vectors px330-grna/p150  (Nepa Gene Co Ltd)

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: CARM1 is required for estrogen-induced gene transcriptional activation. (A) MCF7 cells were transfected with control siRNA (siCTL) or siRNA specific against CARM1 (siCARM1) in stripping medium for three days, and then treated with or without estrogen (E , 10 -7 M, 6 hr) followed by RNA-seq. Genes regulated by estrogen were shown (fold change (FC) (siCTL (E )/siCTL (CTL)) ≥ 1.5). (B) Venn diagram showing genes induced by estrogen and dependent on CARM1 for expression (fold change (FC) (siCTL (E )/siCARM1 (E )) ≥ 1.5). (C, D) Heat map (C) and box plot (D) representation of the expression levels for genes induced by estrogen and dependent on CARM1 as described in (B). Heat map: z-score normalized FPKM; box plot: FPKM (log2). (E) MCF7 cells were infected with control shRNA (shCTL) or shRNA specific against CARM1 (shCARM1) in stripping medium for three days, and treated with or without estrogen (E , 10 -7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± s.e.m., **P<0.01, ***P<0.001). (F, G) Wild type (WT) and CARM1 knockout (KO) MCF7 cells were maintained in stripping medium for three days before treating with or without estrogen (E , 10 -7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes (F) and cognate enhancer RNAs (eRNAs) (G) as indicated (± s.e.m., **P<0.01, ***P<0.001). (H, I) MCF7 cells were transfected with siCTL or siCARM1 in stripping medium for three days, and treated with or without estrogen (E , 10 -7 M, 1 hr) followed by RNA Pol II ChIP-seq analysis. The distribution of Pol II was shown for specific genes, as indicated. Boxed regions indicated cognate active enhancers (also see Figure H and I).

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Activation Assay, Transfection, Stripping Membranes, RNA Sequencing Assay, Expressing, Infection, shRNA, RNA Extraction, Quantitative RT-PCR, Knock-Out, ChIP-sequencing

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: CARM1 is recruited onto ERα-bound active enhancers in the presence of estrogen. (A) MCF7 cells treated with or without estrogen (E 2 , 10 -7 M, 1 hr) were subjected to ChIP-seq with anti-CARM1 specific antibody. CARM1 ChIP-seq binding sites in the presence or absence of estrogen was shown by venn diagram (Fold change (FC) (E 2 /CTL) larger than 4 was considered as E 2 specific). (B) CARM1 ChIP-seq tag density distribution centered on estrogen-induced CARM1 sites (± 3,000 bp). (C) Box plot representation of the CARM1 ChIP-seq tag density on estrogen-induced CARM1 sites (± 3,000 bp). (D) Genomic distribution of estrogen-induced CARM1 sites. (E) Motif analysis of estrogen-induced CARM1 distal sites. (F) Pie chart showing estrogen-induced CARM1 distal sites with or without ERα. (G) Heat map representation of CARM1, ERα, H3K4me1, H3K4me2, H3K4me3, H3K27Ac, P300, MED1, H3K9me3 and H3K27me3 ChIP-seq tag density in the presence or absence of estrogen centered on estrogen-induced CARM1 distal sites (± 3,000 bp). (H, I) UCSC Genome browser views of CARM1, ERα, H3K4me1, H3K4me2, H3K4me3, H3K27Ac, p300, MED1, MED12, H3K9me3 and H3K27me3 ChIP-seq in the presence or absence of estrogen on selected active enhancer regions as indicated were shown. Boxed regions indicated cognate active enhancers. ChIP-seq views, except for CARM1, on GREB1 have been shown in our previous study .

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: ChIP-sequencing, Binding Assay

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: Global mapping of CARM1 substrates. (A) Table showing the number of (the sum of mono- and di-methylation sites after removing duplicates) all arginine methylation sites detected (column 1), arginine methylation sites could be quantified (column 2), arginine methylation sites with methylation signals decreased at least two-fold (column 3) or abolished (column 4) in CARM1 knockout (KO) cells detected from mass spectrometry analysis. The number of proteins encompass all these methylation sites was also shown (bottom lane). Proteins with at least one methylation site on which methylation signal was abolished in CARM1 KO cells were referred as “CARM1 methylome”. (B) Overlap between proteins in CARM1 methylome and proteins which abundance was decreased at least two fold in CARM1 KO cells. (C) The distribution of proteins in CARM1 methylome with different number of arginine methylation sites. All the proteins with more than four methylation sites were shown in oval. (D-F) Motif analysis using iceLogo for arginine methylation sites with methylation signals abolished (D), decreased at least two-fold (E) and unchanged (F) in CARM1 KO compared to control cells. (G) Gene ontology (GO) analysis using Metascape for CARM1 methylome. Representative terms from the top 20 enriched GO term clusters were shown. GO term “intracellular estrogen receptor signaling pathway” was highlighted in orange. (H) The distribution of proteins (exclusively methylated by CARM1) with different number of arginine methylation sites. Representative examples with implications in estrogen receptor-mediated transcriptional control were shown in oval.

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Methylation, Knock-Out, Mass Spectrometry

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: MED12 is hypermethylated by CARM1. (A) Experimental flowchart for detecting post translational modifications (PTMs) of C-terminus of MED12 (1616-2177) in wild type (WT) or CARM1 knockout (KO) MCF7 cells. (B) Cell lysates as described in (A) were subjected to immunoblotting (IB) analysis with antibodies as indicated. Actin was served as a loading control. (C) Schematic representation of the domain architecture of MED12 protein. Leucine-rich (L) domain (light green); Leucine-serine-rich (LS) domain (yellow); Proline-glutamine-leucine (PQL) domain (light blue); Poly-glutamine (Opa) domain (purple). Arginine methylation sites identified in the PQL domain were shown by matchsticks. (D) Methylated arginine residues identified in the C-terminus of MED12 following the protocol as described in (A). me1: mono-methylation; me2: di-methylation. PSM: peptide spectrum match. (E) WT and CARM1 KO cells were infected with lenti-viral vectors expressing Flag-tagged MED12 C-terminus (1616-2177), lysed and subjected to immunoprecipitation using anti-Flag antibody followed by immunoblotting (IB) analysis with antibodies as indicated. Anti-H3R17me2(a) antibody was used to detect methylated MED12.

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Knock-Out, Western Blot, Methylation, Infection, Expressing, Immunoprecipitation

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: CARM1-mediated MED12 methylation is involved in estrogen-induced gene transcriptional activation. (A) Heat map representation of CARM1 and MED12 ChIP-seq tag density centered on estrogen-induced CARM1 sites (± 3,000 bp). (B) Correlation between the ChIP-seq tag density (log2) of CARM1 and MED12 on estrogen-induced CARM1 sites. (C) MCF7 cells were transfected with siCTL, siCARM1 or siMED12, and treated with or without estrogen (E 2 , 10 -7 M, 6 hrs) followed by RNA-seq analysis. Estrogen-induced genes which were dependent on both CARM1 and MED12 were shown by Pie chart. (D, E) Heat map (D) and box plot (E) representation of the expression levels (FPKM) for genes induced by estrogen and dependent on both CARM1 and MED12 as described in (C). Heat map: z-score normalized FPKM; box plot: FPKM (log2). (F) MCF7 cells were transfected with siCTL or siMED12 in stripping medium for three days, and treated with or without estrogen (E 2 , 10 -7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced coding genes as indicated (± s.e.m., **P<0.01, ***P<0.001). (G) MCF7 cells were transfected with siCTL or siCARM1 in stripping medium for three days, and treated with or without estrogen (E 2 , 10 -7 M, 1 hr) followed by ChIP-seq with anti-MED12 antibody. MED12 ChIP-seq tag density distribution centered on estrogen-induced CARM1 sites was shown (± 3,000 bp). (H, I) The binding of MED12 as described in (G) was shown for specific genes, as indicated. (J) MCF7 cells were infected with lenti-viral vectors expressing shRNA targeting MED12 together with or without Flag-tagged wild type (WT) MED12 or MED12 mutants with arginine 1899 replaced by alanine (R1899A), and then treated with or without estrogen (E 2 , 10 -7 M, 6 hrs) followed by RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± s.e.m., **P<0.01, ***P<0.001). Data was presented as fold induction by estrogen.

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Methylation, Activation Assay, ChIP-sequencing, Transfection, RNA Sequencing Assay, Expressing, Stripping Membranes, RNA Extraction, Quantitative RT-PCR, Binding Assay, Infection, shRNA

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: TDRD3 reads MED12 methylation and is involved in estrogen-induced gene transcriptional activation. (A-E) Wild type (WT) and CARM1 KO MCF7 cells treated with estrogen were subjected to immunoprecipitation (IP) with anti-TDRD3 (B), SMN (C), SND1 (D) or SPF30 (E) antibody followed by immunoblotting (IB) with antibodies as indicated. Input was also shown (A). (F) MCF7 cells treated with or without estrogen (E 2 , 10 -7 M, 1 hr) were subjected to ChIP with anti-TDRD3 antibody followed by qPCR analysis with primers specifically targeting enhancer (e) regions as indicated. A control (CTL) region was also included. ChIP signals were presented as percentage of inputs (± s.e.m., ***P<0.001). (G, I) MCF7 cells were transfected with siCTL, siTDRD3, siSMN, siSND1 or siSPF30 (G), or infected with lenti-viral vectors expressing TDRD3, SMN, SND1 or SPF30 (I) in stripping medium for three days, and treated with or without estrogen (E 2 , 10 -7 M, 6 hrs), followed by RNA extraction and RT-qPCR analysis to examine the expression of selected estrogen-induced genes as indicated (± s.e.m., **P<0.01, ***P<0.001). Data for siSMN, siSND1, siSPF30, SMN, SND1 and SPF30 were shown in A, S6B, S6C, S6D, S6E and S6F, respectively. (H) MCF7 cells as described in (G) were subjected to immunoblotting analysis with antibodies as indicated. Actin was served as a loading control.

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Methylation, Activation Assay, Immunoprecipitation, Western Blot, Transfection, Infection, Expressing, Stripping Membranes, RNA Extraction, Quantitative RT-PCR

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: CARM1 is required for estrogen-induced breast cancer cell growth and tumorigenesis. (A) MCF7 cells transfected with siCTL or siCARM1 were subjected to immunoblotting using antibodies as indicated. (B, C) MCF7 cells as described in (A) were subjected to cell proliferation assay (B) and FACS analysis (C) (± s.e.m., **P<0.01, ***P<0.001). (D) Colony formation assay was performed in WT and CARM1 KO MCF7 cells. (E) MDA-MB-231 cells transfected with siCTL or siCARM1 for 48 hrs were both re-seeded at full confluence and then subjected to wound-healing assay. (F) Quantification of wound closure shown in (E) (± s.e.m., *P<0.05). (G) MDA-MB-231 cells as described in (E) were both re-seeded at the same confluence and then subjected to trans-well assay. (H) Quantification of (G) (± s.e.m., **P<0.01). (I) WT and CARM1 KO MCF7 cells were injected subcutaneously into female BALB/C nude mice for tumor xenograft experiments. (J) Tumor weight as shown in (I) (± s.e.m., *P<0.05, **P<0.01).

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Transfection, Western Blot, Proliferation Assay, Colony Assay, Wound Healing Assay, Injection

Journal: Theranostics

Article Title: A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor α-dependent transcriptional activation and breast carcinogenesis

doi: 10.7150/thno.39241

Figure Lengend Snippet: A proposed model of CARM1 function in estrogen-induced gene transcriptional activation, breast cancer cell growth and tumorigenesis. Based on the findings in this manuscript along with our previous report , we proposed that, upon estrogen stimulation, a coactivator complex constituting BRD4, JMJD6, CARM1, CARM1 substrates (represented by MED12), TDRD3 and others, was recruited to ERα-bound active enhancers, leading to the transcriptional activation of active enhancers as well as cognate estrogen/ERα-target genes. During the process of this gene activation event, CARM1 was found to hypermethylate a cohort of proteins, such as MED12, with implications in intracellular ERα-mediated signaling. CARM1-mediated methylation was critical for the recruitment of coactivator protein TDRD3 to activate estrogen/ERα-target genes. Prolonged exposure to high levels of estrogen will lead to the constitutive activation of this gene program, uncontrolled breast cancer cell growth and eventually breast cancer.

Article Snippet: MCF7 cells were then transfected with pX330-gRNA (CARM1) and pIREShyg3 vector (Clontech, 631620) (for selection purpose), followed by hygromycin (0.2 mg/mL) selection.

Techniques: Activation Assay, Methylation

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: (A) Domain structure of the two ADAR1 isoforms, p150 and p110. (B) Schematic representation of the CRISPR/Cas9-mediated disruption of ADAR1 p150 and p150/p110. The target sequence and corresponding vectors containing either a reverse-oriented puromycin- or neomycin- resistance cassette are illustrated. (C) Western blot analysis of ADAR1 isoforms. Whole-cell extracts from WT, p150 KO clones (#1, #2, #3), and p150/p110 KO clones (#1, #2, #3) were resolved by 10% SDS-PAGE. GAPDH was used as a loading control. (D) Clustering analysis of genes exhibiting A- to-I editing. Based on inosine peak scores (fold-enrichment ratio) in WT and p150 KO cells, genes were categorized as strongly p150 dependent (WT/(p150KO+1)≧4; purple), mildly p150 dependent (11, cyan). Asterisk (*) indicates the genes categorized as p150/p110 KO-specific (WT=0, p150KO=0, p150/p110KO>1).

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques: CRISPR, Disruption, Sequencing, Western Blot, Clone Assay, SDS Page, Control

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: Read coverage profiles are shown for representative DNA repair-associated genes: (A) ATM (3′UTR), (B) POLH (3′UTR), (C) POLH (intron between exon4-5), (D) ATR (intron between exon1-2), (E) FANCA (intron between exon5-6), (F), FANCA (intron between exon14-15), and (G) XPA (intron between exon5-6) in wild-type (cyan), p150 KO (pink), and p150/p110 KO cells (green). The y-axis represents read counts and the x-axis indicates genomic coordinates. The scale bar corresponds to 1 kb. “Coverage data range” in the top-right corner of each panel indicates the maximum coverage value. Colored regions within coverage tracks highlight A-to-I RNA editing sites. Strand-specific colors compositions are shown: green (Adenosine) and orange (Guanosine) for forward reads; red (Thymidine) and blue (Cytidine) for reverse reads, facilitating visual estimation of nucleotide ratios at individual position.

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques:

Journal: bioRxiv

Article Title: Epitranscriptome-wide profiling identifies RNA editing events regulated by ADAR1 that are associated with DNA repair mechanisms in human TK6 cells

doi: 10.1101/2025.07.11.664482

Figure Lengend Snippet: (A) Gene expression levels of ATM, ATR , and FANCA were quantified by RT-qPCR in WT (black), p150 KO (gray), and p150/p110 KO (white) cells. Data represent six independent experiments (n = 6). Statistical significance was determined by Student’s t-test (p < 0.05). (B) RNA-seq read coverage profiles of the XPA gene in WT#1, WT#2, p150 KO#1, p150 KO#2, p150/p110 KO#1, and p150/p110 KO#2 cells. Arrows indicate novel splicing peaks between exon 5 and 6 of the main splicing variant. (C) Detection of XPA splicing variants. PCR was performed using cDNA from WT#1, p150 KO#1, p150 KO#2, p150/p110 KO#1, and p150/p110 KO#2, with primers targeting exons 2–6 and 5–6. Amplified products were resolved on 1.0% agarose gels. For amplification between exons 2–6, expected band sizes were 946 bp for variant 1 and 1,260 bp for a potential alternative variant. For amplification between exons 5–6, expected sizes were 544 bp for variant 1 and 964 bp for alternative variants.

Article Snippet: The vectors pX330-gRNA/p150 (6 μg) and the p150 target plasmid (2 μg) were transfected into TK6 cells by a NEPA21 electroporator (Nepa Gene Co. Ltd.) following the manufacturer’s instructions.

Techniques: Gene Expression, Quantitative RT-PCR, RNA Sequencing, Variant Assay, Amplification