Journal: Nature aging

Article Title: Characterization of cellular senescence in aging skeletal muscle

doi: 10.1038/s43587-022-00250-8

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: When appropriate, a mouse-on-mouse detection kit (Vector Laboratories, Burlingame, CA, USA) was used to block endogenous mouse IgG staining to improve the specificity of secondary anti-mouse antibodies binding to primary mouse antibodies used on mouse sections.

Techniques: Western Blot, Plasmid Preparation, Variant Assay, Sequencing, Recombinant, Lysis, Multiplex Assay, Sample Prep, Immunodetection, Avidin-Biotin Assay, Blocking Assay, Protease Inhibitor, DC Protein Assay, Isolation, Software

Journal: Cell reports

Article Title: Development of allergen-specific IgE in a food-allergy model requires precisely timed B cell stimulation and is inhibited by Fgl2

doi: 10.1016/j.celrep.2022.110990

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Wells were blocked with 1% BSA for at least 1 h at room temperature and diluted serum was added and incubated at room temperature for 2 h. Peanut protein extract was labeled with biotin (Sigma) and added into wells for one hour (2 μg/mL) followed by adding poly-HRP streptavidin (Pierce Endogen) for 0.5 h (1:2000).

Techniques: Activation Assay, Marker, Purification, Recombinant, Cell Isolation, Avidin-Biotin Assay, Binding Assay, Knock-Out, Software

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 1. Schematic representation of interaction between various PC2 and a-actinin segments revealed by yeast two-hybrid system. Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘ þ ’ and ‘ 2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Construct, Library Screening, Residue

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 2. Interaction between various PC2 segments and a-actinins by GST pull-down assay. (A) E. coli extracts expressing GST-tagged PC2 polypeptides PC2N, PC2C, PC2CA, PC2CB, PC2CC, PC2CD or GST alone or containing only the binding buffer were visualized by the GST antibody. (B and C) Fusion proteins were incubated with purified non-muscle a-actinin (NM ACTN) from chicken gizzard (B) and muscle a-actinin protein (M ACTN) from rabbit skeletal muscle (C). Glutathione–agarose beads were used to pre- cipitate GST epitope binding proteins. The resultant protein samples were immunoblotted with a-actinin antibodies BM75.2 (non-muscle) or EA53 (muscle). The molecular mass markers (in kDa) are shown.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Pull Down Assay, Expressing, Binding Assay, Incubation

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 3. Binding of PC2 with a-actinins by dot blot overlay assay. Purified non-muscle or muscle a-actinin were spotted on nitrocellulose membranes, incubated with total protein lysates of HEK293 and MDCK cells in a blocking buffer containing 1 mM Ca2þ or 1 mM EGTA and PBS buffer (Control). After washes, the membranes were immunoblotted by 1A11. PC2 signals were detected on dots spotted with chicken gizzard non-muscle a-actinin, rabbit skeletal muscle a-actinin or PC2C (positive control), but not on negative con- trols spotted with bovine albumin (BSA) or PBS binding buffer.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Binding Assay, Dot Blot, Overlay Assay, Incubation, Blocking Assay, Control, Positive Control

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 4. Characterization of polycstin-2 antibodies G20 and 1A11. (A) Left panel, total protein from MDCK cells was precipitated with goat G20, non-immune goat IgG (2G20), mouse 1A11 or non-immune mouse IgG (21A11) and detected with 1A11. Right panel, the same membrane was stripped off and re-probed with 1A11 after incubation with the fusion protein GST–PC2C as the blocking peptide. (B) Precipitated samples in panel A were detected with G20 (left panel) or with G20 following pre-incubation with its antigen peptide (right panel). (C and D) MDCK cells stably expressing GFP–PC2 were stained with G20 (red) or visualized for GFP (green, no staining), in the absence (C) and in the presence (D) of the blocking peptide. (E) Ciliated MDCK cells (5 days post-confluency) were co-stained for acetylated a-tubulin and for PC2 (with G20).

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Membrane, Incubation, Blocking Assay, Stable Transfection, Expressing, Staining

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 5. Association between endogenous PC2 and a-actinins in HEK293 and MDCK cells and rat kidney and heart tissues. (A) Total protein from rat heart was precipitated with either muscle a-actinin antibody EA53 or non-immune mouse IgG (2EA53) and detected with antibody 1A11. (B) Reciprocal to A, total protein from rat heart was precipitated with 1A11 antibody or non-immune mouse IgG and probed with EA53. Total cell lysates from HEK293 (C) and MDCK cells (E) and total protein from rat kidney (G) were precipitated with non-muscle a-actinin antibody BM75.2 or non-immune mouse IgG (2). The precipitates were detected with1A11. In reciprocal co-IP experiments, cell lysates from HEK293 (D) and MDCK cells (F) and total protein from rat kidney (H) were pre- cipitated with 1A11 or non-immune mouse IgG (2). The precipitates were detected with BM75.2. Total protein from rat kidney and heart (I and J) was pre- cipitated with 1A11or non-immune mouse IgG (21A11). These precipitates and the total lysates from rat kidney and heart were detected by the spectrin antibody.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Co-Immunoprecipitation Assay

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 6. Subcellular co-localization of PC2 and a-actinin in NIH 3T3, MDCK and IMCD cells and hST vesicles. (A) Localization of endogenous non-muscle a-actinin and transiently transfected PC2 in 3T3 cells, revealed using IF with antibodies BM75.2 and G20, respectively. (B and C) Localization of endogenous non-muscle a-actinin and PC2 in subconfluent MDCK (B) and IMCD (C) cells. Lower panels are expanded pictures of cells from within the square boxes shown in B3 and C3 upper panels. Triangles and arrows in B3 and C3 lower panels indicate the plasma membrane and cell–cell junction localizations, respectively. (D) Cell surface biotinylation in MDCK and IMCD cells. Cells were first labeled with Sulfo-NHS-biotin and precipitated with avidin beads (upper panel, Surface). The remaining flow-through samples were then precipitated with 1A11 and protein G beads and detected by 1A11 in western blotting. Total lysates were also loaded as shown. IMCD cell extracts (Lysate, Surface and Flow-thru without enrichment) were also detected for Naþ/Kþ ATPase a1 subunit, as a plasma mem- brane marker (positive control), and calreticulin, as an endoplasmic reticulum marker (negative control) (lower panels). (E) Interaction and distribution of endogenous non-muscle a-actinin and PC2 determined in hST apical membrane vesicles in the absence and presence of cytochalasin D (CD) (10 mg/ml for 24 h) treatment. For co-IP, vesicles were lysed with CellLytic-M buffer, precipitated with BM75.2 and detected by 1A11. Total lysates were loaded on the left two lanes to indicate the PC2 protein levels. For IF, vesicles were co-stained with goat anti-PC2 G20 (green) and mouse anti-a-actinin BM75.2 (red). Repre- sentative merged images (green plus red) with and without cytochalasin D treatment were shown. Horizontal bars ¼ 20 mm.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Transfection, Clinical Proteomics, Membrane, Labeling, Avidin-Biotin Assay, Western Blot, Marker, Positive Control, Negative Control, Co-Immunoprecipitation Assay, Staining

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 7. Effect of a-actinin on in vitro translated PC2 channel activity in a lipid bilayer system. (A) Representative tracings of in vitro translated, reconstituted PC2 at þ40 mV before and after addition of 250 nM (final concentration) of non-muscle a-actinin to the cis chamber. Cis chamber, 150 mM Kþ; trans chamber, 15 mM Kþ (Materials and Methods). Tracings at sites (a–c) are shown below with an expanded scale. (B) Representative tracings recorded at þ40 and þ60 mV before and after a-actinin addition. (C) Averaged PC2 single-channel currents recorded at þ40 mV before (N ¼ 7) and after (N ¼ 6, paired) addition of a-actinin.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: In Vitro, Activity Assay, Concentration Assay

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 8. Effect of a-actinin on the current amplitude and subconductance states of in vitro translated PC2 channel in a lipid bilayer system. (A) Representative single-channel tracings and corresponding all-point histograms (below each tracing) indicating the increase in single-channel conductance after the addition of non-muscle a-actinin. Data were obtained at þ40 mV. (B) Current–voltage relationship of the largest single-channel conductance in PC2. Dashed and solid lines correspond to the control condition (no a-actinin, as previously reported) (16) and the presence of a-actinin (N ¼ 5), respectively.

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: In Vitro, Control

Journal: Human molecular genetics

Article Title: Alpha-actinin associates with polycystin-2 and regulates its channel activity.

doi: 10.1093/hmg/ddi167

Figure Lengend Snippet: Figure 9. Regulation of hST PC2 channel function by a-actinin. (A) Representative single-channel tracing (left panels) at þ40 mV of reconstituted hST apical membranes in asymmetrical KCl solutions (Materials and Methods). The corresponding all-point histograms are shown on the right panels. Upper panels, in the absence of a-actinin; lower panels, in the presence of non-muscle a-actinin in the cis chamber. (B) Upper left panel, representative tracings recording at þ40 mV before and after the addition of a-actinin (as indicated). Lower left panel, averaged tracing from six experiments. Right panel, channel open probability (NPo) averaged from the six experiments before and after the application of a-actinin. (C) Cation channel activity in the presence of trans amiloride (100 mM) averaged from six membrane patches, under the same ionic conditions as in (A) and (B) and at þ40 mV. This is compared with channel activity in the absence of amiloride averaged from eight patches of membrane (P , 0.0002).

Article Snippet: Solid bars indicate the bait or interacting candidate constructs in the initial library screening. (A) Human PC2 segments with marked starting and ending amino acid residue numbers and their association with human a-actinin-1 (ACTN1) and a-actinin-2 (ACTN2) indicated by ‘þþþ’, ‘þþ’, ‘þ ’ and ‘2 ’ for development of blue color within 1, 3 and 24 h and no development of blue color within 24 h, respectively. (B) ACTN2 segments and their association with PC2N, PC2CA and PC2CC. at E ast T ennessee State U niversity on M ay 29, 2015 http://hm g.oxfordjournals.org/ D ow nloaded from Co-immunoprecipitation of endogenous PC2 and a-actinins We first tested the specificity of the two anti-PC2 antibodies used in this study, the custom-made mouse monoclonal 1A11 and the commercially available goat polyclonal G20 (Santa Cruz), by immunoprecipitation (IP) using MDCK cells.

Techniques: Activity Assay, Membrane

Journal: Nucleic Acids Research

Article Title: Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

doi: 10.1093/nar/gkw027

Figure Lengend Snippet: The effect of circ-Foxo3 on cell proliferation. ( A ) Left, Real-time PCR showed that circ-Foxo3 was highly expressed in the non-cancer cell lines of mouse embryo fibroblast (MEF), mouse cardiac fibroblast (MCF) and NIH3T3, as compared with the cancer cell lines 67NR, 66C14, 4T07, 4T1 and B16. Right, The levels of Foxo3 linear mRNA were not correlated with circ-Foxo3. Asterisks indicate significant differences. ** P < 0.001, Error bars, SD ( n = 4). ( B and C ) Different cell lines as indicated (B) or NIH3T3 fibroblasts (C) were seeded at the cell density of 1 × 10 5 cells/well on 6-well dishes in 10% FBS/DMEM medium until 50, 80, 100% or over-confluence based on the coverage of the surface of the tissue culture plates, followed by determination of circ-Foxo3 levels and cell cycle distribution. Increased cell densities expressed higher levels of circ-Foxo3 (B) and more cells were detected in the G1 phase (C). ( D ) NIH3T3 fibroblasts were incubated in basal medium with EGF (0, 2, 10 and 50 ng/ml, left) or AG1478 (0, 0.5, 1.5 and 5 μM, right) for 24 h. Real-time PCR showed circ-Foxo3 expression decreased after EGF treatment but increased after AG1478 treatment. ** P < 0.001, Error bars, SD ( n = 4). ( E ) Left, A siRNA was designed to specifically target circ-Foxo3. Right, Cells were transfected with siRNA targeting circ-Foxo3 or a control oligo. RNAs isolated were subject to real-time PCR to confirm downregulation of circ-Foxo3 in the siRNA-transfected cells. ( F ) MCF cells transfected without (wild-type) or with circ-Foxo3 siRNA or 2 oligos with random sequences were cultured in DMEM supplemented with 5% FBS for up to 5 days. The siRNA-transfected cells grew fast compared to the controls. ** P < 0.001, Error bars, SD ( n = 4). ( G ) B16 cells transfected without or with circ-Foxo3 siRNA or the control oligos were cultured in DMEM with 2.5% FBS for up to 5 days. Cell proliferation assays showed that circ-Foxo3 siRNA-transfected cells grew fast compared to the controls. ** P < 0.001, Error bars, SD ( n = 4). ( H ) Silencing circ-Foxo3 decreased the number of cells in G1 phase, but increased the number of cells in S and G2 phase. ** P < 0.01. Error bars, SD ( n = 4).

Article Snippet: In brief, 10 7 cells were washed in ice-cold phosphate-buffered saline, lysed in 500 μl co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μl washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated at room temperature for another hour.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Expressing, Transfection, Control, Isolation, Cell Culture

Journal: Nucleic Acids Research

Article Title: Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

doi: 10.1093/nar/gkw027

Figure Lengend Snippet: circ-Foxo3 enhanced the interaction between p21 and CDK2. ( A ) Lysates prepared from NIH3T3 cells transfected with circ-Foxo3 or mock control were subject to IP with anti-CDK2 antibody, followed by Western blotting. CDK2 precipitation pulled down more p21, and less cyclin A and cyclin E in the circ-Foxo3-transfected cells than the control. ( B ) Left, the lysates were subject to IP with anti-cyclin A antibody, followed by Western blotting. Cyclin A precipitation pulled down less CDK2 in the circ-Foxo3-transfected cells than in the control. Right, the lysates were subject to IP with anti-cyclin E antibody, followed by western blotting. Cyclin E precipitation pulled-down less CDK2 in the circ-Foxo3-transfected cells than in the control. ( C ) Circ-Foxo3 transfection did not change expression of cyclin A and cyclin E. ( D ) Cell lysates prepared from NIH3T3 cells transfected with GFP vector, Foxo3, circular RNA vector, circ-Amotl1 and circ-Foxo3, or over-confluence culture were subject to western blot probed with antibodies against CDK2, p21 and β-action. The lysates were also subject to immunoprecipitation with antibody against p21 or CDK2. Anti-p21 antibody pulled-down more Cdk2 and anti-CDK2 antibody pulled-down more p21 in the cells transfected with circ-Foxo3 or overgrown. ( E ) Lysates prepared from NIH3T3 cells transfected with circ-Foxo3 and a vector, or over-grown cells were mixed with biotinylated probes against circ-Foxo3 or an oligo. Western blotting showed that levels of CDK2 and p21 were not affected (upper). However, the circ-Foxo3 probe pulled down more CDK2 and p21 in the cells transfected with circ-Foxo3 or over-grown relative to the controls (lower). ( F ) Lysates prepared from NIH3T3 cells sorted into G1, S or G2 phase were subject to Western blotting. The levels of p21 and CDK2 were similar in cells of different phases (upper). Anti-p21 and anti-CDK2 antibodies pulled down more CDK2 and p21, respectively, in the G1 phase cells. ( G ) The lysates prepared from circ-Foxo3- and mock-transfected cells were subject to native gradient gel electrophoresis followed by western blotting probed with antibodies against p21 or CDK2. ( H ) Diagram of our hypothesis showing the effect of circ-Foxo3 on cell cycle progression. Circ-Foxo3 retards cell cycle entry via enhancing interaction between p21 and CDK2, which repressed CDK2–cyclin complex formation.

Article Snippet: In brief, 10 7 cells were washed in ice-cold phosphate-buffered saline, lysed in 500 μl co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μl washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated at room temperature for another hour.

Techniques: Transfection, Control, Western Blot, Expressing, Plasmid Preparation, Immunoprecipitation, Nucleic Acid Electrophoresis

Journal: Nucleic Acids Research

Article Title: Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

doi: 10.1093/nar/gkw027

Figure Lengend Snippet: Silencing circ-Foxo3 enhanced cell cycle entry and decreased the interaction between p21 and CDK2. ( A ) Lysates prepared from subconfluence NIH3T3 cells transfected with circ-Foxo3 siRNA or a control oligo were subject to western blotting. Silencing circ-Foxo3 did not change expression of p21 and CDK2. ( B ) The lysates were subject to IP with anti-p21 antibody followed by western blotting. p21 precipitation pulled-down less CDK2 in the circ-Foxo3 siRNA-transfected cells relative to the control. ( C ) The lysates were subject to IP with anti-CDK2 antibody, followed by western blotting. CDK2 precipitation pulled-down less p21, but more cyclin A and cyclin E in the siRNA-transfected cells compared to the control. ( D ) Silencing circ-Foxo3 did not change expression of cyclin A and cyclin E. ( E ) Cyclin A and cyclin E precipitations pulled down more CDK2 in the circ-Foxo3 siRNA-transfected cells. ( F ) The lysates were subject to native gradient gel electrophoresis followed by western blotting probed with antibodies against p21 or CDK2. ( G ) Cell lysates prepared from p21 siRNA- and a control oligo-transfected NIH3T3 cells were subject to immunoprecipitation with anti-rabbit IgG, mouse IgG, p21 and Cdk2 antibodies, followed by real-time PCR. Anti-p21 antibody pulled-down less circ-Foxo3 in the p21 siRNA-transfected cells. ** P < 0.01. Error bars, SD ( n = 4). ( H ) The lysates were hybridized with circ-Foxo3 probe or a control oligo for RNA pull-down assays. Real-time PCR showed that the circ-Foxo3 probe pulled down same levels of circ-Foxo3 in the cells transfected with p21 siRNA. Error bars, SD ( n = 4). ( I ) Silencing p21 did not affect CDK2 expression. However, anti-p21 antibody pulled-down less Cdk2 and p21 in the p21 siRNA-transfected cells. Anti-CDK2 antibody pulled-down less p21, but the same amount of Cdk2 in the p21 siRNA-transfected cells. ( J ) Silencing CDK2 did not affected circ-Foxo3 levels. ( K ) Anti-CDK2 antibody pulled-down less circ-Foxo3 in the Cdk2 siRNA-transfected cells. ** P < 0.01. Error bars, SD ( n = 4). ( L ) Silencing CDK2 did not affect p21 expression. However, anti-CDK2 antibody pulled-down less Cdk2 and p21 in the CDk2 siRNA-transfected cells. Anti-p21 antibody pulled-down less CDK2, but the same amount of p21 in the CDK2 silencing cells.

Article Snippet: In brief, 10 7 cells were washed in ice-cold phosphate-buffered saline, lysed in 500 μl co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μl washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated at room temperature for another hour.

Techniques: Transfection, Control, Western Blot, Expressing, Nucleic Acid Electrophoresis, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2

doi: 10.1093/nar/gkw027

Figure Lengend Snippet: Pulling down circ-Foxo3 precipitated both p21 and CDK2. ( A ) Lysates prepared from NIH3T3 cells transfected with circ-Foxo3 or a control vector, were subject to RNA pull-down assays. Real-time PCR showed that the circ-Foxo3 probe pulled down high levels of circ-Foxo3 in the cells transfected with circ-Foxo3. ** P < 0.01. Error bars, SD ( n = 4). ( B ) Left: the lysates were mixed with biotinylated probes against circ-Foxo3 or a control oligo and subject to western blot with antibodies against CDK2, p21 and β-action. Right: the pull-down mixture was subject to Western blotting. Pulling down circ-Foxo3 also pulled down CDK2 and p21 in cells transfected with circ-Foxo3. ( C ) Lysates prepared from NIH3T3 cells transfected with circ-Foxo3 siRNA or a control oligo, were subject to RNA pull down assays. Real-time PCR showed that the probe pulled down less circ-Foxo3 in the cells transfected with circ-Foxo3 siRNA. ** P < 0.01. Error bars, SD ( n = 4). ( D ) Left, transfection with circ-Foxo3 siRNA did not affect expression of CDK2 and p21. Right, Pulling down circ-Foxo3 precipitated less CDK2 and p21 in the cells transfected with circ-Foxo3 siRNA than those transfected with the control oligo. ( E ) Silencing p21 had little effect on circ-Foxo3 pulling down CDK2. ( F ) Silencing Cdk2 had little effect on circ-Foxo3 pulling down p21.

Article Snippet: In brief, 10 7 cells were washed in ice-cold phosphate-buffered saline, lysed in 500 μl co-IP buffer, and incubated with 3 μg biotinylated DNA oligo probes against endogenous or ectopically expressed circ-Foxo3, at room temperature for 2 h. A total of 50 μl washed Streptavidin C1 magnetic beads (Invitrogen) were added to each binding reaction and further incubated at room temperature for another hour.

Techniques: Transfection, Control, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Expressing