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:

Journal: Molecular cell

Article Title: MISTERMINATE Mechanistically Links Mitochondrial Dysfunction with Proteostasis Failure

doi: 10.1016/j.molcel.2019.06.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The original human C-I30 CDS sequence was from the pPM-N-D-C-His (PV394217, abm) plasmid. pcDNA3.1(+)-C-I30-FLG and pcDNA3.1(+)-C-I30-FLG-TEV plasmids were generated by cloning the human C-I30 CDS with different tags into pcDNA3.1(+) vector via KpnI and Xba I sites. pcDNA3.1(+)-C-I30-TEV-FLG; pcDNA3.1(+)-C-I30-FLG-AT5; pcDNA3.1(+)-C-I30-FLG-AT23; pcDNA3.1(+)-C-I30-FLG-non-AT25; pcDNA3.1(+)-C-I30(mMTS)-FLG (36R/A); pcDNA3.1(+)-C-I30-FLG-Astop; pcDNA3.1(+)-C-I30-FLG-AKstop; pcDNA3.1(+)-C-I30-FLG-AKKstop and pcDNA3.1(+)-HA-C-I30-FLG; were modified based on the pcDNA3.1(+)-C-I30-FLG-TEV plasmid via the Q5 Site-Directed Mutagenesis Kit (cat#: E0554S, NEB). pCMV-FLAG-ABCE1 was a gift from Dr. Ramanujan Hegde. pCMV6-FLAG-NOT4 and pCMV6-ANKZF1 was obtained from OriGene Inc (cat#: RC217418 and RC201054 TrueORF). pCMV-SPORT6.1-eRF1 (cat#: MHS6278–202804766, DharmaconTM) and pCMV-SPORT6.1-VCP (cat#: MHS6278–202760239, DharmaconTM) were from GE healthcare.

Techniques: Recombinant, Protease Inhibitor, FLAG-tag, ATP Bioluminescent Assay, Activity Assay, Western Blot, Autoradiography, Negative Control, Software