(A) RNA-DNA FISH images of ASAR6-141 expression in six individual HTD114 cells. ASAR6-141 RNA (green; green arrows) and chromosome 6 DNA (chromosome paint, red), and DNA was stained with DAPI. (B) …
The location of significant peaks of eCLIP reads that map within ASAR and XIST genes.
eCLIP reads for 120 RBPs in 10 kb windows within ASARs.
Fosmids, BACs, and primers.
(A) Integrative Genomics Viewer view showing the genomic location of ASAR6-141. The genomic locations of RNAseq and eCLIP reads from HepG2 and K562 (from ENCODE) are shown. The zoomed in view shows …
Original image file of PCR products shown in Figure 1—figure supplement 1C.
Figure 1-figure supplement 1C, the original image file of the PCR products shown in panel C. Figure 1-figure supplement 1C (labels), the original image file for panel C with labels and regions of the image used are marked by the black boxes. Figure 1-figure supplement 1D, the original image file of the PCR products shown in panel D. Figure 1-figure supplement 1D (labels), the original image file for panel D with labels and regions of the image used are marked by the black boxes.
(A) Schematic illustration of the BrdU terminal label protocol (Smith and Thayer, 2012). Cells were treated with BrdU (green) for 5 hr and then harvested for mitotic cells. (B) BrdU incorporation in …
(A) DNA FISH on mouse chromosomes from clone ~7 kb(+)A5. Mitotic spread showing DNA FISH using the ~7 kb RBPD (magenta; arrow) and a mouse chromosome 5 BAC (RP24-107D17; red; arrowheads). (B and C) …
(A–J) shRNA mediated depletion of RBPs. K562 cells were transfected with empty vector (A, C, E, G, and I) or vectors expressing shRNAs directed against HNRNPU (B), HNRNPUL1 (D), HNRNPC (F), HNRNPM (H…
K562 or HTD114 cells were transfected with empty vector (-) or shRNA against RBPs and cell extracts were processed for western blots using antibodies (Key Resource Table) as indicated: (A and B) …
Original image files for the western blots.
Figure 3-figure supplement 1A, the original image file for the western blot with HNRNPU (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPU. Figure 3-figure supplement 1A (labels), the same image as above showing labels for the location of HNRNPU and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1B, the original image file for the western blot with HNRNPU (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPU. Figure 3-figure supplement 1B (labels), the same image as above showing labels for the location of HNRNPU and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3—figure supplement 1C, the original image file for the western blot with UCHL5 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against UCHL5. Figure 3-figure supplement 1C (labels), the same image as above showing labels for the location of UCHL5 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1D, the original image file for the western blot with HNRNPC (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPC. Figure 3-figure supplement 1C (labels), the same image as above showing labels for the location of HNRNPC and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3—figure supplement 1E, the original image file for the western blot with HNRNPC (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPC. Figure 3-figure supplement 1E (labels), the same image as above showing labels for the location of HNRNPC and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1F, the original image file for the western blot with HNRNPL (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPL. Figure 3—figure supplement 1F (labels), the same image as above showing labels for the location of HNRNPL and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1G, the original image file for the western blot with HNRNPM (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPM. Figure 3-figure supplement 1G (labels), the same image as above showing labels for the location of HNRNPM and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1H, the original image file for the western blot with HNRNPM (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPM. Figure 3-figure supplement 1H (labels), the same image as above showing labels for the location of HNRNPM and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1I, the original image file for the western blot with HNRNPL (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPL. Figure 3-figure supplement 1I (labels), the same image as above showing labels for the location of HNRNPL and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1J, the original image file for the western blot with KHSRP (green) and tubulin (red) antibodies on K562 cells with and without shRNA against KHSRP. Figure 3-figure supplement 1J (labels), the same image as above showing labels for the location of KHSRP and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1K, the original image file for the western blot with KHSRP (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against KHSRP. Figure 3-figure supplement 1K (labels), the same image as above showing labels for the location of KHSRP and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1L, the original image file for the western blot with HNRNPUL1 (green) and tubulin (red) antibodies on HTD114 cells with and without shRNA against HNRNPUL1. Figure 3-figure supplement 1L (labels), the same image as above showing labels for the location of HNRNPUL1 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1M, the original image file for the western blot with HLTF (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HLTF. Figure 3-figure supplement 1M (labels), the same image as above showing labels for the location of HLTF and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1N, the original image file for the western blot with SAFB2 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against SAFB2. Figure 3-figure supplement 1N (labels), the same image as above showing labels for the location of SAFB2 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1O, the original image file for the western blot with PTBP1 (red) antibody on K562 cells with and without shRNA against PTBP1. Figure 3-figure supplement 1O (labels), the same image as above showing labels for the location of PTBP1, and the region of the image used is highlighted in white. Molecular weight standards are shown in red. Figure 3-figure supplement 1P, the original image file for the western blot with HNRNPA1 (green) and tubulin (red) antibodies on K562 cells with and without shRNA against HNRNPA1. Figure 3-figure supplement 1P (labels), the same image as above showing labels for the location of HNRNPA1 and tubulin, and the region of the image used is highlighted in white. Molecular weight standards are shown in red.
(A–D) K562 cells were transfected with empty vector (A) or shRNA expression vectors for MATR3 (B), PTBP1 (C), or PTBP2 (D). Cells were processed for RNA FISH using probes for ASAR6-141 (green; …
(A) Two color RNA FISH assay for expression of the sense ~7 kb RBPD transgene plus Xist RNA. Examples of three different cells showing colocalization of the ASAR RNA and Xist RNA. Individual cells …
shRNA-mediated depletion of RBPs in K562 cells (A–K) or HTD114 cells (L–Q). Cells were transfected with empty vector (A–C and L–N) or vectors expressing shRNAs directed against HNRNPA1 (D), HNRNPC (E…
HTD114 cells were transfected with empty vector (A) or shRNA expression vectors for HNRNPA1 (B), HNRNPC (C), HNRNPL (D), HNRNPM (E), HNRNPUL1 (F), KHSRP (G), HLTF (H), or UCHL5 (I). Cells were …
(A) BrdU incorporation in parental HTD114 cells. Cells were exposed to BrdU for five hours and processed for BrdU incorporation using an antibody against BrdU. This panel represents chromosomes from …
(A and B) HTD114 cells were transfected with PTBP1 shRNA expression vector, exposed to BrdU for 5 hr, harvested for mitotic cells, and processed for BrdU incorporation (green) and DNA FISH using a …
HTD114 cells were transfected with shRNA expression vectors for HNRNPA1 (A), HNRNPC (B), HNRNPL (C), HNRNPM (D), HLTF (E), KHSRP (F), HNRNPU (G), or UCHL5 (H), exposed to BrdU for 5 hr, harvested …
(A and B) RNA-DNA FISH on mouse cells containing an ASAR6 BAC transgene integrated into mouse chromosome 3 Donley et al., 2013. (A) RNA-DNA FISH using an ASAR6 fosmid probe to detect ASAR6 RNA …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Anti-HLTF (rabbit polyclonal) | Sigma | RRID:AB_1857263 | 1:1000 |
Antibody | Anti-HNRNPA1 (rabbit polyclonal) | Sigma | HPA004276 | 1:500 |
Antibody | Anti-HNRNPC (rabbit polyclonal) | Sigma | RRID:AB_2681335 | 1:1000 |
Antibody | Anti-HNRNPL (rabbit polyclonal) | Sigma | RRID:AB_2681588 | 1:500 |
Antibody | Anti-HNRNPM (rabbit polyclonal) | Sigma | RRID:AB_1850879 | 1:2000 |
Antibody | Anti-HNRNPU (rabbit polyclonal) | Sigma | RRID:AB_2683796 | 1:500 |
Antibody | Anti-HNRNPUL1 (rabbit polyclonal) | Sigma | RRID:AB_2679616 | 1:1000 |
Antibody | Anti-KHDRBS1 (rabbit polyclonal) | Sigma | RRID:AB_2681417 | 1:500 |
Antibody | Anti-KHSRP (rabbit polyclonal) | Sigma | RRID:AB_10601582 | 1:500 |
Antibody | Anti-PTPB1 (mouse monoclonal) | Invitrogen | RRID:AB_2533082 | 1:250 |
Antibody | Anti-SAFB (rabbit polyclonal) | Sigma | RRID:AB_1850622 | 1:500 |
Antibody | Anti-SAFB2 (rabbit polyclonal) | Sigma | RRID:AB_2681266 | 1:500 |
Antibody | Anti-UCHL5 (rabbit polyclonal) | Sigma | RRID:AB_1858585 | 1:2000 |
Antibody | Anti-alpha-tubulin (mouse monoclonal) | Sigma | RRID:AB_2617116 | 1:10,000 |
Antibody | Anti-BrdU FITC (mouse monoclonal) | Roche | RRID:AB_11042627 | 50 µg/ml |
Antibody | Anti-mouse IgG, Dylight 680 (goat polyclonal) | Invitrogen | RRID:AB_1965956 | 1:10,000 |
Antibody | Anti-mouse IgG, Dylight 650 (goat polyclonal) | Thermo Fisher | RRID:AB_10942301 | 1:10,000 |
Antibody | Anti-rabbit IgG, Dylight 800 (goat polyclonal) | Thermo Fisher | RRID:AB_2556775 | 1:10,000 |
Antibody | Anti-rabbit IgG, Alexa Fluor Pus 405 (goat polyclonal) | Invitrogen | RRID:AB_2890548 | 1:100 |
Antibody | Anti-goat IgG, Alexa Fluor 594 (rat monoclonal) | Invitrogen | RRID:AB_2536080 | 1:100 |
Recombinant DNA reagent | shRNA:HLTF (plasmid) | Sigma | TRCN0000272562 | |
Recombinant DNA reagent | shRNA:HNRNPA1 (plasmid) | Sigma | TRCN0000006586 | |
Recombinant DNA reagent | shRNA:HNRNPC (plasmid) | Sigma | TRCN0000006644 | |
Recombinant DNA reagent | shRNA:HNRNPL (plasmid) | Sigma | TRCN0000017246 | |
Recombinant DNA reagent | shRNA:HNRNPM (plasmid) | Sigma | TRCN0000314949 | |
Recombinant DNA reagent | shRNA:HNRNPU (plasmid) | Sigma | TRCN0000350256 | |
Recombinant DNA reagent | shRNA:HNRNPUL1 (plasmid) | Sigma | TRCN0000074732 | |
Recombinant DNA reagent | shRNA:KHDRBS1 (plasmid) | Sigma | TRCN0000428104 | |
Recombinant DNA reagent | shRNA:KHDRBS1 (plasmid) | Sigma | TRCN0000428752 | |
Recombinant DNA reagent | shRNA:KHSRP (plasmid) | Sigma | TRCN0000013256 | |
Recombinant DNA reagent | shRNA:PTBP1 (plasmid) | Sigma | TRCN0000231420 | |
Recombinant DNA reagent | shRNA:SAFB (plasmid) | Sigma | TRCN000022099 | |
Recombinant DNA reagent | shRNA:SAFB2 (plasmid) | Sigma | TRCN0000075057 | |
Recombinant DNA reagent | shRNA:UCHL5 (plasmid) | Sigma | TRCN0000234907 |