Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a Microarray data generated from a survey of RMS primary tumors by Williamson et al. , in addition to healthy human quadriceps (muscle) data, were used to determine the fold change in HNRNPH1 expression (muscle, n = 40; ERMS, n = 32; ARMS, n = 57). b Quantitative RT-PCR of HNRNPH1 expression in LHCN-M2 (LHCN) and RMS cells (RD, RH30, and RH41). Data are expressed relative to LHCN cells as the mean ± SD ( n = 3). c Immunoblot analysis and quantification of whole-cell lysates prepared from LHCN and RMS cells detected with antibodies against HNRNPH1 and β-actin (Actin, loading control). Data were quantified with ImageJ software (mean ± SD, n = 3). d–f Cell growth curves of lipofectamine alone (lipo), siControl (siCon), and si#1–3 (3 individual HNRNPH1 siRNAs) were obtained by using the Incucyte Zoom live-cell imaging system and data are expressed as cell confluence (%; mean ± SD, n = 2). g–i Immunoblot analysis of whole-cell lysates prepared from HNRNPH1 siRNA–transfected g RD, h RH30, and i RH41 cells with antibodies against HNRNPH1 and β-actin. HRNRPH1 quantification is shown below gels and normalized to actin; relative intensity for the sample treated with lipo alone was set as 1.0. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001, all data were compared to the control group using one-way ANOVA

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Microarray, Generated, Expressing, Quantitative RT-PCR, Western Blot, Control, Software, Live Cell Imaging, Transfection

Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a Flow cytometric analysis of EdU incorporation 48 h after HNRNPH1 siRNA transfection in RMS cells. RD cells were labeled with EdU for 3 h. RH30 and RH41 cells were labeled with EdU for 2 h. b The percentage of EdU + cells were quantified (mean ± SD, n = 4). c Flow cytometric analysis of Annexin V + cells 96 h after HNRNPH1 siRNA transfection in RMS cells. d The percentage of Annexin V + cells were quantified (mean ± SD, n = 4). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Transfection, Labeling

Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a Immunoblot analysis of whole-cell lysates prepared from RMS cells (RD, RH30, and RH41) 2 and 4 days post- HNRNPH1 siRNA transfection with antibodies against CDK2/4/6, HNRNPH1, and β-actin. b Immunoblot analysis of whole-cell lysates prepared from RMS cells (RD, RH30, and RH41) 4 and 7 days post- HNRNPH1 siRNA transfection with antibodies against PARP, cleaved PARP, caspase-3, cleaved caspase-3, HNRNPH1, and β-actin. All quantification is shown below the gels and normalized to actin. CDK4 was quantified using the short exposure for RH30 cells

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Western Blot, Transfection

Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a RD and b RH30 Tet-On HNRNPH1 shRNA stable cell lines were induced with 200 ng/mL doxycycline (dox). Incucyte Zoom was used to quantify cell growth, and data are expressed as percentage of cell confluence (%; mean ± SD, n = 2). c , d Mice were treated with 0.4 mg/mL dox in their drinking water immediately after cell transplantation. c RD and d RH30 xenograft volumes were measured at the indicated days after cell transplantation. e , g RD and f , h RH30 xenografts were collected and weighed at the end of the experiments. plko, Tet-pLKO-puro vector control; sh1, HNRNPH1 shRNA#1 in the pLKO-Tet-On vector

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: shRNA, Stable Transfection, Transplantation Assay, Plasmid Preparation, Control

Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a Quantitative RT-PCR of HNRNPH1 expression in HNRNPH1 siRNA-transfected RMS cells (48 h). b Volcano plots of two individual HNRNPH1 siRNAs vs. siCon in RD, RH30, and RH41 cells. Genes with P > 3 (–log10) and absolute logFC < 1.5 are highlighted in green (downregulated) or red (upregulated). Dark grey dots indicate genes with a false discovery rate <5%. c Gene set enrichment analysis of the highlighted genes in b . Genes that were upregulated by HNRNPH1 siRNAs were compared with those downregulated by PAX-FOXO1, as described in Davicioni et al. 2007 . Genes downregulated by HNRNPH1 siRNAs were compared with those downregulated in G1-arrested cells, as described in Zhou et al. 2007

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Quantitative RT-PCR, Expressing, Transfection

Journal: Oncogenesis

Article Title: HNRNPH1 is required for rhabdomyosarcoma cell growth and survival

doi: 10.1038/s41389-017-0024-4

Figure Lengend Snippet: a – c Two individual siRNAs targeting HNRNPH1 were compared against control siRNA, with gene expression (logFC) plotted on the X-axis and exon junctions (logFC) on the Y-axis. Significant changes in exon junction expression (|logFC exon junction| >1 and |logFC gene| <0.05) were classified as candidate alternative splicing events (black). d Summary of the number and type of statistically significant alternative splicing events, as determined by multivariate analysis of transcript splicing (MATS) (false discovery rate <0.05). A3SS, alternative 3´ ends; A5SS, altered 5´ ends; SE, cassette exons; MXE, mutually exclusive exons; and RI, retained introns. e – g Bar charts show gene ontology categories significantly enriched (false discovery rate <5%) in the alternatively spliced genes induced by HNRNPH1 siRNA, as determined by MATS

Article Snippet: HNRNPH1 antibody (NB100-385) was purchased from Novus Biologicals (Littleton, CO).

Techniques: Control, Gene Expression, Expressing, Alternative Splicing