quality control strains staphylococcus aureus atcc 2913  (ATCC)

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Inhibition of AKT or mTOR downregulates PTEN expression in AKT1 E17K MEFs (A) The protein level of PTEN in AKT1 E17K and control MEFs. (B) The PTEN level in AKT1 E17K MEFs after treatment with 5 μM MK2206 for 24 h. (C) The PTEN level in AKT1 E17K MEFs after treatment with 10 nM rapamycin for 24 h. DMSO was used as a vehicle control. (D) The PTEN level in AKT1 E17K MEFs after treatment with 50 nM Torin2 for 24 h. (E) Quantification of the relative protein level of PTEN. Data are expressed as the mean±SD ( n=3). * P<0.05, *** P<0.001. ns, not significant.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Expressing, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Inhibition of mTOR reduces PTEN levels in TSC2 −/− MEFs (A) The protein level of PTEN in TSC2 −/− and TSC2 +/+ MEFs. (B) The protein level of PTEN in TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h. (C) The protein level of PTEN in TSC2 −/− MEFs after treatment with 50 nM Torin2 for 24 h. (D) Quantification of the relative protein level of PTEN. Data are expressed as the mean±SD ( n=3). (E) Relative mRNA levels of PTEN in AKT1 E17K and control MEFs after treatment with 5 μM MK2206, 10 nM rapamycin and 50 nM Torin2 for 24 h. (F) Relative mRNA level of PTEN in TSC2 −/− and TSC2 +/+ MEFs after treatment with 10 nM rapamycin and 50 nM Torin2 for 24 h, respectively. *** P<0.001. ns, not significant.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Inhibition of mTOR does not affect the subcellular localization of PTEN (A) The protein level of PTEN in the cytoplasm and nucleus of AKT1 E17K MEFs after treatment with 10 nM rapamycin for 24 h. (B) The protein level of PTEN in the cytoplasm and nucleus of TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h. Lamin A/C and GAPDH are markers of nuclear and cytoplasmic proteins, respectively. (C) Immunofluorescence microscopy was used to visualize the subcellular localization of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin or 50 nM Torin2 for 24 h. Cy3-labelled PTEN and DAPI were used to stain the nuclei. (D) Quantification of the fluorescence intensity in the cytoplasm. (E) Quantification of the fluorescence intensity in the nucleus. *** P<0.001.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Intervening with 4E-BP1/eIF4E-dependent translation initiation is not able to rescue the decreased PTEN mediated by mTOR inhibition (A,C) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h after 4E-BP1 knockdown. (B,D) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h after eIF4E knockdown.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Knockdown

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Suppression of proteasome or lysosome degradation cannot rescue the downregulated PTEN level (A,C) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 10 nM rapamycin combined with 10 μM chloroquine (CQ), 5 mM NH 4Cl and 5 μM MG132 for 24 h. P62 was used as a positive control. (B,D) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after treatment with 50 nM Torin2 combined with 10 μM CQ, 5 mM NH 4Cl and 5 μM MG132 for 24 h. (E,G) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs pretreated with 100 μg/mL CHX for different time and then incubated with 10 nM rapamycin for 24 h. β-Catenin was used as a positive control. (F,H) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs pretreated with 100 μg/mL CHX for different time and then incubated with 50 nM Torin2 for 24 h. *** P<0.001. ns, not significant.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Positive Control, Incubation

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Inhibition of mTOR upregulates the expressions of miR-23a-3p, miR-23b-3p, miR-25-3p and miR-26a-5p (A) Construction of four mouse PTEN-3′UTR dual-luciferase reporter plasmids. (B) A dual-luciferase reporter assay confirmed the potential miRNA binding sites in the mouse PTEN-3′UTR. (C) Heatmap of differentially expressed miRNAs in TSC2 −/− MEFs after treatment with 10 nM rapamycin. Red triangles indicate upregulated miR-23a-3p, miR-23b-3p, miR-25-3p and miR-26a-5p. (D,E) Real-time PCR analysis of miRNA expression in TSC2 −/− MEFs after treatment with 10 nM rapamycin for 24 h. (F) Real-time PCR confirmed the 4 upregulated miRNAs in AKT1 E17K MEFs after rapamycin treatment. * P<0.05, ** P<0.01, *** P<0.001. ns, not significant.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Luciferase, Reporter Assay, Binding Assay, Real-time Polymerase Chain Reaction, Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: Inhibition of the AKT/mTOR pathway negatively regulates PTEN expression via miRNAs

doi: 10.3724/abbs.2022159

Figure Lengend Snippet: Inhibition of mTOR suppresses PTEN expression by upregulating miRNAs (A,B) Dual-luciferase reporter assay after miR-23a-3p, miR-23b-3p, miR-25-3p and miR-26a-5p mimics were cotransfected with PTEN-3′UTR-F1 and F2. (C,D) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after miR-23a-3p, miR-23b-3p, miR-25-3p and miR-26a-5p mimics were transfected. (E,F) The protein level of PTEN in AKT1 E17K and TSC2 −/− MEFs after the 4 miRNA inhibitors were transfected followed by treatment with rapamycin. (G,H) PTEN protein level in AKT1 E17K and TSC2 −/− MEFs after cotransfection with the 4 miRNA inhibitors followed by rapamycin treatment. ** P<0.01, *** P<0.001.

Article Snippet: AKT1 E17K , TSC2 –/– and control MEFs were isolated from the pregnant mice by the collagen digestion method and preserved in our lab. All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Carlsbad, USA) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO 2 , and 95% humidity in 12-well plates (Corning, New York, USA), untile the cell confluence reached 70%–80%.

Techniques: Inhibition, Expressing, Luciferase, Reporter Assay, Transfection, Cotransfection

Journal: PLOS Genetics

Article Title: STIL overexpression shortens lifespan and reduces tumor formation in mice

doi: 10.1371/journal.pgen.1011460

Figure Lengend Snippet: ( A ) Proliferation of B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Cells were enumerated every 24 h for a total of 5 days. Data are means ± SEM from three independent experiments. ( B ) Immunoblotting showing p S18 -p53 and p53 protein expression levels in protein extracts from p2-6 CMV-STIL +/- and B6-STIL control MEFs. β-actin served as a loading control; DMSO- and etoposide-treated NIH3T3 cell lysate extracts as controls for p53 increase/phosphorylation after DNA damage. The right panel shows the changes in p S18 -p53 and p53 expression compared to passage 2 (p2) of B6-STIL control MEFs. ( C ) Representative images of B6-STIL control, CMV-STIL +/- and CMV-STIL +/+ MEFs, stained for β-galactosidase-cleaved x-Gal (lower panel). In the upper panel differential contrast interference (DIC) images of the cells are shown. Scale bar, 1 μm. ( D ) Percentage of β-galactosidase-positive, senescent B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Data are means ± Clopper-Pearson 95%-CI. Comparisons were made using the two-sided Fisher’s exact test. For space reasons, only statistically significant differences are displayed. * p <0.05, ** p <0.01, *** p <0.001. ( E ) Immunoblotting showing p16 INK4A protein expression levels in protein extracts from p2-6 CMV-STIL +/- and B6-STIL control MEFs. β-actin served as a loading control. The right panel shows the changes in p16 INK4A expression compared to passage 2 (p2) of B6-STIL control MEFs. ( F ) RNA sequencing showing p16 INK4A mRNA levels in CMV-STIL +/- and CMV-STIL +/+ relative to B6-STIL control MEFs. ( G , H) Percentage of ( G ) Apo-15-positive and ( H ) sub-G 1 phase, apoptotic B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs. Data are means ± SEM from three independent experiments. The differences between groups were tested using the one-way ANOVA with post-hoc Tukey test for multiple comparison. No statistical significance was found. For space reasons, only statistically significant differences are displayed. ( I ) Genotyping of p3-5 CMV-STIL +/- and CMV-STIL +/+ MEFs. All passages harbor the STIL transgene. ( J ) RNA sequencing showing STIL mRNA levels in p3-5 B6-STIL control, CMV-STIL +/- and CMV-STIL +/+ MEFs. ( K ) Immunoblotting showing STIL protein expression levels in p1-5 of B6-STIL control and CMV-STIL +/- MEFs. The STIL band at 170 kDa is marked with an asterisk. β-actin served as a loading control. The lower panel shows the changes in STIL expression compared to p1 of CMV-STIL +/- MEFs. ( L ) Percentage of p3 and p6 B6-STIL control, CMV-STIL +/- , CMV-STIL +/+ , CMV-p53-R172H +/- and CMV-STIL +/- /p53-R172H +/- MEFs with supernumerary centrioles. Comparisons were made using the two-sided Fisher’s exact test. Note : for reasons of space, only statistically significant differences were marked. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: As positive and mock controls wildtype MEFs were treated with 100 nM paclitaxel and DMSO, respectively for 48 h. Thereafter, MEFs were stained using the Senescence β-Galactosidase Staining Kit (Cell Signaling) according to manufacturer’s instructions and eosin as cytoplasmic counter staining to detect senescent cells.

Techniques: Control, Western Blot, Expressing, Phospho-proteomics, Staining, RNA Sequencing, Comparison

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Electroporation of alphavirus RNA translational reporters into fibroblastic and myeloid cells as a tool to study the innate immune system

doi: 10.1007/978-1-4939-3625-0_8

Figure Lengend Snippet: Electroporation Conditions for Various Cell Types

Article Snippet: Repeat electroporation for a total of two pulses ( Note 5 ) ( ). table ft1 table-wrap mode="anchored" t5 Table 1: caption a7 Electroporator Cell Type Voltage 1 (V) Capacitance (uF) Pulse Width (ms) Pulses Alphavirus Translation Reporter Concentration (μg) Host Translation Reporter Concentration (μg) BioRad Electroportor BHK 220 1000 N/A 2 2 7.5 0.75 MEF 220 1000 N/A 2 7.5 7.5 C7/10 360 1000 N/A 1 ND 3 ND Neon® Electroporator BHK 1200 N/A 30 1 ND ND RAW264.7 1750 N/A 25 1 7.5 0.75 RAJI 1375 N/A 30 1 ND ND Open in a separate window 1 See Note 3 2 Not applicable 3 Not determined Electroporation Conditions for Various Cell Types Transfer the cells to 15-mL conical tube containing 13 mL of pre-warmed complete media Rinse the cuvette with complete media to recover remaining cells.

Techniques: Electroporation, Concentration Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b is not required for mitochondrial fusion but ensues mitochondrial bioenergetic recovery. (A) Labeling of one subset of mitochondria by photoactivation of PAGFP in cells expressing both mtPAGFP and mtDsRed, as indicated on the top (scale bar: 10 μm). (B) Quantitation of mitochondrial fusion events, including kiss-and-run and complete fusion, per run ( n = 10 runs). (C) Δψm of WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. The mean TMRM FI of mitochondria in WT cells was normalized to 1. The TMRM FI per mitochondrion below 0.3 denoted low (white arrow). (D) Quantitation of percentage of mitochondria with low Δψm in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (≥20 cells for three biological replicates). (E) Quantitation of percentage of mitochondria with low Δψm in Opa1 KO cells expressing ShLuc plus Flag and Opa1 KO cells expressing OPA1-iso5-Flag plus ShLuc, Sh Mfn 1, Sh Mfn 2 or Sh Mfn 1/2 (≥20 cells for three biological replicates). ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Labeling, Expressing, Quantitation Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1 Exon-4b and Exon4 partially recover mitochondrial respiration activity. (A) OCR measurements in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. (B) Relative ATP production, basal, and maximal respiration using OCR measurements in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag ( n = 2, biological replicates). WT MEF cells were used as control, whose ATP production, basal, and maximal respiration were normalized to 1. (C) Cellular ATP levels in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag, with or without oligomycin treatment ( n = 3, biological replicates). * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Activity Assay, Expressing, Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b maintains normal TFAM distribution. (A) N-SIM Images of TFAM distribution in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. (B) Quantification of the percentage of mitochondria with diffuse TFAM in panel (A) (≥15 cells for three biological replicates, scale bar: 10 μm). (C) Anti-DNA immunofluorescence in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (scale bar: 10 μm). (D) Quantification of mtDNA nucleoid number per cell in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag (≥20 cells for three biological replicates). * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Expressing, Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: OPA1-Exon4b Binds to mtDNA D-Loop for Transcriptional and Metabolic Modulation, Independent of Mitochondrial Fusion

doi: 10.3389/fcell.2020.00180

Figure Lengend Snippet: OPA1-Exon4b modulates mtDNA transcription. (A,B) Anti-Flag ChIP was carried out using WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag. The precipitated DNA was analyzed by qPCR using primer pairs for the D-loop region (A) or Cox1 (B) . n = 3, biological replicates. (C) Anti-Flag ChIP was carried out using WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, NT-Exon4-Flag, or NT-Exon4/4b-Flag. The precipitated DNA was analyzed by qPCR using primer pairs for the D-loop region. n = 3, biological replicates. (D) Relative mRNA levels of 9 mtDNA genes in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag, or OPA1-iso5-Flag ( n = 3, biological replicates). (E) Relative mRNA levels of three nuclear genes encoding respiratory subunits in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag ( n = 3, biological replicates). (F) Western blotting analysis of mtDNA-encoded Cox2 in WT MEF cells expressing Flag and Opa1 KO cells expressing Flag, OPA1-iso1-Flag or OPA1-iso5-Flag. Band densities were quantified using ImageJ, and relative band densities are shown on the bottom. n = 3, biological replicates. * p < 0.05, ** p < 0.01, one-way ANOVA.

Article Snippet: Opa1 KO and control WT MEF cells were purchased from ATCC (Manassas, United States).

Techniques: Expressing, Western Blot

Journal: Molecular cell

Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

doi: 10.1016/j.molcel.2018.08.047

Figure Lengend Snippet:

Article Snippet: Mouse IgG2a Isotype Control from murine myeloma antibody (Pre-clear for RPA ChIP on MEF 4–3 cells) , Sigma Aldrich , M5409; RRID:AB_1163691.

Techniques: Recombinant, Purification, Transfection, DNA Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, Plasmid Preparation, Software