Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 2. Tipin directly interacts with Mta2. ( A ) Table indicating the Mta2 peptides identified via mass spectrometry analysis in Tipin immunoprecipitates. ( B ) Equal amounts of extract were immunoprecipitated with either anti-Tipin, anti-Mta2 antibodies or pre-immune serum. Purified proteins were immunoblotted with the indicated antibodies to detect associated proteins. ( C ) Pull down assay using His-Tipin and in vitro translated 35 S-labeled Mta2. Mta2 was detected by autoradiograph.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques: Mass Spectrometry, Immunoprecipitation, Purification, Pull Down Assay, In Vitro, Labeling, Autoradiography

Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 3. Mta2 is required for DNA replication under minimum licensing conditions. ( A ) Immunoblot to assess Tipin or Mta2 depletion from egg extract. ( B ) Scheme of minimum licensing conditions experiments and replication assays. ( C ) DNA replication in “maximum” or “minimum licensed” mock, Tipin or Mta2 depleted extract at 60 min. ( D ) The efficiency of DNA replication in “minimum licensed” mock (blue), Tipin (red), Mta2 (yellow), and Tipin/Mta2 (green) co-depleted extracts at 60 min from nuclei addition to egg extract was tested. The defect in DNA replication observed in the Mta2 depleted extract (yellow) was rescued by the addition of 400 nM Mta2 recombinant protein (purple). Three independent experiments are averaged in the bar graphs. The error bars are standard deviation from the mean value.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques: Western Blot, Recombinant, Standard Deviation

Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 4. Mta2 is required for Tipin loading on the chromatin. ( A ) Immunoblot analysis to detect the level of Mta2, Tipin, Orc1, and Histone H2B on the chromatin at different time points (30, 60, 90 min) in extract untreated or treated with geminin or aphidicolin. ( B ) Immunoblot to detect the levels of Mta2, Tipin, and Orc1 on the chromatin at 60 min from nuclei addition.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques: Western Blot

Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 5. Mta2 is required for the efficient association Pol α to the chromatin in minimum licensing conditions. Sperm nuclei were added to mock or Mta2 depleted Xenopus egg extract. Geminin was supplemented 3 min post-nuclei addition to generate a minimum licensing system and chromatin was harvested at different times. Chromatin-bound proteins were analyzed by SDS-PAGE and immunoblot analysis was performed with the indicated antibodies.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques: SDS Page, Western Blot

Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 6. Mta2 prevents the formation of reversed forks. ( A ) Example of reversed replication fork in Mta2 depleted extract. ( B ) Quantification of replication intermediates with reversed forks in mock or Mta2-depleted extract performed as in Figure 1D . Experiments were repeated at least 3 times. The graph shows the results of a typical experiment where at least 100 forks were scored in mock or Mta2-depleted extracts.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques:

Journal: Cell Cycle

Article Title: Mta2 promotes Tipin-dependent maintenance of replication fork integrity

doi: 10.4161/cc.29157

Figure Lengend Snippet: Figure 8. Model for Tipin–Mta2 function in maintaining fork integrity. ( A ) Tipin–Tim1 complex might create a bridge between replisome components such as Cdc45, GINS, Pol α, and the MCM complex necessary for the stable binding of Pol α to the replication fork. Mta2 might facilitate Tipin function at specific genome loci containing repetitive DNA sequences. The presence of Tipin might promote Pol α-mediated replication restart downstream lesions or structures that halt polymerases progression. ( B ) In the absence of Mta2 and/or Tipin inefficient re-priming and inability to resume fork progression leads to fork reversal.

Article Snippet: The plasmid (pCMV-SPORT6) carrying Xenopus laevis Mta2 full-length cDNA was obtained from ImaGenes, IRBHp990G0784D.

Techniques: Binding Assay

Journal: Journal of Cell Communication and Signaling

Article Title: ACK1 upregulated the proliferation of head and neck squamous cell carcinoma cells by promoting p27 phosphorylation and degradation

doi: 10.1007/s12079-022-00670-6

Figure Lengend Snippet: ACK1 promoted the ubiquitin-mediated proteasomal degradation of p27.The ACK1-knockdown a OECM-1, b HSC-3, or c SAS cells were stably expressing ACK1 and treated with 0.01 or 0.05 µM MG132 for 48 h. These cells were then harvested and total lysates were prepared for western blot analyses. d ACK1-knockdown (shACK1) or ACK1-rescued (shACK1+ACK1) SAS cells were transfected with the control vector or the Flag-tagged wild-type (WT) of p27 plasmids. After 48 h of culture, transfected cells were harvested and total lysates were immunoprecipitated with anti-Flag Abs. The supernatants of immunoprecipitated complexes were then immunoblotted with anti-ubiquitin (Ub) or anti-Flag Abs. Input indicates about 10% of protein amounts used for co-immunoprecipitation (co-IP). e SAS cells were infected with viruses carrying control shRNA (shCt) or COP1-, Skp2-, or KPC1-targeted shRNA (i.e., shCOP1, shSkp2, or shKPC1). Total lysates were prepared for western blot analyses (upper panel) and the quantification result of p27 expression was shown (lower panel). Data were expressed as the mean ± SD from three independent experiments. **<0.01 vs. the ratio of shCt control cells. f The shCt control, ACK1-knockdown, and ACK1-rescued SAS cells were harvested and the total lysates were prepared for western blot analyses

Article Snippet: For the construction of p27-containing plasmid, we have purchased the pCMV-SPORT6 expression vector carrying the wild-type human p27 cDNA from Bioresource Collection and Research Centre (BCRC, Hsinchu, Taiwan).

Techniques: Stable Transfection, Expressing, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Co-Immunoprecipitation Assay, Infection, shRNA

Journal: Journal of Cell Communication and Signaling

Article Title: ACK1 upregulated the proliferation of head and neck squamous cell carcinoma cells by promoting p27 phosphorylation and degradation

doi: 10.1007/s12079-022-00670-6

Figure Lengend Snippet: ACK1 phosphorylated and interacted with p27. a 293T cells were co-transfected with the control vector (vec) or ACK1 plasmids plus the Flag-tagged wild-type (WT), Y74F, Y88F, or Y89F mutant of p27 plasmids. b ACK1-knockdown (shACK1) or ACK1-rescued (shACK1+ACK1) SAS cells were transfected with the control vector, or the WT, Y74F, Y88F, or Y89F mutant of p27 plasmids. After 48 h of culture, transfected cells were harvested and total lysates were immunoprecipitated with anti-Flag Abs. The supernatants of immunoprecipitated complexes were then immunoblotted with anti-ACK1, anti-phosphotyrosine, or anti-Skp2 Abs. Input indicates about 10% of protein amounts used for co-IP

Article Snippet: For the construction of p27-containing plasmid, we have purchased the pCMV-SPORT6 expression vector carrying the wild-type human p27 cDNA from Bioresource Collection and Research Centre (BCRC, Hsinchu, Taiwan).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: International Journal of Molecular Sciences

Article Title: A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity

doi: 10.3390/ijms24021278

Figure Lengend Snippet: Subnetwork of genes associated with SOCE and connected to SURF4 .

Article Snippet: pCMV-SPORT6-SURF4 (SURF4) plasmid was obtained from BioCat (Heidelberg, Germany).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity

doi: 10.3390/ijms24021278

Figure Lengend Snippet: SURF4 contributes to Aβ 1–42 toxicity on neuroblastoma cells. ( A ) Human neuroblastoma cells were transfected with SURF4 siRNA or with a non-active control siRNA, and after 48 h the levels of SURF mRNA were quantified by semi-quantitative rtPCR. Data are the mean ± SEM of 3 independent experiments. **** p < 0.0001 vs. control by Student’s t-test. ( B , C ) Cells transfected with SURF4 siRNA were treated with 5 µM ( B ) or 10 µM ( C ) oAβ 1–42 for 24 h. Data are the mean ± SEM of 3–10 independent experiments. * p < 0.05 vs. control by Student’s t-test. ( D ) Human neuroblastoma cells were transfected with a plasmid containing the sequence of human SURF4 or with a non-coding control ( pcDNA3 ), and after 48 h the levels of SURF mRNA were quantified by semi-quantitative rtPCR. Data are the mean ± SEM of 3 independent experiments. **** p < 0.0001 vs. control by Student’s t-test. ( E , F ) Cells transfected with the plasmid to overexpress SURF4 were treated with 5 µM ( B ) or 10 µM ( C ) oAβ 1–42 for 24 h. Data are the mean ± SEM of 4 independent experiments. ** p < 0.01 and * p < 0.5 vs. control by Student’s t-test.

Article Snippet: pCMV-SPORT6-SURF4 (SURF4) plasmid was obtained from BioCat (Heidelberg, Germany).

Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity

doi: 10.3390/ijms24021278

Figure Lengend Snippet: SURF4 conditions SOCE activity. ( A , B ) Human neuroblastoma cells were transfected with SURF4 siRNA ( A ) or with a plasmid to overexpress SURF4 ( B ), and after 48 h the levels of calcium were measured by using FURA2. Data are the mean ± SEM of 4–7 independent experiments. *** p < 0.001 vs. control by Student’s t-test. ( C , D ) Cells transfected with SURF4 siRNA ( C ) or SURF4 plasmid ( D ) were exposed to 0 extracellular calcium, and intracellular Ca 2+ changes in response to ER depletion by thapsigargin were measured using FURA2. Data are the mean ± SEM of 4–6 independent experiments. ( E , F ) Cells were transfected with SURF4 siRNA ( E ) or SURF4 plasmid ( F ) during 48 h, and SOCE activity (induced by ER Ca 2+ release with thapsigargin) was evaluated with FURA2 following re-addition of Ca 2+ to the bathing solution. Data are the mean ± SEM of 5–7 independent experiments. *** p < 0.001 vs. control by Student’s t-test.

Article Snippet: pCMV-SPORT6-SURF4 (SURF4) plasmid was obtained from BioCat (Heidelberg, Germany).

Techniques: Activity Assay, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: A Genome-Wide Functional Screen Identifies Enhancer and Protective Genes for Amyloid Beta-Peptide Toxicity

doi: 10.3390/ijms24021278

Figure Lengend Snippet: SURF4 affects SOCE proteins. ( A ) Neuroblastoma cells were treated with 10 µM oAβ 1–42 in the presence of the SOCE inhibitor Ro for 24 h. Data are the mean ± SEM of 3 independent experiments. ** p < 0.01, non-significant (ns) vs. the respective controls by ANOVA plus Bonferroni as post-hoc test. ( B ) SOCE is dependent in the interaction of STIM, located in ER, with ORAI, located in the plasmatic membrane that opens to allow the entrance of calcium.

Article Snippet: pCMV-SPORT6-SURF4 (SURF4) plasmid was obtained from BioCat (Heidelberg, Germany).

Techniques: