Review

bsd p2a maid sequence (Addgene inc)

Addgene inc is a verified supplier

About

News

Press Release

Team

Advisors

Partners

Contact

Bioz Stars

Bioz vStars

Buy from Supplier

Structured Review

Addgene inc bsd p2a maid sequence
Bsd P2a Maid Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/bsd p2a maid sequence/product/Addgene inc
Average 93 stars, based on 6 article reviews

bsd p2a maid sequence - by Bioz Stars, 2026-06

93/100 stars

Images

Image Search Results

Figure 1. m6A regulators are dynamically expressed during satellite cell (SC) lineage progression and YTHDC1 is induced upon SC activation/ proliferation. (A) Schematic illustration of SC collection from Pax7- nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC), and cultured SCs (ASC) were subject to RNA- seq, western blotting (WB), and Immunofluorescence (IF) analyses. (B) The expression dynamics of m6A writer, reader, and eraser proteins in the above cells from analyzing the RNA- seq data. (C) Representative RNA- seq tracks showing the expression dynamics of the selected m6A regulators. (D) The expression dynamic of Ythdc1 mRNA (FPKM) from RNA- seq. (E) WB showing the induction of YTHDC1, YTHDF1, and YTHDF2 proteins upon SC activation and proliferation. * denotes the correct position of YTHDC1. Histone H3 was used as a loading control. (F) IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar = 20 μm. (G) WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 1. m6A regulators are dynamically expressed during satellite cell (SC) lineage progression and YTHDC1 is induced upon SC activation/ proliferation. (A) Schematic illustration of SC collection from Pax7- nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC), and cultured SCs (ASC) were subject to RNA- seq, western blotting (WB), and Immunofluorescence (IF) analyses. (B) The expression dynamics of m6A writer, reader, and eraser proteins in the above cells from analyzing the RNA- seq data. (C) Representative RNA- seq tracks showing the expression dynamics of the selected m6A regulators. (D) The expression dynamic of Ythdc1 mRNA (FPKM) from RNA- seq. (E) WB showing the induction of YTHDC1, YTHDF1, and YTHDF2 proteins upon SC activation and proliferation. * denotes the correct position of YTHDC1. Histone H3 was used as a loading control. (F) IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar = 20 μm. (G) WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Article Snippet: Two donor plasmids were generated by overlap PCR ~ 500 bp Ythdc1 genomic sequence flanking BSD/HygR- P2A- mAID- mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20- T vectors by T- A ligation.

Techniques: Activation Assay, Isolation, Cell Culture, RNA Sequencing, Western Blot, Immunofluorescence, Expressing, Control, Staining

Figure 2. Inducible YTHDC1 deletion in satellite cells (SCs) abolishes acute injury- induced muscle regeneration. (A) Breeding scheme for generating YTHDC1- inducible knockout (iKO) and control (Ctrl) mice. (B) Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of YTHDC1 deletion on barium chloride (BaCl2)- induced muscle regeneration process. (C) Left: western blotting (WB) showing the deletion of YTHDC1 in ASC- 48 hr from iKO but not Ctrl mice. Right: no obvious morphological difference was detected in iKO vs. Ctrl mice. (D) H&E staining of the above injured muscles at 0, 5, and 7 days post injury (dpi). Scale bar = 100 μm. (E) Left: immunostaining of eMyHC (red) and laminin (green) of the above injured tibialis anterior (TA) muscles at 5 and 7 dpi. Scale bar = 100 μm. Right: quantification of eMyHC- positive fibers per field. n = 3 mice per group. (F) Immunostaining of Pax7 (red) and laminin (green) on TA muscle sections at 0, 5, and 7 dpi. Scale bar = 100 μm. (G) Quantification of Pax7- positive SCs per field at 0, 5, and 7 dpi. n = 4 mice per group for 0 dpi, n = 3 mice per group for 5 and 7 dpi. Bars represent mean ± SD for all graphs. Statistical significance was determined using a two- tailed Student’s t- test.

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 2. Inducible YTHDC1 deletion in satellite cells (SCs) abolishes acute injury- induced muscle regeneration. (A) Breeding scheme for generating YTHDC1- inducible knockout (iKO) and control (Ctrl) mice. (B) Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of YTHDC1 deletion on barium chloride (BaCl2)- induced muscle regeneration process. (C) Left: western blotting (WB) showing the deletion of YTHDC1 in ASC- 48 hr from iKO but not Ctrl mice. Right: no obvious morphological difference was detected in iKO vs. Ctrl mice. (D) H&E staining of the above injured muscles at 0, 5, and 7 days post injury (dpi). Scale bar = 100 μm. (E) Left: immunostaining of eMyHC (red) and laminin (green) of the above injured tibialis anterior (TA) muscles at 5 and 7 dpi. Scale bar = 100 μm. Right: quantification of eMyHC- positive fibers per field. n = 3 mice per group. (F) Immunostaining of Pax7 (red) and laminin (green) on TA muscle sections at 0, 5, and 7 dpi. Scale bar = 100 μm. (G) Quantification of Pax7- positive SCs per field at 0, 5, and 7 dpi. n = 4 mice per group for 0 dpi, n = 3 mice per group for 5 and 7 dpi. Bars represent mean ± SD for all graphs. Statistical significance was determined using a two- tailed Student’s t- test.

Techniques: Knock-Out, Control, Western Blot, Staining, Muscles, Immunostaining, Two Tailed Test

Figure 3. Inducible YTHDC1 knockout impairs satellite cell (SC) activation/proliferation. (A) Left: EdU (red) staining of ASC- 24 hr and ASC- 48 hr from inducible knock out (iKO) and Ctrl mice. Scale bar = 200 μm. Right: quantification of the percentage of EdU+ cells. n = 3 mice per group for ASC- 24 hr, n = 4 mice per group for ASC- 48 hr. (B) Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO mice and cultured for 48 hr. Scale bar = 100 μm. Right: quantification of the percentage of Pax7+ EdU+ SCs. n = 3 mice per group. (C) Top: immunofluorescence (IF) staining of Pax7 (green) and MyoD (red) of freshly isolated SCs (FISC) (0 hr), ASC- 24 hr and ASC- 48 hr from Ctrl and iKO mice. Scale bar = 100 μm. Bottom: quantification of the percentage

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 3. Inducible YTHDC1 knockout impairs satellite cell (SC) activation/proliferation. (A) Left: EdU (red) staining of ASC- 24 hr and ASC- 48 hr from inducible knock out (iKO) and Ctrl mice. Scale bar = 200 μm. Right: quantification of the percentage of EdU+ cells. n = 3 mice per group for ASC- 24 hr, n = 4 mice per group for ASC- 48 hr. (B) Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO mice and cultured for 48 hr. Scale bar = 100 μm. Right: quantification of the percentage of Pax7+ EdU+ SCs. n = 3 mice per group. (C) Top: immunofluorescence (IF) staining of Pax7 (green) and MyoD (red) of freshly isolated SCs (FISC) (0 hr), ASC- 24 hr and ASC- 48 hr from Ctrl and iKO mice. Scale bar = 100 μm. Bottom: quantification of the percentage

Techniques: Knock-Out, Activation Assay, Staining, Isolation, Cell Culture, Immunofluorescence

Figure 4. LACE- seq defines transcriptome- wide YTHDC1 binding profiles in myoblasts. (A) Schematic illustration of the experimental design for performing LACE- seq and subsequent combination with MeRIP- seq, bulk RNA- seq, subcellular RNA- seq for defining and elucidating YTHDC1 splicing/ export targets and post- transcriptional regulation. (B) LACE- seq was performed in both ASC- 48 hr and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. (C) Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. (D) Left: Go analysis for the identified YTHDC1 targets in C2C12.

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 4. LACE- seq defines transcriptome- wide YTHDC1 binding profiles in myoblasts. (A) Schematic illustration of the experimental design for performing LACE- seq and subsequent combination with MeRIP- seq, bulk RNA- seq, subcellular RNA- seq for defining and elucidating YTHDC1 splicing/ export targets and post- transcriptional regulation. (B) LACE- seq was performed in both ASC- 48 hr and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. (C) Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. (D) Left: Go analysis for the identified YTHDC1 targets in C2C12.

Techniques: Binding Assay, RNA Sequencing

$Figure 6. YTHDC1 loss inhibits mRNA nuclear export. (A) Subcellular RNA- seq was performed using cytoplasmic and nuclear fractions isolated from ASC- 48 hr of Ctrl and YTHDC1 inducible knock out (iKO) mice. The log2(cyto/nuc) expression change was calculated for YTHDC1 targets. On the top and right, the density plot of log2(cyto/nuc) expression changes is depicted. (B) Quantification of log2(cyto/nuc) expression changes in iKO vs. Ctrl. (C) Overlapping between YTHDC1 mRNA export targets and splicing targets in ASCs. (D) Genome tracks of two selected export targets. (E–I) The$

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 6. YTHDC1 loss inhibits mRNA nuclear export. (A) Subcellular RNA- seq was performed using cytoplasmic and nuclear fractions isolated from ASC- 48 hr of Ctrl and YTHDC1 inducible knock out (iKO) mice. The log2(cyto/nuc) expression change was calculated for YTHDC1 targets. On the top and right, the density plot of log2(cyto/nuc) expression changes is depicted. (B) Quantification of log2(cyto/nuc) expression changes in iKO vs. Ctrl. (C) Overlapping between YTHDC1 mRNA export targets and splicing targets in ASCs. (D) Genome tracks of two selected export targets. (E–I) The

Techniques: RNA Sequencing, Isolation, Knock-Out, Expressing

Figure 7. Co- immunoprecipitation/mass spectrometry (co- IP/MS) leads to the identification of YTHDC1 interacting partners. (A, B) Schematic illustration of the co- IP/MS procedure. An empty vector or Flag- tagged YTHDC1 plasmid was expressed in C2C12 myoblasts followed by pull- down with Flag beads; the retrieved proteins were subject to MS with Bruker timsTOF Pro. (B) Overexpression of the Flag- tagged YTHDC1 was confirmed by western blotting (WB) using anti- Flag antibody. (C) 912 proteins were uniquely retrieved in YTHDC1 but not Vector- expressing cells. (D) GO functions of the above proteins are shown. (E–G) The above- identified interacting proteins with RNA splicing, mRNA export, or potential transcriptional regulatory functions are shown in the lists. (H) Flag- tagged YTHDC1 was overexpressed in C2C12 and Flag- beads- based IP was performed followed by WB to verify the retrieved hnRNPG protein. (I) IP of endogenous YTHDC1 protein in C2C12 myoblasts followed by WB to examine retrieved hnRNPG protein. (J) Flag- hnRNPG and HA- YTHDC were overexpressed in 293T cells; IP with YTHDC1 or hnRNPG protein flowed by WB to confirm the interaction

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 7. Co- immunoprecipitation/mass spectrometry (co- IP/MS) leads to the identification of YTHDC1 interacting partners. (A, B) Schematic illustration of the co- IP/MS procedure. An empty vector or Flag- tagged YTHDC1 plasmid was expressed in C2C12 myoblasts followed by pull- down with Flag beads; the retrieved proteins were subject to MS with Bruker timsTOF Pro. (B) Overexpression of the Flag- tagged YTHDC1 was confirmed by western blotting (WB) using anti- Flag antibody. (C) 912 proteins were uniquely retrieved in YTHDC1 but not Vector- expressing cells. (D) GO functions of the above proteins are shown. (E–G) The above- identified interacting proteins with RNA splicing, mRNA export, or potential transcriptional regulatory functions are shown in the lists. (H) Flag- tagged YTHDC1 was overexpressed in C2C12 and Flag- beads- based IP was performed followed by WB to verify the retrieved hnRNPG protein. (I) IP of endogenous YTHDC1 protein in C2C12 myoblasts followed by WB to examine retrieved hnRNPG protein. (J) Flag- hnRNPG and HA- YTHDC were overexpressed in 293T cells; IP with YTHDC1 or hnRNPG protein flowed by WB to confirm the interaction

Techniques: Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Plasmid Preparation, Over Expression, Western Blot, Expressing

Figure 8. A working model of YTHDC1 function in satellite cells (SCs). YTHDC1 is induced upon SC activation and proliferation to promote SC activation and proliferation upon acute injury- induced muscle regeneration. Mechanistically, it functions through both facilitating mRNA splicing synergistically with hnRNPG and promoting mRNA export possibly by binding with the THO nuclear export complex.

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 8. A working model of YTHDC1 function in satellite cells (SCs). YTHDC1 is induced upon SC activation and proliferation to promote SC activation and proliferation upon acute injury- induced muscle regeneration. Mechanistically, it functions through both facilitating mRNA splicing synergistically with hnRNPG and promoting mRNA export possibly by binding with the THO nuclear export complex.

Techniques: Activation Assay, Binding Assay

A. Schematic illustration of SC collection from Pax7-nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC) and cultured SCs (ASC) were subject to RNA-seq, western blotting (WB), and Immunofluorescence (IF) analyses. B. The expression dynamics of m6A writer, reader and eraser proteins in the above cells from analyzing the RNA-seq data. C. Representative RNA-seq tracks showing the expression dynamics of the selected m6A regulators. D. The expression dynamic of YTHDC1 mRNA (FPKM) from RNA-seq. E. WB showing the induction of YTHDC1, YTHDF1 and YTHDF2 proteins upon SC activation and proliferation. *denotes the correct position of YTHDC1. Histone H3 was used as a loading control. F. IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar=20μm. G. WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Schematic illustration of SC collection from Pax7-nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC) and cultured SCs (ASC) were subject to RNA-seq, western blotting (WB), and Immunofluorescence (IF) analyses. B. The expression dynamics of m6A writer, reader and eraser proteins in the above cells from analyzing the RNA-seq data. C. Representative RNA-seq tracks showing the expression dynamics of the selected m6A regulators. D. The expression dynamic of YTHDC1 mRNA (FPKM) from RNA-seq. E. WB showing the induction of YTHDC1, YTHDF1 and YTHDF2 proteins upon SC activation and proliferation. *denotes the correct position of YTHDC1. Histone H3 was used as a loading control. F. IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar=20μm. G. WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Article Snippet: Two donor plasmids were generated by overlap PCR ∼500bp Ythdc1 genomic sequence flanking BSD/HygR-P2A-mAID-mCherry2 sequence (Addgene plasmid #121180, #121183) and cloning into pMD20-T vectors by T-A ligation.

Techniques: Isolation, Cell Culture, RNA Sequencing, Western Blot, Immunofluorescence, Expressing, Activation Assay, Control, Staining

A. Breeding scheme for generating YTHDC1 inducible knockout (iKO) and control (Ctrl) mice. B. Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of YTHDC1 deletion on barium chloride (BaCl 2 ) induced muscle regeneration process. C. Left: WB showing the deletion of YTHDC1 in ASC-48h from iKO but not Ctrl mice. Right: no obvious morphological difference was detected in iKO vs. Ctrl mice. D. H&E staining of the above injured muscles at 0, 5 and 7 dpi. Scale bar=100μm. E. Left: Immunostaining of eMyHC (red) and laminin (green) of the above injured TA muscles at 5 and 7 dpi. Scale bar=100μm. Right: Quantification of eMyHC positive fibers per field. n =3 mice per group. F. Immunostaining of Pax7 (red) and laminin (green) on TA muscle sections at 0, 5 and 7 dpi. Scale bar=100μm. G. Quantification of Pax7 positive SCs per field at 0, 5 and 7 dpi. n =4 mice per group for 0dpi, n =3 mice per group for 5 and 7dpi. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Breeding scheme for generating YTHDC1 inducible knockout (iKO) and control (Ctrl) mice. B. Schematic outline of the tamoxifen (TMX) administration used in the study and experimental design for testing the effect of YTHDC1 deletion on barium chloride (BaCl 2 ) induced muscle regeneration process. C. Left: WB showing the deletion of YTHDC1 in ASC-48h from iKO but not Ctrl mice. Right: no obvious morphological difference was detected in iKO vs. Ctrl mice. D. H&E staining of the above injured muscles at 0, 5 and 7 dpi. Scale bar=100μm. E. Left: Immunostaining of eMyHC (red) and laminin (green) of the above injured TA muscles at 5 and 7 dpi. Scale bar=100μm. Right: Quantification of eMyHC positive fibers per field. n =3 mice per group. F. Immunostaining of Pax7 (red) and laminin (green) on TA muscle sections at 0, 5 and 7 dpi. Scale bar=100μm. G. Quantification of Pax7 positive SCs per field at 0, 5 and 7 dpi. n =4 mice per group for 0dpi, n =3 mice per group for 5 and 7dpi. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Techniques: Knock-Out, Control, Staining, Muscles, Immunostaining, Two Tailed Test

A. Left: EdU (red) staining of ASC-24h and ASC-48h from iKO and Ctrl mice. Scale bar=200μm. Right: Quantification of percentage of EdU+ cells. n =3 mice per group for ASC-24h, n =4 mice per group for ASC-48h. B. Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO and cultured for 48h. Scale bar=100μm. Right: Quantification of the percentage of Pax7+EdU+ SCs. n =3 mice per group. C. Top: IF staining of Pax7 (green) and MyoD (red) of FISC (0h), ASC-24h and ASC-48h from Ctrl and iKO mice. Scale bar=100μm. Bottom: Quantification of the percentage of Pax7+MyoD+ cells. n =3 mice per group. D. Top: IF staining of Pax7 (green) and MyoD (red) on EDL myofibers at 0h (freshly isolated), 24h and 48h. Scale bar=100μm. Bottom: Quantification of the number of Pax7+MyoD+ cells per fiber. ( n =4 mice per group for 0 and 24h, n =5 mice per group for 48h) E. Schematic illustration of in vivo EdU assay in Ctrl and iKO mice. F. Top: EdU staining of the above freshly isolated and fixed SCs at 3 dpi. Scale bar=200μm. Bottom: Quantification of the percentage of EdU+ cells in iKO vs. Ctrl. n =3 mice per group. G-H . Left: RNA-seq was performed in ASC-24h or −48h from iKO and Ctrl. Volcano plot showing the down- and up-regulated genes in iKO vs. Ctrl. Right: GO analysis for the down-regulated genes. I. Schematic illustration of generating a C2C12 cell line with inducible YTHDC1 degradation using the auxin-inducible degron (AID2) system. J. Two independent mAID-YTHDC1 cell lines were treated with DMSO or 5-Ph-IAA(IAA) for the indicated time. EdU assay was performed and the percentage of EdU+ cells were quantified at the designated time points. n =3 replicates. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Left: EdU (red) staining of ASC-24h and ASC-48h from iKO and Ctrl mice. Scale bar=200μm. Right: Quantification of percentage of EdU+ cells. n =3 mice per group for ASC-24h, n =4 mice per group for ASC-48h. B. Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO and cultured for 48h. Scale bar=100μm. Right: Quantification of the percentage of Pax7+EdU+ SCs. n =3 mice per group. C. Top: IF staining of Pax7 (green) and MyoD (red) of FISC (0h), ASC-24h and ASC-48h from Ctrl and iKO mice. Scale bar=100μm. Bottom: Quantification of the percentage of Pax7+MyoD+ cells. n =3 mice per group. D. Top: IF staining of Pax7 (green) and MyoD (red) on EDL myofibers at 0h (freshly isolated), 24h and 48h. Scale bar=100μm. Bottom: Quantification of the number of Pax7+MyoD+ cells per fiber. ( n =4 mice per group for 0 and 24h, n =5 mice per group for 48h) E. Schematic illustration of in vivo EdU assay in Ctrl and iKO mice. F. Top: EdU staining of the above freshly isolated and fixed SCs at 3 dpi. Scale bar=200μm. Bottom: Quantification of the percentage of EdU+ cells in iKO vs. Ctrl. n =3 mice per group. G-H . Left: RNA-seq was performed in ASC-24h or −48h from iKO and Ctrl. Volcano plot showing the down- and up-regulated genes in iKO vs. Ctrl. Right: GO analysis for the down-regulated genes. I. Schematic illustration of generating a C2C12 cell line with inducible YTHDC1 degradation using the auxin-inducible degron (AID2) system. J. Two independent mAID-YTHDC1 cell lines were treated with DMSO or 5-Ph-IAA(IAA) for the indicated time. EdU assay was performed and the percentage of EdU+ cells were quantified at the designated time points. n =3 replicates. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Techniques: Staining, Isolation, Cell Culture, In Vivo, EdU Assay, RNA Sequencing, Two Tailed Test

A. Schematic illustration of the experimental design for performing LACE-seq and subsequent combination with MeRIP-seq, bulk RNA-seq, subcellular RNA-seq for defining and elucidating YTHDC1 splicing/export targets and post-transcriptional regulation. B. LACE-seq was performed in both ASC-48h and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. C. Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. D. Left: Go analysis for the identified YTHDC1 targets in C2C12. Right: genome tracks for three selected genes. E. Top: Overlapping between the above identified C2C12 and ASC peaks. Bottom: Genome tracks for two selected genes. F-G. Left: the genome distribution of YTHDC1 binding peaks in C2C12 or ASC. Right: detailed distribution of the YTHDC1 binding peaks on repeat regions. H. Left: Integrating C2C12 MeRIP-seq data with the C2C12 LACE-seq identified 5356 regions (1771 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above targets. I. Left: GO analysis of the above 1771 targets. Right: Genomic tracks of a selected target, Phlda3. J. Left: Integrating the C2C12 MeRIP-seq data with the ASC LACE-seq identified 56 regions (50 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above identified target mRNAs. K. Left: GO analysis of the above 50 targets. Right: Genomic tracks of a selected target, Ybx1.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Schematic illustration of the experimental design for performing LACE-seq and subsequent combination with MeRIP-seq, bulk RNA-seq, subcellular RNA-seq for defining and elucidating YTHDC1 splicing/export targets and post-transcriptional regulation. B. LACE-seq was performed in both ASC-48h and C2C12 myoblasts and the number of identified peaks in each technical (T) or biological replicate (B) and the shared number of peaks (genes) between the replicates are shown. C. Left: Go analysis for the identified YTHDC1 targets in ASCs. Right: genome tracks for three selected genes. D. Left: Go analysis for the identified YTHDC1 targets in C2C12. Right: genome tracks for three selected genes. E. Top: Overlapping between the above identified C2C12 and ASC peaks. Bottom: Genome tracks for two selected genes. F-G. Left: the genome distribution of YTHDC1 binding peaks in C2C12 or ASC. Right: detailed distribution of the YTHDC1 binding peaks on repeat regions. H. Left: Integrating C2C12 MeRIP-seq data with the C2C12 LACE-seq identified 5356 regions (1771 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above targets. I. Left: GO analysis of the above 1771 targets. Right: Genomic tracks of a selected target, Phlda3. J. Left: Integrating the C2C12 MeRIP-seq data with the ASC LACE-seq identified 56 regions (50 mRNAs) as m6A-YTHDC1 targets. Right: Distribution of YTHDC1 binding on the above identified target mRNAs. K. Left: GO analysis of the above 50 targets. Right: Genomic tracks of a selected target, Ybx1.

Techniques: RNA Sequencing, Binding Assay

A. Splicing analysis based on bulk RNA-seq data from ASC-24h or −48h defined five types of splicing events. The total number of each event in Ctrl, and the differential spliced events (DSE) in iKO vs. Ctrl are shown. B. Left: Combining the above ASC-48h DSEs with the ASC LACE-seq targets identified a total of 189 YTHDC1 splicing target mRNAs. Right: genome tracks of three selected targets. C . Top: RT-PCR assay was performed in ASC-48h from YTHDC1-iKO and Ctrl to verify altered splicing of the three selected target mRNAs, Palb2, Lrp8 and Scn5a. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. Exon inclusion level was defined as the percentage of transcripts which includes the specific exon. Included / (Included + Skipped). n =3 mice per group for Lrp8 and Scn5a. D. Left: combining the above ASC-48h DSE with m6A-YTHDC1 targets uncovered 11 m6A-YTHDC1 splicing targets. Right: genome tracks of two selected targets. E. Top: RT-PCR assay was performed in ASC-48 from YTHDC1-iKO and Ctrl to verify altered splicing of the two selected target mRNA, Itgb3bp, and Nek1. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. n =3 mice per group. F. Left: Splicing analysis based on bulk RNA-seq data from C2C12-mAID-YTHDC1 cells with or without YTHDC1 degradation. Right: Combining the above C2C12 DSEs with the C2C12 LACE-seq targets identified a total of 1040 YTHDC1 splicing target mRNAs. G. Overlapping between the above identified YTHDC1 splicing targets in ASC-48h and C2C12. H. Combining the above C2C12 DSEs with m6A-YTHDC1 targets uncovered 350 m6A-YTHDC1 splicing targets. I . Overlapping of m6A-YTHDC1 splicing targets in C2C12 and ASC-48h. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Splicing analysis based on bulk RNA-seq data from ASC-24h or −48h defined five types of splicing events. The total number of each event in Ctrl, and the differential spliced events (DSE) in iKO vs. Ctrl are shown. B. Left: Combining the above ASC-48h DSEs with the ASC LACE-seq targets identified a total of 189 YTHDC1 splicing target mRNAs. Right: genome tracks of three selected targets. C . Top: RT-PCR assay was performed in ASC-48h from YTHDC1-iKO and Ctrl to verify altered splicing of the three selected target mRNAs, Palb2, Lrp8 and Scn5a. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. Exon inclusion level was defined as the percentage of transcripts which includes the specific exon. Included / (Included + Skipped). n =3 mice per group for Lrp8 and Scn5a. D. Left: combining the above ASC-48h DSE with m6A-YTHDC1 targets uncovered 11 m6A-YTHDC1 splicing targets. Right: genome tracks of two selected targets. E. Top: RT-PCR assay was performed in ASC-48 from YTHDC1-iKO and Ctrl to verify altered splicing of the two selected target mRNA, Itgb3bp, and Nek1. GAPDH was used as a control. Bottom: Quantification of exon inclusion level. n =3 mice per group. F. Left: Splicing analysis based on bulk RNA-seq data from C2C12-mAID-YTHDC1 cells with or without YTHDC1 degradation. Right: Combining the above C2C12 DSEs with the C2C12 LACE-seq targets identified a total of 1040 YTHDC1 splicing target mRNAs. G. Overlapping between the above identified YTHDC1 splicing targets in ASC-48h and C2C12. H. Combining the above C2C12 DSEs with m6A-YTHDC1 targets uncovered 350 m6A-YTHDC1 splicing targets. I . Overlapping of m6A-YTHDC1 splicing targets in C2C12 and ASC-48h. Bars represent mean ± s.d. for all graphs. Statistical significance was determined using a two-tailed Student’s t test.

Techniques: RNA Sequencing, Reverse Transcription Polymerase Chain Reaction, Control, Two Tailed Test

$A. Subcellular RNA-seq was performed using cytoplasmic and nuclear fractions isolated from ASC-48h of Ctrl and YTHDC1 iKO. The log2 (cyto/nuc) expression change was calculated for YTHDC1 targets. On the top and right, the density plot of log2 (cyto/nuc) expression changes is depicted. B. Quantification of log2 (cyto/nuc) expression changes in iKO vs. Ctrl. C. Overlapping between YTHDC1 mRNA export targets and splicing targets in ASCs. D. Genome tracks of two selected export targets. E-I. The above assay/analysis was performed in DMSO or IAA treated mAID-YTHDC1 C2C12 myoblasts to identify YTHDC1 regulated export targets in C2C12. J-N. The above analyses were conducted using m6A-YTHDC1 targets to identify m6A-YTHDC1 mRNA export targets in C2C12.$

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A. Subcellular RNA-seq was performed using cytoplasmic and nuclear fractions isolated from ASC-48h of Ctrl and YTHDC1 iKO. The log2 (cyto/nuc) expression change was calculated for YTHDC1 targets. On the top and right, the density plot of log2 (cyto/nuc) expression changes is depicted. B. Quantification of log2 (cyto/nuc) expression changes in iKO vs. Ctrl. C. Overlapping between YTHDC1 mRNA export targets and splicing targets in ASCs. D. Genome tracks of two selected export targets. E-I. The above assay/analysis was performed in DMSO or IAA treated mAID-YTHDC1 C2C12 myoblasts to identify YTHDC1 regulated export targets in C2C12. J-N. The above analyses were conducted using m6A-YTHDC1 targets to identify m6A-YTHDC1 mRNA export targets in C2C12.

Techniques: RNA Sequencing, Isolation, Expressing

A-B. Schematic illustration of the Co-IP/MS procedure. An empty vector or Flag tagged YTHDC1 plasmid was expressed in C2C12 myoblasts followed by pull down with Flag beads; the retrieved proteins were subject to MS with Bruker timsTOF Pro. B. Overexpression of the Flag tagged YTHDC1 was confirmed by WB using anti-Flag antibody. C. 912 proteins were uniquely retrieved in YTHDC1 but not Vector expressing cells. D. GO functions of the above proteins are shown. E-G. The above identified interacting proteins with RNA splicing, mRNA export, or potential transcriptional regulatory functions are shown in the lists. H. Flag tagged YTHDC1 was overexpressed in C2C12 and Flag-beads based IP was performed followed by WB to verify the retrieved hnRNPG protein. I. IP of endogenous YTHDC1 protein in C2C12 myoblasts followed by WB to examine retrieved hnRNPG protein. J. Flag-hnRNPG and HA-YTHDC were overexpressed in 293T cells; IP with YTHDC1 or hnRNPG protein flowed by WB to confirm the interaction between the two proteins.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: A-B. Schematic illustration of the Co-IP/MS procedure. An empty vector or Flag tagged YTHDC1 plasmid was expressed in C2C12 myoblasts followed by pull down with Flag beads; the retrieved proteins were subject to MS with Bruker timsTOF Pro. B. Overexpression of the Flag tagged YTHDC1 was confirmed by WB using anti-Flag antibody. C. 912 proteins were uniquely retrieved in YTHDC1 but not Vector expressing cells. D. GO functions of the above proteins are shown. E-G. The above identified interacting proteins with RNA splicing, mRNA export, or potential transcriptional regulatory functions are shown in the lists. H. Flag tagged YTHDC1 was overexpressed in C2C12 and Flag-beads based IP was performed followed by WB to verify the retrieved hnRNPG protein. I. IP of endogenous YTHDC1 protein in C2C12 myoblasts followed by WB to examine retrieved hnRNPG protein. J. Flag-hnRNPG and HA-YTHDC were overexpressed in 293T cells; IP with YTHDC1 or hnRNPG protein flowed by WB to confirm the interaction between the two proteins.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Over Expression, Expressing

YTHDC1 is induced upon SC activation and proliferation to promote SC activation and proliferation upon acute injury induced muscle regeneration. Mechanistically, it functions through both facilitating mRNA splicing synergistically with hnRNPG and promoting mRNA export possibly by binding with the THO nuclear export complex.

Journal: bioRxiv

Article Title: Nuclear m6A Reader YTHDC1 Promotes Muscle Stem Cell Activation/Proliferation by Regulating mRNA Splicing and Nuclear Export

doi: 10.1101/2022.08.07.503064

Figure Lengend Snippet: YTHDC1 is induced upon SC activation and proliferation to promote SC activation and proliferation upon acute injury induced muscle regeneration. Mechanistically, it functions through both facilitating mRNA splicing synergistically with hnRNPG and promoting mRNA export possibly by binding with the THO nuclear export complex.

Techniques: Activation Assay, Binding Assay

Review

Structured Review

Images

Similar Products

Image Search Results