Journal: Nature Communications

Article Title: Increased global transcription activity as a mechanism of replication stress in cancer

doi: 10.1038/ncomms13087

Figure Lengend Snippet: ( a ) TBP mRNA quantification by quantitative reverse transcriptase–PCR in BJ-HRAS V12 cells 72 h after RAS induction. TBP mRNA levels were normalized to GAPDH and control. ( b ) Protein levels of TBP and β-ACTIN after RAS induction for the times indicated. ( c ) Densitometry quantification of TBP levels based on western blotting as in b after RAS induction for the times indicated. Values were normalized to 72 h control. N =3 (24 and 48 h), N =6 (72 h). ( d ) Twenty-four hours after RAS induction, cells were transfected with TBP siRNA (TBPsi #1) or control siRNA (nonTsi). Cells were processed for DNA fibre analysis or western blotting 48 h later and for 53BP1 staining 24 h later. ( e ) Protein levels of TBP, HRAS and GAPDH (loading control) 72 h after RAS induction and 48 h after siRNA transfection. ( f ) Quantification of nascent RNA synthesis by EU incorporation ±TBPsi #1 72 h after RAS induction. N =3. ( g ) Distribution of replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( h ) Median replication fork speeds ±TBPsi #1 72 h after RAS induction. N =3. ( i ) Percentages of cells containing more than eight 53BP1 foci, ±TBPsi #1 96 h after RAS induction. N =3. ( j ) Median replication fork speeds in cells treated with TBPsi #1 and DRB 72 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( k ) Percentages of cells treated with TBPsi #1 and DRB containing more than eight 53BP1 foci after 96 h after RAS induction, compared with TBPsi #1 or DRB alone. N =3. ( l ) Model for the role of TBP in HRAS V12 -induced replication stress. Means ±s.e.m. (bars) are shown. Student's t -test, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: For BJ-TBPind cells, human TBP complementary DNA (Origene, SC118124) was inserted into a pInducer20 lentivirus construct to generate TBP-pInducer20 and infected BJ-hTert cells were selected with 500 μg ml −1 G418 (Gibco).

Techniques: Reverse Transcription, Control, Western Blot, Transfection, Staining

Journal: Nature Communications

Article Title: Increased global transcription activity as a mechanism of replication stress in cancer

doi: 10.1038/ncomms13087

Figure Lengend Snippet: ( a ) Protein levels of TBP, p53 and TUBULIN (loading control) in BJ-TBPind cells treated with doxycycline for 1–6 days, to induce TBP overexpression. No doxycycline was used as a control. ( b ) Nascent RNA synthesis as measured by EU incorporation after TBP induction for 3 days. Scale bars, 10 μm. ( c ). Quantification of nuclear EU intensity after TBP induction for 1–4 days. N =4 (con, day 1, 2 and 4), N =5 (day 3). ( d ) Median replication fork speeds in after TBP induction. N =2 (day 1, 2 and 4), N =4 (con, day 3). Asterisks compare with control. ( e ) Nascent RNA synthesis as measured by EU incorporation in cells treated with DRB or dimethylsulfoxide (DMSO) (control) for 100 min, 72 h after TBP induction. N =3. ( f ) Median replication fork speeds in BJ-TBPind cells treated with DRB 72 h after TBP induction. N =3. ( g ). Percentage of cells displaying more than eight 53BP1 foci or micronuclei after TBP induction. Right panel: representative images of cells with 53BP1 foci and micronuclei. Asterisks compare with control. N =2–7. ( h ) Cell cycle distribution of 53BP1-positive cells 96 h after TBP induction as determined by co-staining with Cyclin A. N =2. ( i ) Representative images and percentages of β-galactosidase staining after TBP induction for 1–6 days. Scale bars, 100 μm. ( j ) Model of how HRASV 12 and other growth factor oncogenes such as epidermal growth factor receptor (EGFR) induce replication stress by increasing transcription through TBP and other transcription factors. Additional mechanisms, such as reactive oxygen species, may also contribute to HRASV 12 -induced DNA damage. Means ±s.e.m. (bars) are shown. Student's t -test, * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: For BJ-TBPind cells, human TBP complementary DNA (Origene, SC118124) was inserted into a pInducer20 lentivirus construct to generate TBP-pInducer20 and infected BJ-hTert cells were selected with 500 μg ml −1 G418 (Gibco).

Techniques: Control, Over Expression, Staining

Journal: Nature Communications

Article Title: Improved in situ characterization of protein complex dynamics at scale with thermal proximity co-aggregation

doi: 10.1038/s41467-023-43526-2

Figure Lengend Snippet: a Dynamics of CORUM-annotated protein complexes under glucose deprivation as profiled by Slim-TPCA. Complexes are grouped based on their functions. The complexes marked in red were selected to do Co-IP experiment. b K562 cells were incubated in presence and absence of glucose followed by quantification of ATP based on luminescence intensity ( n = 3 independent biological experiments). Data are presented as mean values ± s.d. (error bars). c K562 cells were incubated in presence and absence of glucose, harvested at different time points and analyzed by immunoblotting using Phospho-Rb (Ser780) antibody, Phospho-Histone H3(Ser10) antibody, and Phospho-CDK Substrate antibody. GAPDH antibody and β-Tubulin antibody were used as loading control ( n = 3 independent biological experiments). d , e K562 cells were incubated in presence and absence of glucose for 48 h, and analyzed by Immunoprecipitation and immunoblotting using MYH9 antibody, Emerin antibody, TAF9B antibody and TADA3L antibody. GAPDH antibody was used as loading control ( n = 3 independent biological experiments). Source data are provided as a file.

Article Snippet: The primary antibodies used in this study were Phospho-Rb (Ser780) (CST, 8180S,1:1000), Phospho-Histone H3(Ser10) (CST, 3377S,1:1000), Phospho-CDK Substrate (CST, 14371S,1:1000), β-Tubulin (CST, 2128S,1:1000), GAPDH (Proteintech, 10494-1-AP,1:5000), MYH9 (Proteintech, 11128-1-AP,1:10,000), Emerin antibody (Proteintech, 10351-1-AP,1:5000), TAF9B antibody (Proteintech, 28713-1-AP,1:3000), TADA3L antibody (Proteintech, 10839-1-AP,1:4500).

Techniques: Co-Immunoprecipitation Assay, Incubation, Western Blot, Control, Immunoprecipitation

Journal: Biology of Sex Differences

Article Title: Sex-specific regulation of cardiac microRNAs targeting mitochondrial proteins in pressure overload

doi: 10.1186/s13293-019-0222-1

Figure Lengend Snippet: Putative miRNAs regulating the mRNA of mitochondrial proteins

Article Snippet: Primary antibodies against Auh (ab155980, Abcam), Crat (ab153699, Abcam), Decr1 (sc-366484, Santa Cruz), Hadha (sc-82185, Santa Cruz), Ndufs4 (ab139178, Abcam), Tim23 (611222, BD; control for mitochondrial structural integrity), Tom40 (sc-365466, Santa Cruz; control for mitochondrial structural integrity), and tubulin (T9026, Sigma-Aldrich; loading control) were used.

Techniques:

Journal: Biology of Sex Differences

Article Title: Sex-specific regulation of cardiac microRNAs targeting mitochondrial proteins in pressure overload

doi: 10.1186/s13293-019-0222-1

Figure Lengend Snippet: Active miRNA binding sites in the 3′-UTRs of mitochondrial targets. Effect of specific miRNA mimics on complete 3′-UTR clones of Auh, Crat, Decr1, Hadha, and Ndufs4 in cardiac muscle cells (HL-1). Black bars indicate the respective non-specific mimic controls. Data present mean ± SEM. n = 5/group; *** P < 0.001, ** P < 0.01, * P < 0.05

Article Snippet: Primary antibodies against Auh (ab155980, Abcam), Crat (ab153699, Abcam), Decr1 (sc-366484, Santa Cruz), Hadha (sc-82185, Santa Cruz), Ndufs4 (ab139178, Abcam), Tim23 (611222, BD; control for mitochondrial structural integrity), Tom40 (sc-365466, Santa Cruz; control for mitochondrial structural integrity), and tubulin (T9026, Sigma-Aldrich; loading control) were used.

Techniques: Binding Assay, Clone Assay