EMBO/soda-nel huggingface dataset:
Cell softness regulates tumorigenicity and stemness of cancer cells (2020)
Figure 6

Figure 6. BCL9L highly expressed in soft tumor cells from cancer pateints.

(A) The stiffness of primary breast (HBC, n = 3) and colon (HCC, n = 3) cancer cells screened from the outlet or backflow inlet was measured by AFM. n = 100 from each patient.

(B) The correlation between the percentage of soft primary tumor cells from breast (n = 11) or colon (n = 23) cancer patients and the grade of breast or colon cancer, respectively.

(C) The stiff and soft tumor cells were isolated from 3 patients with breast cancer and stained with anti-BCL9L antibody. The expression of BCL9L was observed under confocal microscope. Scale bar, 10 μm.

(D) The tissue sections from 6 melanoma patients with high grades of cancer and 3 melanoma patients with low grade of malignancy were immunohistochemical staining with anti-BCL9L antibody. Scale bar, 50 μm.

(E) The correlation between the expression of BCL9L from 18 melanoma patients and the grade of melanoma.

(F) Overall survival compared to the BCL9L level in breast cancer (BRCA, n = 536) or melanoma (n = 376) patients.

Data information: Mann-Whitney test (A), Paired Student's t test (C), Pearson's correlation test (B and E) or Log-rank survival analysis (F). The data represent mean ± SD.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 1

Figure 1. METTL14 C-terminal IDR is arginine methylated in vitro and in cells.

Schematic representation of the domain structure of METTL14. The C-terminal IDR, containing an array of RGG motifs, is highlighted. NHM: N-terminal α-helical motif; MTase: Methyltransferase domain; IDR: Intrinsically disordered region.

METTL14 is arginine methylated in vitro. In vitro methylation assays were performed by incubating recombinant PRMTs (1-8) with purified GST-tagged METTL14.

METTL14 is arginine methylated at its C-terminal IDR in vitro. GST-tagged full-length (FL) and C-terminal IDR-truncated (1-400) METTL14 were incubated with recombinant PRMT1 and PRMT6.

METTL14 is arginine methylated in cells. Endogenous METTL14 was immunoprecipitated from HEK293 cells under denaturing conditions and detected using the ADMA antibody ASYM26.

Inhibiting type I PRMT activity reduces METTL14 arginine methylation. HEK293 cells were treated with the type I PRMT inhibitor MS023 (1 μM, 48 h). METTL14 was immunoprecipitated from the cells and detected by Western blot analysis using anti-METTL14 and anti-ASYM26 antibodies.

METTL14 is arginine methylated at its C-terminal IDR in cells. HEK293 cells expressing Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14 were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was analyzed by Western blot using two different antibodies that recognize ADMA.

Data information: In both (B) and (C), Ponceau staining shows the loading of the recombinant proteins. The black dots indicate PRMT enzymes; black triangles indicate arginine methylated-METTL14; open triangles indicate recombinant METTL14 proteins. In (D-F), the black triangles indicate arginine methylated-METTL14; open triangles indicate the immunoprecipitated METTL14 proteins.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 2

Figure 2. PRMT1 catalyzes METTL14 C-terminal IDR arginine methylation.

PRMT1 interacts with METTL14 in cells. HEK293 cells expressing GFP-tagged PRMT1, PRMT3, or PRMT6 were lysed and immunoprecipitated with an anti-METTL14 antibody, followed by Western blot analysis using an anti-GFP antibody. * indicates the location of the IgG heavy chain.

Endogenous METTL14 interacts with PRMT1. The reciprocal endogenous co-immunoprecipitation (IP) assays were performed using the METTL14 antibody for IP and the PRMT1 antibody for Western blot detection (left panel) and using the PRMT1 antibody for IP and the METTL14 antibody for Western blot detection (right panel).

The C-terminal IDR of METTL14 is essential for its interaction with PRMT1. HEK293 cells were transfected with GFP-tagged PRMT1 and Flag-tagged FL or C-terminal IDR-truncated (1-400) METTL14. IP was performed using an anti-GFP antibody, and Western blot analysis was performed using anti-GFP and anti-Flag antibodies.

GST pull-down detection of the interactions of PRMT1 with GST-tagged FL and truncated (1-400) recombinant METTL14. The black triangles indicate recombinant METTL14 proteins.

Overexpression of PRMT1 enhances METTL14 arginine methylation. HEK293 cells were transfected with either GFP vector or GFP-PRMT1, together with Flag-METTL14. The level of METTL14 methylation was detected by IP using an anti-Flag antibody, followed by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.

Knockdown of PRMT1 expression reduces METTL14 arginine methylation. HEK293 cells were transfected with control siRNA (siCtrl) and the siRNA targeting PRMT1 (siPRMT1). METTL14 was immunoprecipitated from these cells, and its methylation level was detected by Western blot analysis using an anti-ASYM26 antibody. * indicates the location of the IgG heavy chain.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 3

Figure 3. C-terminal IDR arginine methylation enhances METTL14-RNA interactions and METTL3/METTL14 RNA methylation activity.

Summary of METTL14 arginine methylated peptides identified by LC-MS/MS.

METTL14 IDR arginine methylation occurs at multiple arginine residues within RGG/RG motifs. Mutation of five arginine sites identified from mass spectrometry reduces METTL14 arginine methylation, but only mutation of all arginine sites to lysine completed blocked METTL14 methylation. Ponceau S staining shows the loading of the recombinant proteins. The black triangles indicate arginine methylated-METTL14; open triangles indicate recombinant METTL14 proteins.

METTL14 is methylated at multiple arginine residues in cells. HEK293 cells expressing Flag-tagged WT or various R-to-K METTL14 mutants were lysed and immunoprecipitated with an anti-Flag antibody. Arginine methylation of immunoprecipitated METTL14 was detected by Western blot analysis using an anti-ADMA antibody. Both short and long exposures of the chemiluminescence signals are shown.

Arginine methylation of the METTL14 IDR enhances its interaction with RNA substrates. RNA pull-down assay was performed by incubating biotin-labeled RNA with WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14. The pull-down samples were detected by Western blot analysis using an anti-Flag antibody. The methylation status of the recombinant proteins was confirmed by Western blot analysis using an anti-ADMA antibody.

EMSA was performed to compare the interactions of WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 with 6-FAM-labeled RNA. Arrow indicates the shift of the RNA probe caused by the protein-RNA interaction. Coomassie staining shows the increasing amounts of recombinant proteins used in the assay.

Fluorescence polarization assays were performed by incubating 6-FAM-labeled RNA with WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14. Each point represents the average of three independent replicates and error bars represent standard deviation (SD). The dissociation constant values (Kd) were listed as mean ± SD.

Arginine methylation of the C-terminal IDR enhances the RNA methylation activity of the METTL14/METTL3 complex in vitro. In vitro RNA methylation assays were performed by incubating biotin-labeled RNA substrates with METTL3/METTL14 methyltransferase complexes containing WT, hypomethylated (MS023), and arginine methylation-deficient (RK) mutant METTL14 in various concentrations (10-100 nM). The methylation status of the METTL3/METTL14 complex was confirmed by Western blot analysis using an anti-ADMA antibody. Coomassie staining shows the purification of the enzyme complex. Enzymatic activity was measured in counts per minute (c.p.m.) using a scintillation counter. Data from three replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01; ns, not significant.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 4

Figure 4. Arginine methylation of the C-terminal IDR enhances the interaction of METTL14 with RNAPII in cells.

Endogenous METTL14 interacts with RNAPII. Endogenous co-immunoprecipitation (IP) was performed using the METTL14 antibody for IP and the RNAPII antibody for Western blot detection (left panel) and using the RNAPII antibody for IP and the METTL14 antibody for Western blot detection (right panel).

Arginine methylation of the METTL14 C-terminal IDR enhances its interaction with RNAPII. HEK293 cells were transfected with HA-tagged WT or arginine methylation-deficient (RK) mutant METTL14. IP was performed using an anti-HA antibody, and Western blot analysis was performed using the indicated antibodies.

Co-IP assays were performed to compare the interactions between METTL14 and RNAPII in control and MS023-treated HEK293 cells. Cells were treated with either DMSO or MS023 (1 μM) for 48 h before they were lysed. IP was performed using control IgG and METTL14 antibodies, respectively. Western blot analysis was performed using anti-RNAPII, anti-ADMA, anti-METTL14, and anti-METTL3 antibodies.

Co-IP assays were performed to examine the involvement of RNA in the METTL14-RNAPII interaction. Total cell lysates were either left untreated or treated with RNase A to remove the RNA component before IP. Western blot analysis was performed using anti-RNAPII, anti-METTL3, and anti-METTL14 antibodies.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 5

Figure 5. Analysis of METTL14 arginine methylation-dependent m6A sites.

Generation of isogenic mESC lines expressing WT and arginine methylation-deficient mutant (RK) METTL14. Mettl14 KO mESCs were transfected with Flag-tagged WT or RK mutant METTL14 using a lentivirus expression system. The expression of METTL14 and METTL3 in these cells was detected by Western blot analysis using anti-METTL14 and anti-METTL3 antibodies. ACTIN was used as a loading control.

m6A levels are reduced in mESCs expressing arginine methylation-deficient (RK) mutant METTL14. The mRNA purified from WT, Mettl14 KO, KO+WT, and KO+RK mESCs was subjected to LC-MS/MS analysis to quantify m6A levels (presented as the m6A/A ratio).

Morphology and alkaline phosphatase (AP) staining of mESCs expressing WT, Mettl14 KO, KO+WT, and KO+RK METTL14.Scale bar: 400 µM

Quantification of AP-positive clones in (C).

Proliferation of mESCs expressing WT, Mettl14 KO, KO+WT, and KO+RK METTL14 over a 5-day period. Each point represents the average of three independent replicates and error bars represent standard deviation (SD).

Sequence motifs of m6A-enriched regions in KO+WT and KO+RK mESCs (upper panels). Topological distribution of normalized m6A peaks across the 5' UTR, CDS and 3' UTR of mRNAs (lower panels).

Cumulative distribution of log2 m6A peak intensity in KO+WT and KO+RK mESCs. Statistical analysis was performed using the Wilcoxon test to measure the median difference of peak intensities between the two groups.

Overlay of m6A distributions across the 5' UTR, CDS, and 3' UTR of mRNAs in KO+WT and KO+RK mESCs (upper panel). Statistical analysis of differential m6A peaks in KO+RK vs. KO+WT mESCs (lower panel). The y-axis represents the q-value (-log10). The dashed grey line indicates q-value = 0.05.

Gene Ontology (GO) analysis of genes harboring METTL14 arginine methylation-dependent m6A sites. Statistical analysis was performed using Hypergeometric test. The p-value for the enrichment of each biological process (GO term) is shown.

Data information: In (B), (D), and (E), data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 6

Figure 6. METTL14 arginine methylation-dependent m6A sites are associated with enhanced translation of DNA repair genes

UCSC Genome Browser custom tracks of m6A-seq reads along the indicated mRNAs in WT, Mettl14 KO, KO+WT, and KO+RK mESCs. The y-axis represents the normalized number of reads. Blue reads are from non-immunoprecipitated input libraries, and red reads are from m6A-IP libraries. Above the custom tracks, the thick blue boxes represent the protein coding regions (CDSs), the thin blue boxes represent the untranslated regions (UTRs), and the blue lines represent introns. The bars at the bottom of the custom tracks indicate the amplicon locations for MeRIP (m6A-IP)-qPCR assays (B) and METTL14 RIP-qPCR assays (C) to detect m6A-positive (red) and negative (blue) regions.

MeRIP (m6A-IP)-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to validate the MeRIP-seq results. m6A-negative regions of the transcripts (blue) were included as negative controls.

METTL14 RIP-qPCR assays were performed for WT, Mettl14 KO, KO+WT, and KO+RK mESCs to compare the binding of WT and RK mutant METTL14 to mRNA targets. Primers (red color) that amplify m6A positive regions of the transcripts were used.

The expression of ICL repair genes is reduced in mESCs expressing arginine methylation-deficient mutant (RK) METTL14. Total cell lysates from WT, Mettl14 KO, KO+WT, and KO+RK mESCs were subjected to Western blot analysis using the indicated antibodies.

Polysome profiling was performed for KO+WT and KO+RK mESCs. Whole-cell extracts were fractionated through centrifugation in a sucrose density gradient. Optical scans (OD260) of the collected fractions are shown.

Quantification of ribosome-bound mRNA for the indicated genes from individual fractions (as in (E)), relative to the amount of the total mRNA in all fractions. Gapdh was included as a negative control.

Data information: In (B), (C), and (F), data from three independent replicates were analyzed by Student's t-test and shown as mean ± SD. *, p < 0.05; **, p < 0.01, ***, p < 0.001.
m6A deposition is regulated by PRMT1-mediated arginine methylation of METTL14 in its disordered C-terminal region (2020)
Figure 7

Figure 7. Loss of METTL14 arginine methylation sensitizes mESCs to DNA damage.

mESCs expressing arginine methylation-deficient mutant (RK) METTL14 are sensitive to ICL damage induced by MMC. WT, Mettl14 KO, KO+WT, and KO+RK mESCs were treated with various concentrations of MMC for 4 days before cell viability was measured.

Similar to (A), except that mESCs were treated with cisplatin, another ICL-inducing chemical, at various concentrations.

Proposed model for METTL14 C-terminal IDR arginine methylation-mediated regulation of m6A RNA modification and its effects on ICL DNA repair. PRMT1-mediated arginine methylation of the C-terminus IDR of METTL14 promotes its interactions with RNA substrates and RNAPII, which enables efficient m6A deposition on transcripts involved in ICL repair. The deposition of m6A enhances the translation efficiency of these DNA repair genes, promoting the recovery of mESCs from DNA damage. Inhibiting METTL14 arginine methylation using the type I PRMT inhibitor MS023 reduces m6A deposition and the protein synthesis of ICL repair genes, thus sensitizing mESCs to DNA damage-induced cell death.

Data information: In both (A) and (B), each point represents the average of three independent replicates and error bars represent standard deviation (SD). Statistical analysis was performed using Student's t-test. *, p < 0.05.

Expanded View Figure legends
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 1

Baseline characterization of mice with cardiac-specific LXRα overexpression

A Murine LXRα DNA construct for the generation of transgenic (LXRα-Tg) mice.

B–D Left ventricular LXRα expression in Wt and LXRα-Tg mice aged 12 weeks. (B) Western blot of LXRα in liver and LV tissue. Quantification of (C) mRNA expression (normalized to 36b4; n = 6–8/group, *P < 0.00001 versus Wt) and (D) protein expression (normalized to GAPDH; n = 4/group, *P = 0.0001 versus Wt).

E LV weight to tibia length ratios (LV/tibia); n = 15/group. *P = 0.02.

F Assessment of mean arterial pressure (MAP) with in situ catheterization shows no significant differences.

G Representative Masson’s trichrome and wheat germ agglutinin (WGA)-FITC staining in cross-sectional LV, scale bars = 100 μm and 10 μm, respectively.

H Relative mRNA levels of known LXRα-associated and target genes assessed with RT–PCR; n = 8/group. *P = 0.0003, #P = 0.0004, §P = 0.043, †P = 0.0009 versus Wt.

Data information: Data are means ± SEM; Student’s paired 2-tailed t-test was used to compare groups. Source data are available online for this figure.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 2

LXRα overexpression attenuates TAC-induced cardiac hypertrophy and fibrosis after 5 weeks

LV/tibia ratios in sham- and TAC-operated Wt and LXRα-Tg mice; n = 21 Wt sham, n = 24 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-Tg TAC. *P < 0.00001 versus respective sham, #P = 0.00004.

Quantification of cardiomyocyte cross-sectional area from WGA-FITC-stained histological sections; n = 5 Wt sham, n = 4 LXRα-Tg sham, n = 5 Wt TAC, n = 3 LXRα-Tg TAC. **P = 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.01.

Representative LV sections stained with WGA-FITC and Masson’s trichrome to assess cardiomyocyte hypertrophy and fibrosis, respectively; scale bars = 1 mm, 100 μm, 10 μm, from top to bottom.

Whole area percentage of fibrosis in Masson’s trichrome-stained sections; n = 8 per group, except n = 7 in Wt TAC group. *P = 0.02 versus Wt sham, #P = 0.02.

Measurement of mRNA levels with RT–PCR to assess induction of fetal gene program, as well as hypertrophy (Rcan1) and fibrosis (Col1a1, Ctgf); n = 8 per group, except n = 7 in Wt TAC group. Myh6/Myh7: *P = 0.0001 versus Wt sham, #P = 0.00001; Anp: *P = 0.0005 versus LXRα-Tg sham, #P = 0.02; Bnp: *P = 0.0006 versus Wt sham, **P = 0.009 versus LXRα-Tg sham, #P = 0.03; Acta1: §P < 0.00001 versus Wt sham, *P = 0.01 versus LXRα-Tg sham, #P = 0.001; Rcan1: **P = 0.00004 versus Wt sham, *P = 0.05 versus LXRα-Tg sham, #P = 0.04; Col1a1: *P = 0.00003 versus Wt sham; Ctgf: §P < 0.00001 versus Wt sham, #P = 0.00001.

Data information: Data are means ± SEM; one-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 3

Reduced cardiac dysfunction in mice with cardiac LXRα activation in response to pathological pressure overload

A Echocardiographic assessment of percent fractional shortening in mice subjected to 5 weeks of TAC; n = 19 Wt sham, n = 22 LXRα-Tg sham, n = 24 Wt TAC, n = 26 LXRα-Tg TAC. **P < 0.00001 versus Wt sham, *P = 0.0005 versus LXRα-Tg sham, #P = 0.009.

B–D Hemodynamic measurements obtained in situ in sham- and TAC-operated mice; n = 6–8/group. (B) Mean arterial pressure (MAP) showed no significant differences. (C) LV end-systolic pressure (LVESP). **P < 0.00001 versus Wt sham, *P = 0.00004 versus LXRα-Tg sham, *P = 0.003. (D) LV end-diastolic pressure (LVEDP). *P = 0.02 versus Wt sham.

Data information: Data are means ± SEM; one-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 4

Cardiac LXRα overexpression enhances myocardial glucose uptake

Classification of genes differentially expressed in LXRα-Tg hearts (see Supplementary Fig S3).

Representative 18F-FDG images with microPET in the coronal and axial planes in Wt and LXRα-Tg mice 5 weeks post-TAC.

Myocardial FDG uptake measured as standard uptake value (SUV); n = 4–6/group. *P = 0.04 versus Wt sham, **P = 0.02 versus LXRα-Tg sham, #P = 0.03, ##P = 0.01.

Quantification of myocardial glycogen content shows no significant differences; n = 5 per sham group, n = 7 per TAC group.

GLUT protein expression in LV tissue normalized to GAPDH; n = 6/group. Glut1: *P = 0.01 versus LXRα-Tg sham, #P = 0.03; Glut4: *P = 0.03 versus LXRα-Tg sham, #P = 0.01.

Data information: Data are means ± SEM; one-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups. Source data are available online for this figure.
Cardiac LXRα protects against pathological cardiac hypertrophy and dysfunction by enhancing glucose uptake and utilization. (2015)
Figure 5

LXRα-null mice manifest reduced myocardial glucose uptake capacity in response to TAC

A LV/tibia ratios in sham- and TAC-operated WT and LXRα−/− mice; n = 10 WT sham, n = 8 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P = 0.006 versus WT sham, **P = 0.00008 versus LXRα−/−sham.

B, C Cardiac functional assessment at 5 weeks post-TAC. (B) Percent fractional shortening determined with echocardiography; n = 10 WT sham, n = 7 LXRα−/− sham, n = 12 WT TAC, n = 10 LXRα−/− TAC. *P < 0.00001 versus respective sham, §P = 0.06. (C) LV end-diastolic pressure (LVEDP) recorded in situ; n = 10 WT sham, n = 7 LXRα−/− sham, n = 10 WT TAC, n = 9 LXRα−/− TAC. *P = 0.001 versus WT sham, **P = 0.00005 versus LXRα−/− sham.

D–G Relative mRNA gene expression as determined by RT–PCR, normalized to 36b4; n = 8/group. Anp: no significant differences; Bnp: *P = 0.01 versus WT sham, **P = 0.03 versus LXRα−/− sham; Myh6/Myh7: *P = 0.0002 versus WT sham, **P = 0.0003 versus LXRα−/− sham; Acta1: *P = 0.001 versus respective sham.

H Western blot detection of GLUT protein expression in LV tissue normalized to GAPDH, expressed as fold change, shows no significant differences among groups; n = 6–8/group.

I Representative 18F-FDG-microPET scans.

J Myocardial FDG uptake quantified as standard uptake value (SUV); n = 6/group. *P = 0.0001 versus WT sham, #P = 0.047.

Data information: Data are means ± SEM; one-way ANOVA with Bonferroni’s multiple comparison test was used to compare groups. Source data are available online for this figure.