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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
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(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline <t>phosphatase</t> (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.
Vector Blue Substrate, supplied by Vector Laboratories, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


(A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline phosphatase (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.

Journal: bioRxiv

Article Title: Co-option of a mouse-specific retrotransposon rewires Ash2l isoform usage to prime developmental promoters

doi: 10.64898/2026.02.25.707914

Figure Lengend Snippet: (A) Quantification of histone H3 lysine 4 mono-, di-, and tri-methylation by Western blot in CRISPRi mESCs (left) and NIH/3T3 fibroblasts (right) targeting Ash2l TSS1 or TSS2. Data represent the mean ± SD from n = 3 biological repeats. (B) Same as (A), except ASH2L (TSS1) truncated and (TSS2) full-length protein isoforms were overexpressed in NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (C) Cell proliferation of CRISPRi mESCs and NIH/3T3 fibroblasts targeting TSS1 or TSS2. Viable cells were counted over a period of 5 days. Data represent the mean ± SD from n = 3 biological repeats. (D) Histone H3 serine 10 phosphorylation levels measured by FACS in CRISPRi NIH/3T3 fibroblasts. Data represent the mean ± SD from n = 3 biological repeats. (E) Bar plot showing the number of alkaline phosphatase (AP) positive colonies that formed during the colony formation assay. Mean+SD shown, n=3 biological replicates. (F) Expression of pluripotency markers Oct4 , Nanog , and Klf2 . CRISPRi mESCs were induced to exit pluripotency by culturing in 2i/LIF withdrawal conditions. Expression was measured by RT-PCR. Data represent the mean ± SD of n = 3 biological replicates.

Article Snippet: Vector Red Alkaline Phosphatase Substrate (Vector Laboratories) was used to assess alkaline phosphatase levels in the mESCs, according to the manufacturer’s protocol.

Techniques: Methylation, Western Blot, Phospho-proteomics, Colony Assay, Expressing, Reverse Transcription Polymerase Chain Reaction