA in C and F, by two-tailed t-test in G. Data presented as mean ± SD. **, p < 0.01, by two-tailed t- test in D, by One-way ANOVA in F.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 7

Figure 7 LINC00115 upregulates ZNF596 to activate EZH2/STAT3 signaling

GSEA of EZH2-targeted signature genes using ranked gene expression changes in 1123 GSCs with ZNF596 sgRNA or a control sgRNA. NES, normalized enrichment score.

qRT-PCR analysis of effect of ZNF596 knockout on EZH2 expression.

ZNF596 knockout impaired EZH2 expression, its downstream H3K27me3, and STAT3 phosphorylation in 1123 and 528 GSCs.

LINC00115 regulated ZNF596/EZH2/STAT3 signaling. 1123 and 528 GSCs were treated with or without EZH2 inhibitor GSK343 (3 μM) for 6 h.

LINC00115 and ZNF596 regulate EZH2 promoter activity.

ChIP-qPCR analysis of ZNF596 binding with the EZH2 promoter.

EZH2 inhibitor GSK343 impaired ZNF596 overexpression-reversed and LINC00115 knockdown-inhibited neuro-like sphere formation. 1123 and 528 GSCs with an inducible ZNF596 shRNA were treated with or without Dox (2 μg/ml) and/or EZH2 inhibitor GSK343 (1 μM) for 7 days.

Growth kinetics of subcutaneous xenograft tumor with indicated genetic modifications after treatment with or without Dox and/or GSK343. Tumor-bearing mice (five mice per group) were fed with 10% sucrose water with or without 2 mg/ml Dox and combined with treatment with or without GSK343 (5 mg/kg body weight from Monday to Friday via intraperitoneal injection) for three weeks beginning five days post-xenograft transplantation. Data were from three independent experiments with five mice per group.

Kaplan-Meier analysis of animal implantation with GSC1123 with indicated modifications. n = 10. Statistical analysis was performed by log-rank test.

Data information: In B to G, data are representative of three independent experiments. Error bars, ± SD. *, p < 0.05, **, p < 0.01, ***, p < 0.001, by One-way ANOVA in B and F, by two-tailed t-test in E, G, and H.
TGF-β-activated LncRNA LINC00115 is a critical regulator of glioma stem-like cell tumorigenicity (2019)
Figure 8

Figure 8 LINC00115 is co-expressed with ZEB1 and ZNF596 in GBM specimens

Representative images of RNAscope for LINC00115 and IHC for ZEB1 and ZNF596 expression in two GBM specimens, Sample #1 (positive) and Sample #2 (negative). Scale bar, 50 μm. Images are representative of two independent experiments.

Correlation of detected expression between ZEB1 or ZNF596, and LINC00115 as shown in A from a separate cohort of a total of 75 paraffin-embedded clinical GBM samples. Statistical analysis was performed by Chi-square test.

Kaplan-Meier survival analysis of patients with GBM tumors expressing high or low levels of LINC00115 with high or low levels of ZEB1 or ZNF596 expression. Median survival (in months): LINC00115hi/ZEB1hi, 5.4; LINC00115lo/ZEB1lo, 27.9; LINC00115hi/ZNF596hi, 4.2; LINC00115lo/ZNF596lo, 23.8. Statistical analysis was performed by log-rank test.

A working model of TGF-β-activated LINC00115-driven GSC tumorigenicity and self-renewal through sequestering miR-200s from its binding targets, resulting in enhanced both ZEB1 signaling and ZNF596/EZH2/STAT3 signal axis.

Expanded View Figures
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 1

Figure 1. Time course expression of surface Cripto in pro- and anti -inflammatory macrophages in acute skeletal muscle injury.

A. Experimental scheme of Cripto expression analysis in macrophage (MP) subpopulations from wild-type skeletal muscles at different time points after Cardiotoxin (CTX) injection.

B. Representative flow cytometry dot plots of CD11b+ and CD11b+/Cripto+ cells at day 2, 3 and 5 after injury. PE (Phycoerythrin/free channel). Data are mean±SEM (n=5 biological replicates; P≤0.00008, Student's t-test).

C. Representative flow cytometry dot plots of CD11b+/F4/80+/Ly6CHigh/Low and CD11b+/Cripto+/F4/80+/Ly6CHigh/Low cells at day 2, 3 and 5 after injury.

D. Quantification of F4/80+/Ly6CHigh/Low cells in the CD11b+ and CD11b+/Cripto+ cell population from injured muscles at the indicated time points. Data represent mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 2

Figure 2. Myeloid-specific Cripto deletion perturbs MP plasticity without affecting monocyte/macrophage accumulation.

A. Representative pictures of immunostaining for F4/80 (green) in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury.

B. Quantification of F4/80 staining/damaged area (μm2) at day 2 (top graph) and 5 (bottom graph) after injury.

C. Representative pictures of immunostaining for CD206 in Control and CriptoMy-LOF TA sections at day 2 (top panel) and 5 (bottom panel) after injury, respectively.

D-E. Quantification of CD206+ MPs per area (mm2) at day 2 (D) and 5 (E) after injury.

Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 3

Figure 3. Cripto controls the proper expansion of CD206+ MPs by modulating TGFβ signaling

A. Representative flow cytometry dot plots showing the percentage of GFP+ cell population in GFP-Control and GFP-CriptoMy-LOF muscles at day 2 (top panel) and 5 (bottom panel) after injury. Percentage of GFP+ cells expressing F4/80 in GFP-Control and GFP-CriptoMy-LOF muscles is shown at day 2 after injury (middle panel). Data are mean±SEM (n=4 biological replicates; P=ns; Student's t-test).

B. qRT-PCR analysis of pro- (Nos2, Mcp1 and Tnfα) and anti- (Arg1, Fizz1, Il10, Il4rα and Tgfβ) inflammatory markers in GFP-CriptoMy-LOF and GFP-Control MPs at day 2 and 5 after injury. Data represent mean±SEM of relative mRNA level normalized with Gapdh (n≥4 biological replicates; ***P<0.001, Student's t-test).

C. Representative pictures of immunostaining for GFP (green), CD206 (red), pSMAD3 (white) in GFP-Control and GFP-CriptoMy-LOF TA sections at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.

D. Quantification of GFP+/CD206±/pSMAD3± cell distribution in TA sections from GFP-Control and GFP-CriptoMy-LOF at day 5 after injury. Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; **P<0.01, Student's t-test).

E. Representative flow cytometry dot plots of F4/80+/Ly6CLow cell population gated on GFP+ cells from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data are mean±SEM (n=3 biological replicates; P=ns; Student's t-test).

(F) Smear plot of RNA-seq data from GFP+/F4/80+/Ly6CLow cells FACS sorted from GFP-Control and GFP-CriptoMy-LOF muscles at day 5 after injury. Data show the expression level as Log2 fold-change (FC) [Log2(GFP-Control/GFP-CriptoMy-LOF)], against Log2(expression) [Log2(average of gene expression across all samples], for each individual gene. The blue lines correspond to LogFC of 1 and -1. (n=3 biological replicates).
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 4

Figure 4. Effect of myeloid-specific Cripto deletion on skeletal muscle regeneration process

A. Schematic representation of the experimental design in the single injury model (CTX-I).

B. Representative pictures of double immunostaining with Laminin (green) and embryonic myosin heavy chain (eMHC; red) at day 5 (left panel) and 30 (right panel) after cardiotoxin (CTX) injection.

C. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after CTX injection. Data are mean±SEM (n=5 biological replicates; P=ns, Student's t-test).

D. Schematic representation of the experimental design in the re-injury model (CTX-II).

E. Representative pictures of double immunostaining with Laminin (green) and eMHC (red) at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II).

F. Minimal Feret's diameter distribution of Control and CriptoMy-LOF myofibers at day 5 (top panel) and 30 (bottom panel) after re-injury (CTX-II).

Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Data are mean±SEM (n=5 biological replicates; *P<0.05, Student's t-test).
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 5

Figure 5. Myeloid-specific Cripto deletion reduced accumulation of CD206+ MPs and worsens the mdx phenotype

A. Schematic representation of the experimental strategy. Four-month-old lethally irradiated mdx mice were transplanted with either GFP-CriptoMy-LOF (mdx-CriptoMy-LOF) or GFP-Control (mdx-Control) bone marrow and sacrificed at 16 weeks (wks) after transplantation.

B. Representative pictures of immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections with GFP (green) and CD206 (white; left panel), pSMAD3 (red; middle panel) and GFP and pSMAD3 (right panel).

C-E. Quantification of GFP (C), GFP/CD206 (D) and GFP/pSMAD3 (E) positive cells per area (mm2) in mdx-Control and mdx-CriptoMy-LOF diaphragms. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; **P<0.01, Student's t-test).

F-G. Representative pictures of Picrosirius red staining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (F) and quantification of picrosirius red staining (G). Data are expressed as percentage of stained area and are box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05, Student's t-test).

H. Hydroxyproline (HOP) concentration in mdx-Control and mdx-CriptoMy-LOF diaphragm muscle sections. Data are expressed as HOP levels (μg) per muscle section volume (mm3) and as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01, Student's t-test).

I-K. Representative images of Laminin (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (I) and minimal Feret's diameter distribution (J) and Variance coefficient (VC; K) of centrally nucleated myofibers. Data are expressed as mean±SEM (n=5 biological replicates; *P<0.05; ***P<0.001; Student's t-test) and box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; **P<0.01; Student's t-test).

L-M. Representative images of eMHC (red) immunostaining of mdx-Control and mdx-CriptoMy-LOF diaphragm sections (L) and quantification of eMHC+ myofibers per area (mm2, M). Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n=5 biological replicates; *P<0.05; Student's t-test).

Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 6

Figure 6. Myeloid-specific Cripto deletion affects vascular remodeling

A. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after single injury (CTX-I).

B-D. Quantification of CD31+ capillary number per area (mm2; B), average of capillary cross-sectional area (µm2; C) and percentage of small (<20 µm2) and large (>100 µm2) capillaries (D) in Control and CriptoMy-LOF injured muscles at indicated time points after single injury (CTX-I). Data are mean±SEM (n=5 biological replicates; *P<0.05; **P<0.01; ***P<0.001, Student's t-test).

E. Representative pictures of CD31 (red) immunostaining of Control and CriptoMy-LOF TA sections at day 5 (left panel) and 30 (right panel) after re-injury (CTX-II).

F. Representative pictures of mdx-Control and mdx-CriptoMy-LOF diaphragm sections immunostained with VEcad (red).

G-H. Quantification of CD31+ capillary number per area (mm2; G) and average of capillary cross-sectional area (µm2; H) in Control and CriptoMy-LOF injured muscles at the indicated time points after re-injury (CTX-II) Data are mean±SEM (n=6 biological replicates; *P<0.05; ***P<0.001, Student's t-test).

I-J. Quantification of capillary number per area (mm2; I) and average of capillary cross-sectional area (µm2; J) in mdx-Control and mdx-CriptoMy-LOF diaphragm muscles. Data are mean±SEM (n=5 biological replicates; ***P<0.001, Student's t-test).

Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Magnification of the boxes is 3.5 x.
Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle (2020)
Figure 7

Figure 7. Cripto modulates endothelial plasticity by restricting Endothelial-to-Mesenchymal Transition

A. Representative pictures of double immunostaining with VEcad (red; left and middle panel) and KLF4 (green, left panel) or TCF4 (green, middle panel), and with CD31 (red; right panels, confocal pictures) and TCF4 (white, right panel) on Control and CriptoMy-LOF TA sections at day 5 after injury.

B-D. Quantification of VEcad/KLF4 (B), VEcad/TCF4 (C) and CD31/TCF4 (D) double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after injury.

E. Representative Confocal pictures of CD31+ (red) and PDGFRα+ (green) immunostaining on Control and CriptoMy-LOF TA sections at day 5 after injury.

F-G. Quantification of CD31+/PDGFRα+ (F) and CD31-/PDGFRα+ (G) cells per area (mm2). H-I. Representative pictures of triple immunostaining with VEcad (red), KLF4 (green) and pSMAD3 (white) on Control and CriptoMy-LOF TA sections at day 5 after injury (H) and quantification of VEcad/KLF4/pSMAD3 triple positive cells per area (mm2; I).

J-K. Representative pictures of double immunostaining with VEcad (red) and KLF4 (green) on Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II; J) and on diaphragm sections of mdx-Control and mdx-CriptoMy-LOF (K).

L-M. Quantification of VEcad/KLF4 double positive cells per area (mm2) in Control and CriptoMy-LOF TA sections at day 5 after re-injury (CTX-II; L) and in diaphragm sections of mdx-Control and mdx-CriptoMy-LOF (M).

Data information: Nuclei were counterstained with DAPI (blue). Scale bar: 100 μm. Confocal pictures scale bar: 25 μm. Magnification of the boxes is 3.5 x. Data are expressed as box plots displaying minimum, first quartile, median, third quartile and maximum (n≥5 biological replicates; *P<0.05; **P<0.01; ***P<0.001; Student's t-test).

Expanded View Figure Legends
Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation (2020)
Figure 1.

Structure of 2019-nCoV S in the prefusion conformation.(A) Schematic of 2019-nCoV S primary structure, colored by domain. Domains that were excluded from the ectodomain expression construct or could not be visualized in the final map are colored white. SS= signal sequence, NTD= N-terminal domain, RBD= receptor-binding domain, SD1= subdomain 1, SD2= subdomain 2, S1/S2= S1/S2 protease cleavage site, S2′= S2′ protease cleavage site, FP= fusion peptide, HR1= heptad repeat 1, CH= central helix, CD= connector domain, HR2= heptad repeat 2, TM= transmembrane domain, CT= cytoplasmic tail. Arrows denote protease cleavage sites. (B) Select 2D class averages of the particles that were used to calculate the 2019-nCoV S reconstruction (left). Side and top views of the prefusion structure of the 2019-nCoV S protein with a single RBD in the "up" conformation (right). The two RBD "down" protomers are shown as cryo-EM density in either white or gray and the RBD "up" protomer is shown in ribbons, colored corresponding to the schematic in Fig 1A.
Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation (2020)
Figure 3.

2019-nCoV S binds human ACE2 with high affinity.(A) SPR sensorgram showing the binding kinetics for human ACE2 and immobilized 2019-nCoV S. Data are shown as black lines and the best fit of the data to a 1:1 binding model is shown in red. (B) Negative-stain EM 2D class averages of 2019-nCoV S bound by ACE2. Averages have been rotated so that ACE2 is positioned above the 2019-nCoV S protein with respect to the viral membrane. A cartoon depicting the ACE2-bound 2019-nCoV S protein is shown (right) with ACE2 in blue and S protein monomers colored tan, pink and green.
Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation (2020)
Figure 4.

Antigenicity of the 2019-nCoV RBD.(A) The SARS-CoV RBD is shown as a white molecular surface (PDB ID: 2AJF), with residues that vary in the 2019-nCoV RBD colored red. The ACE2 binding site is outlined with a black dotted line. (B) A biolayer interferometry sensorgram that shows binding to ACE2 by the 2019-nCoV RBD-SD1. Binding data are shown as a black line and the best fit of the data to a 1:1 binding model is shown in red. (C) Biolayer interferometry to measure cross-reactivity of the SARS-CoV RBD-directed antibodies S230, m396 and 80R. Sensortips with immobilized antibodies were dipped into wells containing 2019-nCoV RBD-SD1 and the resulting data are shown as a black line.
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 1

Figure 1. Expression of RBM15 and AS-RBM15 in hematopoietic cells.

A Genomic organization of the human RBM15 locus. Exons are represented by black boxes. Arrows indicate transcriptional direction. 5’UTR and 3’UTR are represented by open boxes.

B Expression levels of RBM15 and AS-RBM15 in different blood lineages by meta-analysis of RNA-seq data [38]: HSC (hematopoietic stem cells), MPP (multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (Granulocyte-macrophage progenitor cells), MEP (megakaryocyte- erythrocyte progenitor cells), MK (megakaryocyte), and EB (erythroid body).

C Relative expression levels of AS-RBM15 (left) and RBM15 (right) in MEG-01 cells treated with PMA were measured by real-time PCR assays (n=4, mean ± SD). SD: standard deviation.

D Relative expression levels of AS-RBM15 and RBM15 in CD34+ cells grown in pro-MK medium were measured by real-time PCR. GAPDH was used as an internal control (n=3, mean ± SD). Also see Fig EV 1.

Data information: P values were calculated using one-way ANOVA (*p<0.05, **p<0.01, and ***p<0.001).
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 2

Figure 2. AS-RBM15 enhances the megakaryocyte terminal differentiation of human umbilical CD34+ cells.

A CFU-MK assays with CD34+ cells expressing AS-RBM15. MK colonies staining for acetylcholinesterase activity were counted (right). P value was calculated using Student’s t test (n=3, mean ± SD).

B The number of CD41+CD42+ mature MK cells was determined by FACS analysis. CD34+ cells expressing sh-AS-RBM15 were grown in pro-MK medium containing thrombopoietin and stem cell factor (TPO/SCF) for five days (n=4, mean ± SD).

C Schematic diagram of AS-RBM15 truncations, FL (full-length), ∆5 (nt463-2611), ∆3 (nt1-471), and Exon1 (nt1-175). Exon 2 (blue) contains repeat element (SINE/Alu).

D FACS analysis of CD34+ cells grown in pro-MK medium and expressing AS-RBM15 full length or ∆5 or ∆3. The CD41+CD42+ cell percentages were analyzed using one way ANOVA, ** P<0.01, (n=4, mean ± SD).

E The percentage of CD41+CD42+ cells in CD34+ cells expressing AS-RBM15 Exon 1 were determined by FACS analysis after these cells were grown in pro-MK medium for five days. P value was calculated by t test, p<0.01, (n=3, mean ± SD).

Also see Figure EV 2.
The AS-RBM15 lncRNA enhances RBM15 protein translation during megakaryocyte differentiation (2016)
Figure 3

Figure 3. AS-RBM15 upregulates RBM15 protein level.

A RBM15 protein levels in leukemia cell lines (MEG-01, K562, HEL) were assessed by Western blotting (WB) (left) (n=3). The mRNA levels of RBM15 in cells overexpressing AS-RBM15 were measured by real-time PCR (right, n=3, mean ± SD). GAPDH was used as an internal control for WB and PCR.

B Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing two different shRNAs against AS-RBM15 respectively were measured using WBs and real-time PCR assays, respectively (n=3, mean ± SD).

C Protein levels (left) and mRNA levels (right) of RBM15 in MEG-01 cells expressing truncated AS-RBM15 RNAs (the schematic as shown on Fig 2C) were measured using WB and real-time PCR respectively (n=3, mean ± SD).

D The role of AS-RBM15 in protein translation was assessed by reporter assays. The reporter under control of the CMV promoter contains the 5’UTR of RBM15 mRNA ligated to firefly luciferase followed by an IRES element controlling Renilla luciferase as an internal control. The reporter plasmid was cotransfected with plasmids expressing full-length, exon 1 and Δ5 AS-RBM15 respectively. The vector reporter without 5’UTR is a negative control. (n=3, mean ± SD). P values were calculated by one-way ANOVA test (*p<0.05, **p<0.01 and ***p<0.001).

Source file is available for this figure.