rr036a cell cycle checkpoint antibody sampler kit cell signaling technology  (Cell Signaling Technology Inc)

Journal: Bioengineered

Article Title: Knockdown of mitochondrial threonyl-tRNA synthetase 2 inhibits lung adenocarcinoma cell proliferation and induces apoptosis

doi: 10.1080/21655979.2022.2037368

Figure Lengend Snippet: TARS2 knockdown inhibits cell cycle progression (a) Downregulation of TARS2 inhibits the transition from G1 phase to S phase, as evidenced by a flow cytometric assay. (b) The protein levels of p-RB (ser807/811), CDK2, cyclin E1 and cyclin E2 were decreased and that of p21 waf1/cip1 was increased in TARS2-shRNA transfected LUAD cells. GAPDH is utilized as a reference protein.

Article Snippet: The following antibodies were used in this study: TARS2 (15,067-1-AP, Proteintech, China); GAPDH (60,004-1-Ig, Proteintech, China); Phospho-RB (ser807/811) (9917, Cell Signaling Technology); Cyclin E1(20,808, Cell Signaling Technology); Cyclin E2 (4132, Cell Signaling Technology); CDK2 (18,048, Cell Signaling Technology); p21 Waf1/Cip1 (9932, Cell Signaling Technology); MT-ND6 (A17991, ABclonal, China); CYTB (55,090-1-AP, Proteintech, China); MT-CO1(62,101; Cell Signaling Technology); MT-CO3 (55,082-1-AP, Proteintech, China); MT-ATP8 (26,723-1-AP, Proteintech, China); TOM20 (11,802-1-AP, Proteintech, China); cleaved caspase-3 and cleaved caspase-8 (92,570, Cell Signaling Technology); cleaved caspase-9 (9930, Cell Signaling Technology) [ ].

Techniques: Knockdown, Flow Cytometry, shRNA, Transfection

Journal: Bioengineered

Article Title: Knockdown of mitochondrial threonyl-tRNA synthetase 2 inhibits lung adenocarcinoma cell proliferation and induces apoptosis

doi: 10.1080/21655979.2022.2037368

Figure Lengend Snippet: Knockdown of TARS2 inhibits LUAD cells growth in vivo. (a) Tumor growth curves of mice inoculated with A549 cells infected with TARS2 lentivirus were measured weekly with vernier calipers. (b) Tumor weights at 6 weeks post injection. (c)Protein expression in tumor tissue sections detected by immunohistochemical staining. a – b, p-RB(Ser807/811) expression was decreased in shTARS2-1 group compared with SCRAMBLE group; c – d p21 expression was increased in shTARS2-1 group compared with SCRAMBLE group; e-f, cleaved-caspase 9 expression was increased in shTARS2-1 group compare with SCRAMBLE group; g-h, cleaved-caspase 8 expression was increased in shTARS2-1 group compare with SCRAMBLE group; i-j, cleaved-caspase 3 expression was increased in shTARS2-1 group compare with SCRAMBLE group. The scale bar represents 200 μm.

Article Snippet: The following antibodies were used in this study: TARS2 (15,067-1-AP, Proteintech, China); GAPDH (60,004-1-Ig, Proteintech, China); Phospho-RB (ser807/811) (9917, Cell Signaling Technology); Cyclin E1(20,808, Cell Signaling Technology); Cyclin E2 (4132, Cell Signaling Technology); CDK2 (18,048, Cell Signaling Technology); p21 Waf1/Cip1 (9932, Cell Signaling Technology); MT-ND6 (A17991, ABclonal, China); CYTB (55,090-1-AP, Proteintech, China); MT-CO1(62,101; Cell Signaling Technology); MT-CO3 (55,082-1-AP, Proteintech, China); MT-ATP8 (26,723-1-AP, Proteintech, China); TOM20 (11,802-1-AP, Proteintech, China); cleaved caspase-3 and cleaved caspase-8 (92,570, Cell Signaling Technology); cleaved caspase-9 (9930, Cell Signaling Technology) [ ].

Techniques: Knockdown, In Vivo, Infection, Injection, Expressing, Immunohistochemical staining, Staining

Journal: Nature Communications

Article Title: Crosstalk between CST and RPA regulates RAD51 activity during replication stress

doi: 10.1038/s41467-021-26624-x

Figure Lengend Snippet: a Crosstalk among CST, RPA, and RAD51 on ssDNA subjected to replication stress. b CST lies in close proximity to RPA upon fork stalling. (i) PLA is a technique that detects the physical proximity of two different proteins. In principle, if the two proteins are <40 nm apart, fluorescence signal can be detected. In brief, the two target proteins are bound by specific primary antibodies. If the target proteins are sufficiently proximal, PLA secondary antibodies hosting oligonucleotides can be ligated by means of two PLUS/MINUS PLA oligos to circularize. The DNA polymerase phi29 then processes rolling-circle amplification, and the resulting copies can be detected by hybridizing the fluorescence-labeled oligonucleotide . (ii), (iii) PLA assays were performed to establish the close proximity of CTC1/STN1 with RPA in HeLa cells treated with hydroxyurea (HU) for 3 h. Representative PLA images of CTC1/RPA32 (ii) and STN1/RPA32 (iii) are shown. Scatter plots from one experiment are shown here. Red lines represent mean values ± SEM. N: the number of cells analyzed in each sample. P values were calculated by one-way ANOVA. NS not significant, *** P < 0.001; **** P < 0.0001. c CST colocalizes with RPA on the same ssDNA in response to fork stalling. HeLa cells expressing Flag-CTC/Myc-STN1/HA-TEN1 were labeled with BrdU and treated with or without HU (3 h), followed by co-immunostaining with anti-Flag (red), anti-RPA32 pS33 (cyan), and anti-BrdU (green) antibodies. N = 3 biologically independent experiments.

Article Snippet: Most importantly, whereas RAD51 only formed fragmented and short filaments (median length 67.83 nm) on RPA-bound ssDNA, we observed longer RAD51 filaments (median length 99.17 nm) on CST-bound ssDNA (Fig. , compare panels iv and v, see quantification in Fig. ).

Techniques: Fluorescence, Amplification, Labeling, Expressing, Immunostaining

Journal: Nature Communications

Article Title: Crosstalk between CST and RPA regulates RAD51 activity during replication stress

doi: 10.1038/s41467-021-26624-x

Figure Lengend Snippet: a Affinity pulldown assay. Flag-CTC1-STN1-TEN1-His 6 (1 μM) was incubated with RAD51 (1 μM), followed by incubation with His-Tag Dynabeads to capture the CST and associated proteins by means of a magnetic bead separator. The supernatant (S) and eluate (E) were analyzed by 15% SDS-PAGE with Coomassie blue staining. RAD51 alone is shown as a control. N = 3 biologically independent experiments. b (i) Schematic of the ssDNA pulldown experiment. (ii) Excessive RPA was preincubated with biotinylated 80-nt ssDNA linked to magnetic streptavidin beads. Upon addition of CST and RAD51, the ssDNA and its associated proteins were captured using a magnetic bead separator. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. (iii) Quantitative plot of amounts of RAD51 in the bound fraction. Data represent mean ± S.D. calculated from three independent experiments. c For ssDNA pulldown analysis, RPA was preincubated with magnetic ssDNA beads. Then CTC1∆700N-ST and RAD51 were added to complete the reaction. The unbound and bound fractions from the reaction were analyzed by 15% SDS-PAGE with Coomassie blue staining. N = 3 biologically independent experiments.

Article Snippet: Most importantly, whereas RAD51 only formed fragmented and short filaments (median length 67.83 nm) on RPA-bound ssDNA, we observed longer RAD51 filaments (median length 99.17 nm) on CST-bound ssDNA (Fig. , compare panels iv and v, see quantification in Fig. ).

Techniques: Incubation, SDS Page, Staining, Control

Journal: Nature Communications

Article Title: Crosstalk between CST and RPA regulates RAD51 activity during replication stress

doi: 10.1038/s41467-021-26624-x

Figure Lengend Snippet: a Schematic of the DNA strand-exchange assay. The 32 P-labeled DNA is marked by an asterisk. b , c RPA ( b ) or CST ( c ) was preincubated with ssDNA at the indicated concentrations of KCl. RAD51 activity was determined by monitoring the formation of strand-exchange products. Note that 5 mM CaCl 2 was included in the reaction to stimulate RAD51 activity. d , e The indicated amounts of RPA and CST were preincubated with ssDNA and 150 mM KCl before adding RAD51. CaCl 2 ( d ) or AMPPNP ( e ) was used to stimulate RAD51 activity. b–e Quantitative plots are shown below the gel images. Data represent mean ± S.D. calculated from three independent experiments. NS not significant, ** P < 0.01; *** P < 0.001; **** P < 0.0001, as calculated by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: Most importantly, whereas RAD51 only formed fragmented and short filaments (median length 67.83 nm) on RPA-bound ssDNA, we observed longer RAD51 filaments (median length 99.17 nm) on CST-bound ssDNA (Fig. , compare panels iv and v, see quantification in Fig. ).

Techniques: Labeling, Activity Assay

Journal: Nature Communications

Article Title: Crosstalk between CST and RPA regulates RAD51 activity during replication stress

doi: 10.1038/s41467-021-26624-x

Figure Lengend Snippet: a Electron microscopy with negative staining was used to observe the formation of RAD51 filaments on CST- or RPA-bound ssDNA under the condition of 150 mM KCl. The 80-nt ssDNA substrate was incubated with CST (i), RPA (ii) or, as a control, RAD51 (iii). RAD51 was then added to CST-bound ssDNA (iv) or RPA-bound ssDNA (v). Representative images from panels (i) to (v) are shown. N = 2 biologically independent experiments. b RAD51 filament length was measured in Image J software. The quantitative graph was generated in GraphPad Prism. Boxes represent the 25th and 75th percentiles and encompass the median, with whiskers showing the minimum and maximum values. N values represent the number of RAD51 filaments identified in numerous electron microscopy images collected from two independent experiments. NS not significant, **** P < 0.0001 as calculated by one-way ANOVA with Kruskal–Wallis test followed by Dunn’s post hoc test. The median values for RAD51, CST→RAD51, RPA→RAD51 are 96.41, 99.17, and 67.83 nm, respectively. Note that some filaments are much longer than expected due to end-to-end association of two filaments . c RAD51 assembly on CST- or RPA-bound ssDNA was monitored by smFRET under the condition of 150 mM KCl. A 35-nt overhang DNA substrate was used for smFRET, as shown in Fig. . The smFRET states of RAD51-, RPA-, and CST-ssDNA are ~0.05 (i), ~0.15 (ii), and ~0.2 (iv), respectively. Although addition of RAD51 did not alter the smFRET state of RPA-bound ssDNA (~0.15, iii), the smFRET state shifted from 0.2 to 0.05 for CST-bound ssDNA (v). This outcome indicates that RAD51 can access CST-bound ssDNA. N values represent the number of individual molecules collected from at least three independent experiments and are displayed in the upper right corner. d Representative time-course of RAD51 assembly on CST-coated ssDNA. RAD51 was introduced to the preformed surface-bound CST-ssDNA (gray). Before transitioning into the RAD51-bound smFRET state (pink), a high-FRET intermediate state (green) was observed. Rastergram of 126 molecules showing a similar pattern. e Kinetics analysis showing that RAD51 assembled on CST-coated ssDNA in 64 ± 2.2 s ( N = 96), whereas it was predicted to take thousands of minutes to do so on RPA-coated ssDNA ( N = 13). Nearly no RAD51 assembly was observed for RPA-coated ssDNA. Data are plotted as a best-fit result ± 95% C.I.

Article Snippet: Most importantly, whereas RAD51 only formed fragmented and short filaments (median length 67.83 nm) on RPA-bound ssDNA, we observed longer RAD51 filaments (median length 99.17 nm) on CST-bound ssDNA (Fig. , compare panels iv and v, see quantification in Fig. ).

Techniques: Electron Microscopy, Negative Staining, Incubation, Control, Software, Generated