EMBO/soda-nel huggingface dataset:
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 4

Figure 4. EWS interacts with PARylated PARP1 using RGG domains at DNA damage sites.

(A) PARP1 and EWS interaction was visualized in cells using the CUPID system. After transfection of mRCD-EWS and GC3-PARP1 (EGFP-PARP1) plasmid, the cells were treated with either H2O2 (1 mM, 10 minutes) or PMA. Scale bar indicates 5 μm. (B) Endogenous interaction between EWS and PARP1 was measured using immunoprecipitation western blot analysis. Cells were treated with H2O2 (1 mM, 10 minutes) with or without Olaparib (5 μM, 7 hours). (C) MMS-treated cells, with or without Olaparib, were immunoprecipitated using an anti-EWS antibody and immunoblotted by anti-PAR antibody (upper). Pre-Olaparib group was treated with Olaparib for 3 hours before MMS treatment (0.02% 30 minutes) and after-Olaparib group treated Olaparib after 10 minutes following treatment of MMS. Lower blot indicates input of anti-EWS antibody. (D) In vitro PAR binding assay. THRAP3 binding to PAR was used as a positive control and BSA binding to PAR was used as a negative control. (E) Schematic map of nine EWS mutants (left) and IP-western blot analysis (right). GFP-PARP1 and siPARG were transfected into HEK-293 cells with each EWS mutants plasmid. After H2O2 treatment, the cells were conducted to IP with anti-Flag antibody. (F) Western blot analysis of PARP1 accumulation on chromatin. EWS-WT and M4 mutants were transfected into EWS depleted HEK-293 cells. After treatment of MMS (0.02%, 1 hour), the proteins were fractionated to either chromatin-bound or soluble fraction. (G) Quantification of relative amount of PARP1 and γH2AX on chromatin, divided by chromatin H3, from three independent western experiments are presented. Data represented as mean ±SEMs, were measured from 3 independent experiments. Significance determined by t-test. * P < 0.05, **P < 0.01. n.s indicate non-significance, (H) WT and Ews-/- cells were transfected with EWS-WT and -M4 mutant and cell viability was measured 24 hours after MMS treatment (indicated concentration). Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, **P < 0.01. n.s indicate non-significance,
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 5

Figure 5. EWS regulates genomic integrity in a PARP1-dependent manner.

(A) Relative cell viability of cells transfected with siCon (WT), siEWS (EWS KD), PARP1 (PARP-1 KD), and double siRNA (DKD) after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. Analysis (***) indicates differences between DKD with EWS KD. (B) Additional Parp1 knockout in Ews knockout cells (DKO) were subjected to viability test after MMS treatment. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. *** indicated differences between DKO with EWS-KO. (C) After transfection of siCon, siEWS, PARP1, and double siRNA, the effect of co-depletion of PARP1 and EWS on the γH2AX and phosphorylated CHK1 upon MMS treatment was analyzed by western blot. (D) After treatment of MMS, the effect of additional Parp1 knockout in Ews knockout cells (Double KO) to the γH2AX and phosphorylated CHK1 were analyzed using western blot. (E) Whole cell expression of PAR in mBA (WT: Ews-WT, KO: Ews-KO, DKO: double KO). (F) Relative NAD+/NADH ratios were measured in WT, Ews-/- and DKO cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. (G) PARGi (5 μM, 24 hours) treated WT and Ews-/- cells were subjected to cellular viability assay. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA. (H) Relative cellular viability was measured after NMN treatment, with or without MMS pretreatment, in WT and Ews-/- cells. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. (I) Chromatin-bound PARP1 was quantified by western blot in WT and Ews-/- cells treated with Olaparib (5 μM, 24 hours) treatment. (J) Relative viability was measured in WT and Ews-/- cells upon treatment of Olaparib for 24 hours. Error bars represent as mean ±SEMs, and technical repeats (n=3). Significance determined by Two-way ANOVA, ***P < 0.001. (K) Table of Ews-/- pups survival number in control (normal water) or NMN supplements (NMN containing water) groups 3 days after birth. Statistics (χ²) were determined by chi-square test.
Ewing sarcoma protein promotes dissociation of poly(ADP-ribose) polymerase 1 from chromatin (2020)
Figure 6

Figure 6. Accumulation of PARP1 or hyper-PARylation in human Ewing Sarcoma patient-derived samples.

(A) Western blot analysis of PARP1 in chromatin. Two Ewing Sarcoma cells (CHP100 and A4573) were treated with MMS (0.02%, 1 hour) with or without release (1 hour) from MMS (N.T: non treat, T: treat, R: release sample). (B) U2OS and two Ewing Sarcoma cells were treated MMS (0.02%, 30 minutes) and subjected to immunofluorescence by PARP1 antibody after chromatin bound fraction. Scale bar indicates 20 μm (left). Red line indicates mean and more than 300 cells were analyzed. Significance determined by Two-way ANOVA, ***P < 0.001. (C) Human Ewing Sarcoma histology samples were analyzed by immuno-histochemistry using PAR antibody. Scale bar represents 100 mm. (D) Model of EWS role in genomic integrity.

Expanded View figure legends
The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy (2013)
f1

a, Starvation (3 h)-induced autophagy results in a downregulation of H4K16ac in histone extracts of MEF cells. b, Upon rapamycin treatment (300 nM) LC3 conversion and downregulation of H4K16ac are observed in WT MEF cells but not in the autophagy-deficient atg5−/− and atg7−/− MEF cells. c, Rapamycin treatment increased the LC3-II/LC3-I ratio and promoted H4K16ac decrease in sirt1−/− and WT MEF cells. d, Rapamycin-induced autophagy led to downregulation of H4K16ac at 48 h in histone extracts of HeLa and U20S cells, and after 6 h in U1810 cells. e, Quantification of H4K16 acetylation by immunoblotting is depicted for rapamycin-treated cells. Data are expressed as mean ± s.e.m. (n = 3–5); *P  0.05; **P  0.01.
The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy (2013)
f3

Rapamycin treatment (48 h) promoted the downregulation of the H4K16 histone acetyltransferase hMOF expression level in MEF (a) and transfected HeLa cells (b). VPA (1 mM) treatment counteracted rapamycin-induced H4K16ac downregulation (c) and decreased the LC3 ratio (d). e, Co-treatment with chloroquine (CQ, 10 µM) showed that the decrease in LC3 ratio was a result of an increase in autophagic flux. f, Inhibition of autophagy by CQ after hMOF overexpression shows that hMOF does not inhibit autophagic flux. g, The yeast homologue of hMOF, Sas2, tagged with 3× HA showed a complete disappearance of HA signal upon autophagy induction after 3 h. h, Overexpression of Sas2 repressed the downregulation of H4K16ac upon rapamycin treatment.
The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy (2013)
f4

a, VPA increases the autophagic flux in rapamycin-treated HeLa cells transfected with the mRFP–GFP–LC3 tandem reporter construct which allows distinction between autophagosomes (GFP+/RFP+ yellow puncta) and autolysosomes (GFP−/RFP+ red puncta). b, Confocal microscopy image of a cell treated with rapamycin and VPA depicting a high ratio of red to green LC3 puncta indicating an increase in autophagic flux. c, Co-treatment with VPA and rapamycin led to increased cell death. d, Co-treatment with CQ abrogated VPA + rapamycin-induced cell death. e, f, Increasing H4K16ac levels by either overexpression of hMOF, inhibition of SIRT1 by siRNA or the chemical inhibitor Ex527 promoted cell death upon rapamycin treatment. Co-treatment with the autophagy inhibitor bafilomycin A1 (BafA, 40 nM) (g) or 3-methyladenine (3MA, 5 mM) (h) abrogated VPA + rapamycin-induced cell death. Treatment of HeLa cells upon amino-acid starvation with VPA induced cell death (i), which was rescued when cells were co-treated with CQ (j). Data are expressed as mean ± s.e.m. (n = 3).
The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA (2020)
Figure 2

Figure 2. TruB1 selectively promotes the expression of let-7 family miRNAs

(A). Volcano plot of TaqMan array showing the mean average expression and p-value of miRNA expression profiles between TruB1 KD and ctrl (scramble). N=3. Significance was assessed using 2‐tailed Student's t‐test. MiRNAs suppressed (Relative expression TruB1 KD / ctrl <0.8 and p-value < 0.1) are enclosed by the red square.

(B). Northern blotting for let-7a, miR-34a and RNU6B in HeLa cells with TruB1 KD or ctrl (siRNA).

(C). Processing rates were quantified and normalized to ctrl based on hybridization intensities of (B). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(D). Relative expression of let-7 family miRNAs and other miRNAs in HEK293FT cells with TruB1 KD or ctrl (siRNA) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(E). Relative expression of primary-let-7 family miRNAs as in (D) determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

Data information: All experiments were performed in triplicate. Error bars show SD.
The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA (2020)
Figure 3

Figure 3. TruB1 promotes let-7 processing independently of its enzymatic activity.

(A). Amino acid sequences encoding for the enzyme activity and RNA binding ability in E.coli TruB and human TruB1 (Top). Design of mutant 1 (mt1) and mutant 2 (mt2) (bottom).

(B). In vitro enzyme activity assay. 32p-UTP-labelled tRNAphe were treated with recombinant TruB1, mt1, or mt2. The strong upper bands represent uridine (U), and the lower weaker bands represent pseudouridine (Ψ) on autoradiographs of the TLC plate.

(C). Relative miRNA expression of let-7a in HEK-293 cells infected with tetracycline-inducible expressing lentiviruses for TruB1, mt1, mt2 or GFP 5 days after doxycycline treatment, as determined by qRT-PCR. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(D). Northern blotting for let-7a and RNU6B in HeLa cells infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP, 5 days after doxycycline treatment.

(E). Hybridization intensities of (D) were quantified and normalized to ctrl (GFP). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(F). Pseudouridylation activity of TruB1 for tRNA and pri-miRNAs. 32p-UTP-labelled tRNAphe, pri-let-7a1 or pri-miR-10a were treated with recombinant TruB1. Upper bands represent uridine (U), lower bands represent pseudouridine (Ψ) in autoradiographs of the TLC plate.

(G). Location of pseudouridine sites detected by the CMC primer extension method. Total RNA purified from HEK-293FT cells were treated with CMC. CMC treated RNA were reverse-transcribed with RI-labelled specific primers for tRNAphe or pri-let-7b. ddATP was used for sequence control. Pseudouridines are indicated by black arrows.

Data information: All experiments were performed in triplicate. Error bars show SD.
The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA (2020)
Figure 4

Figure 4. TruB1 binds to tRNA and primary-let-7

(A). RIP analysis of pri-let-7a1 and Flag-TruB1 from HEK-293FT cells. RNA was extracted from IP material and analyzed by qRT-PCR. HEK-293FT cells were infected with lentiviruses encoding tetracycline-inducible expression of TruB1, mt1, mt2, or GFP and treated with doxycycline. Error bars show SD; n=3. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(B). EMSA of 32p-ATP-labelled pri-let-7a1 or pri-let-7a1 loop mt mixed with recombinant TruB1, mt1 or mt2 at several doses. RNP: Ribonucleoprotein complexes.

(C). Design of Flag-labelled TruB1 knock-in (KI) cells. HiBIT and 3 x Flag sequences were inserted into the N-terminus of the TruB1 gene in HEK-293FT cells.

(D) Sequencing clusters obtained from HITS-CLIP experiment. Left are for let-7, and right for tRNAs.

(E). Tag densities represented as dots. Density of miRNA clusters in HITS-CLIP were normalized by miRNA expression as determined by TaqMan array.

(F). The proportions of different types of mapped transcripts from the HITS-CLIP experiment.

(G). Sequence motifs from reads mapped to tRNAs from the HITS-CLIP experiment.

(H). TruB-modified site in tRNAs. Location of pseudouridine is colored in red.

(I). Sequence of the terminal loop in pri-let-7a1. Lin28B binding site is colored in red.
The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA (2020)
Figure 5

Figure 5. TruB1 promotes the microprocessing of primary let-7 and enhances binding of the microprocesser to primary let-7

(A). In vitro processing assay for RI-labelled pri-let-7a1. Autoradiographs of gels showing pri-let-7a1 treated with whole cell lysate (WCL) from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right). Dicer KD was used to show the height of pre-let-7a1.

(B) RI intensities of (A) were quantified and normalized to ctrl. Relative processing rate of pri-let-7a1 into pre- and mature- are shown. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(C). RIP assay of pri-let-7a1 and DGCR8 from HEK-293FT cells. Western blotting for input or immunoprecipitate (IP) using anti-DGCR8 antibody and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(D). RIP assay of pri-let-7a1 and Flag-tagged TruB1 from HEK-293FT cells with Lin28B KD or ctrl (siRNA). Western blotting for input or IP material using anti-Flag antibody, anti-Lin28B antibody, and anti-actin antibody are shown on top. RNA was extracted from IP material and analyzed by qRT-PCR (bottom). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

Data information: All experiments were performed in triplicate. Error bars show SD.
The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA (2020)
Figure 6

Figure 6. TruB1 suppresses cell growth by regulating let-7

(A). Design of luciferase reporter vector comprising the let-7 target region of KRAS mRNA (KRAS reporter).

(B). Relative luciferase activity of KRAS reporter or Ctrl (empty) reporter in HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1, mt1, mt2, or GFP 5 days after doxycycline treatment. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(C). Protein expression of KRAS. Protein expression was evaluated by Western blotting. Protein was isolated from HEK-293FT cells transfected with GFP, TruB1, mt1, or mt2 (overexpression, left), and TruB1 KD or ctrl (siRNA, right).

(D). Relative expression of let-7 families determined by qRT-PCR in HEK-293FT cells with KD of let-7 family members or ctrl (using 2'-O-methylated antisense inhibitor). Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(E). Real-time glo assay for HEK293FT cells infected with lentiviruses expressing tetracycline-inducible TruB1 or GFP, 5 days after doxycycline treatment, with or without KD of let-7 family members. GFP: GFP without let-7 KD. WT: overexpression of wild type TruB1 without let-7 KD. WT + let7 KD: WT: overexpression of wild type TruB1 with let-7 KD. GFP + let-7KD: GFP with let-7 KD. Significance was assessed using 2‐tailed Student's t‐test, < 0.05*.

(F). Schematic model for TruB1-dependent induction of let-7.

Data information: All experiments were performed in triplicate. Error bars show SD.

Expanded View Figure Legends
ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export (2021)
Figure 1

Figure 1. Depletion of Zfp207 leads to growth defects of mouse ESCs

A, B. RT-qPCR analysis of Zfp207 and Oct4 in mouse ESCs during (A) retinoic acid (RA)-induced and (B) embryoid body (EB)-mediated differentiation. Nestin was used as a neuronal differentiation marker to monitor RA-mediated differentiation. mRNA levels are relative to the expression at day 0.

C, D. Western blot of ZFP207 and OCT4 of mouse ESCs during (C) RA-induced and (D) EB-mediated differentiation.

E, F. (E) RT-qPCR and (F) western blot to monitor the knockdown efficiency of Zfp207 (sh1 and sh2) (shScr).

G. Cell proliferation rate of shScr, sh1 and sh2 ESCs assessed over a period of 6 days.

H. Percentage of live (Annexin V-) and apoptotic cells (Annexin V+) in sh1 and sh2 mouse ESCs compared to shScr.

I. Bar chart displaying the cell cycle distribution in sh1 and sh2 mouse ESCs relative to shScr.

J, K. (J) AP staining of shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Scale bars, 20 µM. (K) Percentage of fully differentiated (FD), partially differentiated (PD) and undifferentiated (UN) ESC colonies in shScr, sh1 and sh2.

L. Immunofluorescence analysis of SSEA1 in shScr, sh1 and sh2 ESCs. DAPI was used as the nuclear marker. Scale bars, 20 μm.

M, N. (M) Western blot of ZFP207, OCT4, NANOG, and SOX2 in shScr, sh1 and sh2 ESCs and (N) RT-qPCR analysis of Oct4, Nanog, and Sox2 in shScr, sh1 and sh2 ESCs; data is relative to shScr.

O, P. (O) Western blot of OCT4 during a 9-h cycloheximide (CHX) time course treatment in shScr, sh1 and sh2 ESCs. (P) Protein degradation curves were made after quantification and normalization of the bands from (O).

Q, R. (Q) Western blot of OCT4 during 4 and 8 h treatment with the proteasome inhibitor MG132. (R) graph bars after quantification and normalization of the bands from (Q).

Data information: data are presented as mean ± SEM or representative images of n ≥ 3 independent biological experiments. §P < 0.05, §§P < 0.01, §§§P < 0.001 (D0 vs. other indicated days). *P < 0.05, **P < 0.01, ***P < 0.001, ns = no significant (shScr vs. sh1 or sh2). A, B, G, H and I: unpaired Student's t-test; E, N, P and R: ordinary one-way ANOVA.
ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export (2021)
Figure 2

Figure 2. Loss of Zfp207 results in defective differentiation

A, B. (A) Bright-field images (10x (left) and 4x (right) magnification) and (B) quantification of embryoid bodies (EB) generated from shScr, sh1 and sh2 at day 8 of differentiation. Scale bars, 200 µM.

C. Quantification of BrdU incorporation in shScr, sh1 and sh2 ESCs at the indicated days of EB differentiation. Data are relative to shScr.

D. RT-qPCR of Zfp207, the pluripotency genes (Oct4, Nanog and Sox2), the mesodermal markers (Msx1 and Brachyury, T), the endodermal markers (Foxa2, Sox17), and the neural-associated genes (Pax6, Sox11 and Nestin) in shScr, sh1 and sh2 ESCs along the time-course of EB-mediated differentiation. mRNA levels are relative to the expression of shScr at day 0.

Data information: data are presented as mean ± SEM or representative images of n ≥ 3 independent biological experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2). B, C: unpaired Student's t-test; D: ratio paired Student's t-test.
ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export (2021)
Figure 3

Figure 3. ZFP207 is essential for neural cell fate specification

A. Schematic depiction for the neuroectodermal-directed differentiation. DMEM/F12 supplemented with N2B27and retinoic acid (RA) was added after two days of EB culture.

B. Immunostaining of NESTIN (green) and TUJ1 (red) of neural progenitors generated from shScr on day 8 and 10 of the neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 μM.

C-E. (C) TUNEL (green), (D) CASPASE 3 (red) staining and (E) quantification of the signal in shScr, sh1 and sh2 at day 5 of neuroectodermal differentiation. Nuclei were counterstained with DAPI. Scale bar, 20 µM.

F. Flow cytometric profile and quantification of BrdU incorporation at day 4 of neuroectodermal differentiation.

G. RT-qPCR of Zfp207 and neural-associated markers in shScr, sh1 and sh2 along the course of neural differentiation. mRNA levels are relative to shScr at day 0.

Data information: data are presented as mean ± SEM or representative images of n ≥ 3 independent biological experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (shScr vs. sh1 or sh2). E, F and G: unpaired Student's t-test.
ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export (2021)
Figure 4

Figure 4. The neural transcriptome is not altered upon depletion of Zfp207

A, B. (A) Volcano plots of common differentially expressed genes in shScr and Zfp207-depleted (sh1 and sh2) mouse ESCs. Up-regulated (Up) and down-regulated (Down) genes are indicated in red and blue, respectively (p < 0.05; > 1.5-fold). Zfp207 is depicted in black. Grey dots indicate non-significant (NS) and < 1.5-fold differential expressed genes. (B) Venn diagram depicting the overlap of down-regulated and up-regulated genes between sh1 and sh2 ESCs (FC >1.5 and p < 0.05). FDR value was calculated with the Benjamini-Hochberg correction.

C, D. Gene ontology (GO) analysis of biological processes associated with common (C) down-regulated and (D) up-regulated genes in Zfp207-depleted ESCs (sh1 and sh2); NHEJ: Non-homologous end joining.

E-H. Heatmap of column z-scores of log2 transformed values of genes related to (E) pluripotency and (G) ectoderm in shScr, sh1 and sh2 ESCs. GSEA plots depicting the expression of (F) ESC-related and (H) ectodermal genes in Zfp207-depleted ESCs compared to shScr. High and low expression of genes is represented in red and blue color, respectively. FDR, false discovery rate; NES, normalized enrichment score.
ZFP207 sustains pluripotency by coordinating OCT4 stability, alternative splicing and RNA export (2021)
Figure 5

Figure 5. Silencing of Zfp207 leads to AS defects

A. Heatmap of column z-scores of log2 transformed values of genes involved in alternative splicing (AS) in shScr, sh1 and sh2 mouse ESCs.

B. RT-qPCR analysis of Rbm3, Mbnl2 and Sf3b3 in shScr, sh1 and sh2 ESCs. mRNA expression is relative to shScr and presented as mean ± SEM of n ≥ 3 independent biological experiments; *P < 0.05, **P < 0.01, ***P < 0.001 (shScr vs. sh1 or sh2); unpaired Student's t-test.

C. Violin plot depicting statistically significant spliced in (PSI) events in each category in sh1 and sh2 related to shScr. Alternative 3´ (A3); Alternative 5´ (A5); Alternative first exon (AF); Alternative last exon (AL); Mutually exclusive exons (MX); Intron retention (RI); Exon skipping (SE). The dotted line indicates ΔPSI = 0.

D, E. (D) Venn diagram showing the overlap of AS in sh1 and sh2. (E) Gene ontology analysis of common genes undergoing AS in sh1 and sh2 mouse ESCs according to the type of AS event.

F. RT-PCR of Zfp207-regulated AS events. For Mta1, AS1 (alternative splicing 1) and AS2 (alternative splicing 2). #1 and #2 indicate distinct biological replicates. *Denotes an isoform that was not taken in consideration for the quantification. The structure of each isoform is indicated (not to scale). Alternative exons are blue. The percent spliced in (PSI) was quantified for each condition.