Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: RNF8 promotes ALT-EJ. ( A ) RNA analysis of an Rnf8-/- mESC line generated using Cas9 and sgRNAs targeting Rnf8 . Shown are RT-PCR amplification products for a region of the RNF8 mRNA from exons 1–6 (Ex1–Ex6), and Actin control, for RNA from WT and Rnf8-/- mESCs, treated with and without reverse transcriptase (RT). ( B ) The Rnf8-/- mESC line shows a defect in 53BP1 foci formation. Shown are representative fluorescent images of WT and Rnf8-/- mESCs treated with 6 Gy and recovered for 4 h, stained with DAPI and antibodies for γH2AX and 53BP1. Scale bar = 20 μm. Also shown are violin plots depicting the number of γH2AX foci per nucleus (left) and 53BP1 foci per nucleus (right) in WT and Rnf8-/- mESC. N = 120. (*) P < 0.001 for WT versus Rnf8-/- measuring γH2AX or 53BP1 foci using the Mann–Whitney test. ( C ) RNF8 promotes ALT-EJ, and inhibits EJ without indels (i.e. C-NHEJ). Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, EJ6-GFP+TREX2, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Rnf8-/- mESCs and Rnf8-/- mESCs transfected with a 3×Flag-tagged RNF8 expression vector (3×F-RNF8). WT EJ7-GFP and EJ6-GFP+TREX2 values are from Figure and shown here for comparison. As with all mESC experiments in this study, the reporters are integrated into chromosome 17 at the Pim1 locus. Error bars represent SD. N ≥ 6. (*) P ≤ 0.038 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for Flag, and Actin control, in Rnf8-/- mESCs transfected with and without 3×F-RNF8. (*) Non-specific bands. ( D ) RNF8 promotes ALT-EJ in mESCs, as measured by the EJ2-GFP reporter. Shown are GFP+ frequencies for EJ2-GFP in WT and Rnf8-/- mESCs with and without complementation vector. N = 12. Error bars represent SD. (*) P ≤ 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) RNF8 promotes ALT-EJ in HEK293 cells, as measured by the EJ2-GFP reporter. Two independent RNF8 knockout ( RNF8-KO ) cell lines ( RNF8-KO clones A and B) were generated in an HEK293 cell line with the EJ2-GFP reporter. Shown are GFP+ frequencies for the EJ2-GFP assay for these two cell lines with and without an RNF8 expression vector, as well as for the HEK293 WT cell line. N = 6. Error bars represent SD. (*) P ≤ 0.001766 for unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot analysis for RNF8, and Actin control, for the two RNF8-KO HEK293 cell lines transfected with RNF8, or only control EV, along with HEK293 WT cells transfected with EV.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Generated, Reverse Transcription Polymerase Chain Reaction, Amplification, Staining, MANN-WHITNEY, Transfection, Expressing, Plasmid Preparation, Western Blot, Knock-Out, Clone Assay

Journal: Nucleic Acids Research

Article Title: RNF8 has both KU-dependent and independent roles in chromosomal break repair

doi: 10.1093/nar/gkaa380

Figure Lengend Snippet: 53BP1 does not have a substantial effect on either EJ without indels or ALT-EJ, whereas POLQ promotes ALT-EJ independently of KU. ( A ) Influence of 53BP1 on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, 53bp1-/- mESCs and 53bp1-/- mESCs transfected with an expression vector for human 53BP1. Also shown are GFP+ frequencies for 4-μHOM Embed in 53bp1-/-Rnf8-/- mESCs transfected with and without 53BP1 and RNF8 (3×F-RNF8) expression vectors, and compared to 53bp1-/- mESCs. WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P ≤ 0.049 using unpaired multiple t -tests with the Holm–Sidak correction. Also shown is an immunoblot staining for 53BP1 and Actin control in WT and 53bp1-/- mESCs transfected with and without 53BP1. (*) represent non-specific bands. ( B ) Influence of POLQ on distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, Polq-/- mESCs and Polq-/- mESCs transfected with human Flag-tagged POLQ (POLQ). WT values for EJ7-GFP and 4-μHOM are from Figures and , respectively, and are shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( C ) Immunoblot analysis of KU70, Flag-POLQ and Actin control. A double mutant Ku70-/-Polq-/- mESC line was generated to examine the influence of POLQ and KU70 on EJ independently of the other factor. Shown are KU70 immunoblot signals for WT, and Ku70-/-Polq-/- mESCs transfected with and without KU70. Also shown are Flag and Actin control immunoblot signals for Ku70-/-Polq-/- mESCs transfected with and without Flag-tagged POLQ. (*) indicates a non-specific band. ( D ) POLQ and KU70 independently mediate distinct EJ events. Shown are the GFP+ frequencies for several chromosomal reporter assays (EJ7-GFP, 4-μHOM Terminal and 4-μHOM Embed) in WT mESCs, and Ku70-/-Polq-/- mESCs without complementation vector, or with expression vectors for KU70 or POLQ. WT values for EJ7-GFP, and 4-μHOM are from Figures and , respectively, and shown for comparison. N = 6, except N = 9 for WT. Error bars represent SD. (*) P < 0.001 for unpaired multiple t -tests with the Holm–Sidak correction. ( E ) Shown is a model for the influence of RNF8 on chromosomal break repair outcomes.

Article Snippet: The pCAGGS-53BP1 (human) expression vector was generated from N-Myc-53BP1 WT pLPC-Puro (Addgene 19836) ( ).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Mutagenesis, Generated

Journal:

Article Title: Induction of p53-Independent Apoptosis by Simian Virus 40 Small t Antigen

doi: 10.1128/JVI.75.19.9142-9155.2001

Figure Lengend Snippet: Protection from apoptosis by Bcl-2, caspase inhibitor, or MEK inhibitor. (A) U2OS cells were transfected with the pIRES2 EGFP-st vector, which expresses st bicistronically with EGFP as a marker (left panel). The right panel shows staining of the same field of cells with Hoechst 33258, thus visualizing cellular DNA. Note the rounded shape of st-expressing cells (left panel) as well as the condensed chromatin (right panel). (B) U2OS cells were transfected with pIRES2 EGFP-st either alone (left panel) or together with a CMV Bcl-2 expression vector in a 1:4 ratio (right panel). The rounded shape of dying cells is reversed by Bcl-2 coexpression to resemble normal cell shape. (C) U2OS cells were cotransfected with pSG5 st and CMV Bcl-2 in a 1:4 ratio. Subsequently, the cells were stained for st with PAb419 and with DAPI to visualize the DNA as described in the legend to Fig. ​Fig.2A.2A. Note the intact nuclear morphology, in comparison to Fig. ​Fig.2B.2B. (D) U2OS cells were transfected with st alone or, alternatively, coexpressed with either Bcl-2 or the broad caspase inhibitor p35. In some instances, the st-transfected cells were treated with potential modulators of apoptosis such as the broad caspase inhibitor z-VAD-Fmk (50 μM) or the MEK inhibitor U0126 (10 μM), and results were compared to that obtained with vehicle (DMSO) alone. Transfected cells expressing st were identified by cell staining with PAb419, and nuclear morphology was assessed as intact or disrupted. The percentage of disrupted nuclei was plotted in the graph and compared to that obtained with LT (negative control). At least two independent experiments, each time counting 100 st-expressing cells, were conducted; for some of the combinations several more experiments were performed. Average values are displayed in the graph. (E) U2OS cells transfected with either 1 μg of st vector plus 4 μg pcDNA3 vector or 1 μg of st vector plus 4 μg of pCMV Bcl-2 were lysed after 48 h and assayed by Western blotting for st expression using the PAb419 antibody. In the right panel, the same samples were analyzed for Bcl-2 expression using a Bcl-2 monoclonal antibody.

Article Snippet: Basically, the colony-forming ability of an empty vector (pLPCX) and that of the same vector directing expression of st from the CMV promoter (pLPCX st) were compared when transfecting U2OS cells and selecting for puromycin resistance inherent in the pLPCX vector (Clontech).

Techniques: Transfection, Plasmid Preparation, Marker, Staining, Expressing, Negative Control, Western Blot

Journal: BMC Biology

Article Title: Targeting of REST with rationally-designed small molecule compounds exhibits synergetic therapeutic potential in human glioblastoma cells

doi: 10.1186/s12915-024-01879-0

Figure Lengend Snippet: REST promotes glioblastoma growth. A Boxplot of REST mRNA expression in TCGA-LGG and TCGA-GBM samples compared to normal brain samples from TCGA and GTEx datasets (* p < 0.001). B Survival analysis using data from TCGA-GBM and TCGA-LGG projects. Analysis was done using GEPIA2 web server ( A , B ). C Basal REST protein amount in a panel of select cell lines. Three to four independent biological replicates are shown as mean ± SD. Statistical difference vs SVGp12 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File A. D Proliferation of GBM cell lines assessed by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments (mean ± SD). Statistical difference vs U251 was tested using ANOVA with post hoc tests. Individual data values are provided in Additional File B. E Western blot confirms the lack of REST in homozygous REST-KO clones of T98G ( left ) and HEK293 ( right ). F Proliferation of WT and REST-KO T98G cells was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three to four independent experiments (mean ± SD). Statistical comparison vs T98G control was performed using ANOVA with post hoc tests. Individual data values are provided in Additional File C. G Proliferation of T98G WT and REST-KO C10 cells (transfected with empty vector pLPC vs REST OE) was examined by counting cells every 24 h. Shown is one representative replicate and quantification of PDT based on three independent experiments. Statistical difference was tested using two-tailed paired t -test. Individual data values are provided in Additional File D. H Wound scratch assay and its quantification using ImageJ. Shown are mean ± SD from three independent biological experiments. Groups were compared using paired t -tests. Individual data values are provided in Additional File E. I Effect of REST loss on GSC marker expression. Shown are fold changes (FC) vs CRISPR Control derived from three independent biological replicates. Comparison vs control was performed using unpaired one-tailed t -tests. Dashed line indicates FC = 1. Individual data values are provided in Additional File F. *** p < 0.001; ** p < 0.01; * p < 0.05; ns—not significant

Article Snippet: On the next day, cells were transiently transfected with 500 ng either REST-WT-expressing pLPC-vector (Addgene, #41903) or empty pLPC vector (Addgene, #12521) with Fugene HD transfection reagent (Promega) following manufacturer’s instructions.

Techniques: Expressing, Western Blot, Clone Assay, Comparison, Transfection, Plasmid Preparation, Two Tailed Test, Wound Healing Assay, Marker, CRISPR, Derivative Assay, One-tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STXBP4 regulates APC/C-mediated p63 turnover and drives squamous cell carcinogenesis

doi: 10.1073/pnas.1718546115

Figure Lengend Snippet: ΔNp63α promotes proliferation and suppresses terminal differentiation in primary human keratinocytes. (A) APC/C degradation-resistant ΔNp63α and Stxbp4 suppress terminal differentiation. Empty vector (Mock), degradation-resistant ΔNp63α (RL7-ΔNp63α), and Stxbp4 retrovirally infected pHKCs were cultured in 1.5 mM CaCl2 to induce differentiation and were imaged by an LSM 700 laser-scanning confocal microscope (Zeiss). (Scale bars: 50 μm.) (B) Retrovirus-infected pHKCs cultured in high-calcium conditions were harvested at the indicated times, and cell lysates were subjected to immunoblotting with the indicated antibodies. (C) Involucrin mRNA level as in B was determined by RT-qPCR. (D) Stxbp4 (ΔCC-Stxbp4) retrovirally infected pHKCs cultured in high-calcium conditions were harvested at the indicated times. The cell lysates were subjected to immunoblotting with the indicated antibodies. Full-length Stxbp4 is shown in B. (E) Involucrin mRNA levels as in D were determined by RT-qPCR. Stxbp4 mRNA is shown in C. (F) Stxbp4 and APC/C-resistant ΔNp63α-infected pHKCs do not undergo terminal differentiation in 3D organotypic raft cultures. 3D skin equivalents were generated using human pHKCs infected with empty vector (Mock), stable RL7-ΔNp63α–, and Stxbp4-expressing retroviruses. Involucrin (Alexa 594: red) and Loricrin (Alexa 488: green) were detected in pLPCX-infected keratinocytes (Left), and human skin epidermis was used for comparison (Lower Right). (Scale bars: 20 μm.) IF, immunofluorescence.

Article Snippet: Plasmids expressing FLAG-HA doubly tagged ΔNp63α, FLAG-tagged wild-type and ubiquitination-resistant (RL7) ΔNp63α, and FLAG-tagged Stxbp4 were cloned into the MSCV or pLPCX retrovirus expression vector (Clontech, Takara Bio).

Techniques: Plasmid Preparation, Infection, Cell Culture, Microscopy, Western Blot, Quantitative RT-PCR, Generated, Expressing, Immunofluorescence