Journal: Cell Reports

Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

doi: 10.1016/j.celrep.2022.110889

Figure Lengend Snippet: JAZF1-SUZ12 lacks interaction with JARID2, EPOP, and PALI1 due to loss of the SUZ12 N terminus (A) Models of FLAG-tagged (F) SUZ12, SUZ12Δ93, JAZF1-SUZ12, and JAZF1 constructs. (B) Immunoblots for FLAG, SUZ12 (N terminus), EZH2, JARID2, AEBP2, PCL2, EPOP, TRRAP, and β-actin in input (In.) and SUZ12 IPs from WT or Suz12 GT/GT ESCs stably expressing FLAG-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent IPs. (C) Immunoblots for FLAG, HA-PALI, and EZH2 in input and Strep-Tactin pull-downs (P) from NIH3T3 cells transfected with HA-PALI1 and FS2-tagged GFP, SUZ12, SUZ12Δ93, JAZF1-SUZ12, or JAZF1. Data are representative of two independent pull-downs. See also Figure S1 .

Article Snippet: Proteins were detected with primary antibodies to FLAG M2 (Sigma, A8592), HA 3F10 (Roche, 12013819001), V5 (Abcam, ab15828), SUZ12 (Santa Cruz sc-46264), EZH2 (CST 3147), JARID2 (CST 13594), AEBP2 (CST 14129), PCL2 (Proteintech 16208-1-AP), EPOP (kind gift of L. Di Croce), TRRAP (Abcam, ab73546), FUS (Novus Biologicals 100–565), β-actin (CST 4967), alpha tubulin (CST 2144), H3K27me3 (Abcam ab192985), H4 pan-acetyl (Thermofisher, PA5-40083) and H3 (Abcam ab1791) and HRP-conjugated secondary antibodies (anti-mouse (Dako, P0447) or anti-rabbit (Dako, P0448).

Techniques: Construct, Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell Reports

Article Title: JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation

doi: 10.1016/j.celrep.2022.110889

Figure Lengend Snippet:

Article Snippet: Proteins were detected with primary antibodies to FLAG M2 (Sigma, A8592), HA 3F10 (Roche, 12013819001), V5 (Abcam, ab15828), SUZ12 (Santa Cruz sc-46264), EZH2 (CST 3147), JARID2 (CST 13594), AEBP2 (CST 14129), PCL2 (Proteintech 16208-1-AP), EPOP (kind gift of L. Di Croce), TRRAP (Abcam, ab73546), FUS (Novus Biologicals 100–565), β-actin (CST 4967), alpha tubulin (CST 2144), H3K27me3 (Abcam ab192985), H4 pan-acetyl (Thermofisher, PA5-40083) and H3 (Abcam ab1791) and HRP-conjugated secondary antibodies (anti-mouse (Dako, P0447) or anti-rabbit (Dako, P0448).

Techniques: Recombinant, Avidin-Biotin Assay, SYBR Green Assay, Sensitive Assay, Western Blot, Software

Journal: bioRxiv

Article Title: The lncRNA Firre functions as a transcriptional activator from a distance

doi: 10.1101/2022.05.15.492001

Figure Lengend Snippet: (A) Timeline of GapmeR-mediated gene knockdown and dox-induced Firre transgene expression. (B) Depiction of possible outcomes for Firre -responsive gene expression (red line) after knockdown of a regulatory factor (blue line) and induction of Firre transgene expression. (C) Relative gene expression of Firre and its targets Adgrg1 and Shf in Firre OE cells transfected with non-targeting control-(NTC), Ezh2 -, Suz12, Jarid2 -, Aebp2 -, Wdr5 -, or G9a -targeting GapmeRs, and treated without (zero hours) or with (six hours) dox as determined by RT-qPCR. (D and E) smRNA FISH-based quantification of the distance between Shf intron and Firre transgene (D) and Shf intron and the five closest Firre exons (distance averaged) (E) in Firre -overexpression and Firre -rescue cells after 0, 2, 4, and 6 hours of dox treatment. The images below depict the rationale of the distance measurements.

Article Snippet: Membranes were blocked in 5% milk in TBS-T (TBS with 0.1% Tween 20) and probed with anti-JARID2 (D6M9X), anti-AEBP2 (D7C6X), anti-EZH2 (D2C9), anti-SUZ12 (Cell Signaling Technology, D39F6), or anti-GAPDH antibody (14C10, all Cell Signaling Technology) at a dilution of 1:1000 in blocking buffer overnight at 4°C or for one hour at room temperature.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Over Expression

Journal: Bioengineered

Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

doi: 10.1080/21655979.2022.2036891

Figure Lengend Snippet: PCR primer sequences

Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

Techniques:

Journal: Bioengineered

Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

doi: 10.1080/21655979.2022.2036891

Figure Lengend Snippet: E2F2 is the target gene of miR-631, and elevated E2F2 facilitates the malignant behavior of NSCLC.

Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

Techniques:

Journal: Bioengineered

Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

doi: 10.1080/21655979.2022.2036891

Figure Lengend Snippet: MiR-631 exerts an influence on the malignant behavior of NSCLC via modulating E2F2.

Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

Techniques:

Journal: Bioengineered

Article Title: MicroRNA-631 deriving from bone marrow mesenchymal stem cell exosomes facilitates the malignant behavior of non-small cell lung cancer via modulating the E2F family of transcription factor 2/phosphatidylinositol 3‐kinase/Akt signaling pathway

doi: 10.1080/21655979.2022.2036891

Figure Lengend Snippet: BMSCs-Exo exerts an influence on the malignant behavior of NSCLC via the miR-631/E2F2 axis.

Article Snippet: A total of 20 g samples were applied to 12% sulfate-polyacrylamide gel, electrophoresis was performed to separate proteins, and then electroblot was onto polyvinylidene fluoride membrane (Invitrogen, Carlsbad, CA, USA), which was sealed with 5% skimmed milk powder for 1 h. Subsequently, incubation of the membrane was with the following primary antibodies: CD9 (ab92726, Abcam), E2F2 (14,129, 1:1000, Cell Signaling Technology), CD81 (ab79559, Abcam BD Biosciences), CD63 (ab134045, Santa Cruz Biotechnology), TSG101 (ab125011, GAPDH (2118, Cell Signaling Technology) and Santa Cruz Biotechnology).

Techniques:

Journal: Nature Communications

Article Title: Joint single-cell multiomic analysis in Wnt3a induced asymmetric stem cell division

doi: 10.1038/s41467-021-26203-0

Figure Lengend Snippet: a Violin plots showing the average expression levels of components of PRC2 in the Mix, Proxi, and Dista clusters. To increase the reproducibility, the gene expressions of H3K27me3 and H3K4me3 scSET-seq were merged for plotting. P values were calculated by Student’s t -test, two-sided. Rbbp7 was not detected in the scSET-seq. n = 291 (Mix), 133 (Proxi), and 121 (Dista) cells. b Western blot showing the indicated protein levels in parental and KO mESCs. Ezh2 KO decreased the total levels of H3K27me3 and increased the H3K27ac. Aebp2 KO didn’t exhibit obvious effects on the total levels of tested histone marks. Cell extracts were analyzed via Western blotting using the indicated antibodies, and two biological replicates were performed for each blot. Star indicated a nonspecific band. See Supplementary Fig. for the Sanger sequencing results of KO clones. c Representative images of divided Nanog - mCherry mESCs cocultured with Wnt3a beads. Wnt3a beads and cells were marked in the immunofluorescence image. Asymmetric, higher mCherry amounts in the bead-proximal cell; Symmetric, similar amounts of mCherry in either cell; Reverse, higher amounts of mCherry in the bead-distal cell. BF, bright field. Scale bar, 15 µm. d The percentages of asymmetrically, symmetrically, and reversely divided cells, as defined by Nanog - mCherry signals, in parental, Ezh2 and Aebp2 KO cells. P values were calculated by chi-squared test. e The enrichments of AEBP2 at cluster marker genes that were identified by gene expression. AEBP2 peaks were first called and annotated to the closet transcription starting sites within 20 Kb. The reads density of Aebp2 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t -test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 90, 108, and 200 Aebp2 peaks at Mix, Proxi and Dista markers, respectively. n = 1000 (Mix), 1000 (Proxi), and 1000 (Dista) bins of Aebp2 at cluster marker genes. f The enrichments of H3K27me3 at cluster marker genes that were identified by gene expression. H3K27me3 peaks were first called in WT and Aebp2 KO cells, merged, and annotated to the closet transcription starting sites within 20 Kb. The reads density of H3K27me3 was then calculated for peaks annotated to each gene. P values were calculated by Student’s t-test, two-sided. The boxes were drawn from lower quartile (Q1) to upper quartile (Q3) with the middle line denoting the median, and whiskers with maximum 1.5 IQR (interquartile range). n = 504, 243, and 371 H3K27me3 peaks at Mix, Proxi, and Dista markers, respectively. Source data are provided as a Source Data file.

Article Snippet: Rabbit polyclonal anti-Histone H3 (Cat.# ab1791, Abcam), 1:5000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K27me3 (Cat.# 9733, Cell Signaling Technology), 1:1000 diluted for Western blot, 1:50 diluted for SET-seq, 1:200 diluted for scSET-seq; Rabbit polyclonal anti-Histone H3K4me3 (Cat.# ab8580, Abcam), 1:1000 diluted for Western blot, 1:50 diluted for SET-seq, 1:200 diluted for scSET-seq; Rabbit polyclonal anti-Histone H3K9me3 (Cat.# ab8898, Abcam), 1:1000 diluted for Western blot; Rabbit polyclonal anti-Histone H3K27ac (Cat.# ab4729, Abcam), 1:1000 diluted for Western blot; Rabbit monoclonal anti-EZH2 (Cat.# 5246, Clone name D2C9, Cell Signaling Technology), 1:1000 diluted for Western blot; Rabbit monoclonal anti-AEBP2 (Cat.# 14129, Clone name D7C6X, Cell Signaling Technology), 1:1000 diluted for Western blot; Rabbit polyclonal anti-JARID2 (Cat.# G-2, Novus Biologicals), 1:1000 diluted for Western blot; Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (Cat.# 111-035-003, Jackson ImmunoResearch Laboratories), 1:1000 diluted for Western blot.

Techniques: Expressing, Western Blot, Sequencing, Clone Assay, Immunofluorescence, Marker

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet: PRC2 sub-complexes differentially contribute to the BAP1-dependent intergenic H3K27me3 deposition (A) Cartoon showing divergent compositions of the PRC2.1 and PRC2.2 complexes and their differing affinities for chromatin features. (B) Western blot analysis with the indicated antibodies on total protein extracts from the Bap1 KO, Aebp2 + Jarid2 ( A+J ) KO, Aebp2 + Jarid2 + Bap1 ( A+J+B ) KO, and matching WT ESCs. (C) Western blot analysis with the indicated antibodies on total protein extracts from the indicated ESC lines. (D) Boxplots representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (E) Boxplots representing H3K27me3 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (F) Boxplots showing the log2 fold change RPKM ratio for H2AK119ub1 or H3K27me3 in the indicated cell lines (A = Aebp2 , J = Jarid2 , P = Pcl1-3 ) at intergenic regions (n = 38,068). (G) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Aebp2/Jarid2/Bap1 KO versus Aebp2/Jarid2 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. (H) Genome-wide comparison of ChIP-seq signal using 5 kb windows. Log2 fold change H2AK119ub1 ChIP-seq for the Pcl1-3/Bap1 KO versus Pcl1-3 KO and the matching Bap1 KO versus WT comparison (x axis) plotted against log2 fold change of H3K27me3 ChIP-seq (y axis). Each dot represents one 5 kb window. See also Figure S4 .

Article Snippet: Rabbit monoclonal anti-AEBP2 (D7C6X) , Cell Signaling , Cat #14129; RRID: AB_2798398.

Techniques: Western Blot, ChIP-sequencing, Genome Wide

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-AEBP2 (D7C6X) , Cell Signaling , Cat #14129; RRID: AB_2798398.

Techniques: Recombinant, Transfection, Purification, Sequencing, Imaging, Plasmid Preparation, Software