Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 1. Knockout of Rnaseh2a gene of HEK293 cells with the CRISPR/Cas9 system.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out, CRISPR

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 2. Screening of Rnaseh2a-/- homozygote HEK293 cells.

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques:

Journal: Journal of Biological Macromolecules

Article Title: Generation of ribonuclease H2 A subunit (RH2A)-knockout HEK293 cells and analysis of the ribonucleotide content of their genomic DNA

doi: 10.14533/jbm.24.33

Figure Lengend Snippet: Fig. 7. Knockout of Rnaseh2a gene of HEK293

Article Snippet: After separation, the proteins were transferred by electroblotting onto a polyvinylidene difluoride (PVDF) membrane Sequi- BlotTM PVDF (BioRad, Hercules, CA) in 25 mM Tris-HCl buffer (pH 8.3), 192 mM glycine, 20% v/v methanol at 25 V for 50 min. After blotting, the membrane was washed with 50 mM Tris-HCl buffer (pH 8.3), 138 mM NaCl, 2.7 mM KCl, 0.05% Tween 20 (TBS-T), blocked with TBS-T containing 2% w/v skim milk, and incubated with mouse anti human RH2A polyclonal antibody, Anti RNASEH2A (Proteintech, Rosemont, IL, 1:1000 in TBS-T containing 1% w/v skim milk).

Techniques: Knock-Out

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: Primer sets used for PCR and targeted sequences for knockdown studies

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Knockdown, Cloning

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: TF expression, activation, and the impact of PAX5 on HCK transcription in MYD88-mutated lymphoma cells. (A) Western blot studies depicting protein expression levels of PAX5, STAT3, NF-kB (NF-κB-p65), and AP-1 complex members (JunB, c-Jun, JunD) predicted by TF promoter-binding assay and PROMO analysis as HCK promoter binding TFs in MYD88-mutated WM and ABC-DLBCL cell lines (BCWM.1, MWCL-1, TMD-8, HBL-1, OCI-Ly3, and SU-DHL-2) and MYD88 wild-type B-cell lymphoma (OCI-Ly7, OCI-Ly19, Ramos) and myeloma cells (RPMI-8226, MM.1S). The HCK protein expression levels and the phosphorylation levels of mutated MYD88–directed TFs STAT3, NF-κB-p65, and AP-1 complex members (JunB, c-Jun) were also detected. GAPDH protein expression was used to demonstrate uniform protein loading. (B) The regulation of HCK transcription by PAX5 was assessed by lentiviral knockdown of PAX5 with 2 distinct shRNAs in MYD88-mutated BCWM.1 and TMD-8 cells and compared with scrambled control vector. Quantitative RT-PCR was performed after day 5 of lentiviral transduction. HCK protein levels and knockdown efficiencies for PAX5 were analyzed by western blot at the same time as the sample collection for HCK mRNA quantification. GAPDH was used for loading control. (C) The regulation of PAX5 by mutated MYD88 was assessed by lentiviral-mediated knockdown of MYD88 in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector. Protein levels of PAX5 are shown, and GADPH served as a protein loading control. (D) Transcriptome analysis depicting PAX5 transcript levels in CD19-selected bone marrow LPCs from MYD88-mutated WM patients, and MYD88 wild-type WM patients; peripheral CD19-selected B cells and CD19- and CD27-selected memory B cells from healthy donors; and CD138-selected bone marrow plasma cells from healthy donors. ***P < .001.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Western Blot, Binding Assay, Phospho-proteomics, Knockdown, Control, Plasmid Preparation, Quantitative RT-PCR, Transduction, Clinical Proteomics

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: ChIP studies assessing STAT3, NF-kB, and AP-1 TF binding to the HCK promoter. The fold enrichments of HCK promoter-specific sequence assessed by quantitative PCR following ChIP with ChIP grade antibodies to STAT3, NF-kB-p65, JunB, and c-Jun in MYD88-mutated WM (BCWM.1, MWCL-1) and ABC-DLBCL (TMD-8, HBL-1, OCI-Ly3) cells, and MYD88 wild-type lymphoma cells (OCI-Ly7, OCI-Ly19). Antibody to GAPDH was used as control antibody.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Binding Assay, Sequencing, Real-time Polymerase Chain Reaction, ChIP-chip, Control

Journal: Blood Advances

Article Title: Expression of the prosurvival kinase HCK requires PAX5 and mutated MYD88 signaling in MYD88-driven B-cell lymphomas

doi: 10.1182/bloodadvances.2019000947

Figure Lengend Snippet: The regulation of JunB by TLR/MYD88 signaling and the impact of JunB on HCK transcription. The phosphorylation of JunB (Ser79) and c-Jun (Ser63) was assessed following TLR4 (by LPS-EB) and TLR9 (by ODN-2006) stimulation (A) as well as the lentiviral cells (C). HCK overexpression of MYD88 L265P mutant vs MYD88 WT (B) in both MYD88-mutated BCWM.1 cells and MYD88 wild-type Ramos cells. The phosphorylation of JunB (Ser79) and c-Jun (Ser63) was determined following MYD88 knockdown in MYD88-mutated BCWM.1 protein levels detected following lentiviral mediated knockdown of JunB in MYD88-mutated BCWM.1 and TMD-8 cells using 2 distinct shRNAs and compared with scrambled control vector (D). Protein levels of MYD88, JunB, c-Jun, and GADPH served as protein expression, knockdown efficiency, and loading controls.

Article Snippet: Western blots were performed for the detection of protein phosphorylation or expression levels in cell lines or following the cell stimulation by LPS (for TLR4) or ODN-2006 (for TLR9); MYD88 overexpression or knockdown; PAX5 knockdown; JunB knockdown or pull-down with biotinylated probes using antibodies for PAX5 (Abcam); phospho-NF-kB-p65(Ser529; Rockland Immunochemicals, Limerick, PA); phospho-JunB (Ser79; OriGene Technologies); phospho-c-Jun (Ser63), phospho-STAT3 (Tyr705), STAT3, NF-kB-p65, JunB, c-Jun, JunD, HCK, MYD88 (Cell Signaling Technology).

Techniques: Phospho-proteomics, Over Expression, Mutagenesis, Knockdown, Control, Plasmid Preparation, Expressing

Journal: BMC Biology

Article Title: Machine learning combined with omics-based approaches reveals T-lymphocyte cellular fate imbalance in abdominal aortic aneurysm

doi: 10.1186/s12915-025-02400-x

Figure Lengend Snippet: Identification of TIRS regulatory mechanisms and key biomarkers. A LASSO-based feature selection, with the optimal lambda determined when the partial likelihood deviance reached the minimum value (left). SVM-RFE-based feature selection, with root mean square error (RMSE) reached the minimum value and R -squared reached the max value (mid). Venn diagram presented the intersection of key biomarkers obtained through both algorithms (right). B Aberrant expression profiles for key biomarkers in Abdominal Aortic Wall Dataset 1 (AAA n = 80 patients, control n = 10 healthy individuals; Student’s t -test). C ROC curve demonstrating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Abdominal Aortic Wall Dataset 1. D Clinical impact plot illustrating the clinical utility of key biomarkers. The “Number high risk” curve closely aligns with the “Number high risk with the event” curve at each threshold probability, indicating exceptional predictive power. E Aberrant expression profiles for key biomarkers in Abdominal Aortic Wall Dataset 2 (AAA n = 9 patients, control n = 10 healthy individuals; Student’s t -test). F ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Abdominal Aortic Wall Dataset 2. G Clinical impact plot demonstrating the clinical utility of key biomarkers. Again, the “Number high risk” curve is closely aligned with the “Number high risk with the event” curve at each threshold probability, highlighting the biomarkers’ strong predictive power. H Aberrant expression profiles for key biomarkers in Perivascular Adipose Tissue Dataset 3 (dilated n = 30, non-dilated n = 30; Student’s t -test). I ROC curve verifying the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in Perivascular Adipose Tissue Dataset 3. J Impact plots reiterated superior predictive performance probability, indicating outstanding predictive capability

Article Snippet: The primary antibodies against FOSB (1:500, catalog No. ab184938, Abcam) and JUNB (1:50, catalog No. 10486–1-AP, Proteintech) were incubated overnight at 4 °C, followed by incubation with horseradish peroxidase conjugated secondary antibodies.

Techniques: Selection, Expressing, Control, Diagnostic Assay

Journal: BMC Biology

Article Title: Machine learning combined with omics-based approaches reveals T-lymphocyte cellular fate imbalance in abdominal aortic aneurysm

doi: 10.1186/s12915-025-02400-x

Figure Lengend Snippet: Verification of key biomarkers. A Abdominal aortic wall and peripheral blood samples obtained from AAA patients. B Aberrant expression profiles for key biomarkers in the abdominal aortic wall (Inhouse Dataset 1; AAA n = 5 patients, control n = 4 healthy individuals). C ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in the abdominal aortic wall (Inhouse Dataset 1). D Clinical impact plot demonstrating the clinical utility of key biomarkers. The “Number high risk” curve closely aligns with the “Number high risk with the event” curve at each threshold probability, indicating exceptional predictive power. E Aberrant expression profiles for key biomarkers in peripheral blood (Inhouse Dataset 2; AAA n = 24 patients, control n = 15 healthy individuals). F ROC curve validating the diagnostic efficacy of FOSB, JUNB, CST7, and TBC1D4 in peripheral blood (Inhouse Dataset 2). G Clinical impact plot illustrating the clinical utility of key biomarkers. Again, the “Number high risk” curve remains closely aligned with the “Number high risk with the event” curve at each threshold probability, highlighting the biomarkers’ strong predictive capability. H Mice were infused with saline or Ang II (1000 ng/kg/min) + BAPN. Gross abdominal aorta images were shown. Scale bar is 1 cm. I Representative images of immunohistochemical stains for elastin fiber (Van Gieson) and representative photomicrographs of hematoxylin and eosin (H&E) staining. Scale bar is 200 μm. J – L Representative immunohistochemical staining of FOSB and JUNB in aortic cross sections. Scale bar is 50 μm. Data are expressed as mean ± SEM (control n = 3 mice, AAA n = 5 or 6 mice). Student’s t -test was utilized to compare continuous variables between the two groups

Article Snippet: The primary antibodies against FOSB (1:500, catalog No. ab184938, Abcam) and JUNB (1:50, catalog No. 10486–1-AP, Proteintech) were incubated overnight at 4 °C, followed by incubation with horseradish peroxidase conjugated secondary antibodies.

Techniques: Expressing, Control, Diagnostic Assay, Saline, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

doi: 10.7554/eLife.08007

Figure Lengend Snippet: ( A ) Genomic DNA was treated with RNase H2 to reveal the presence of ribonucleotides as single-strand nicks. An untreated control was used to reveal background single-stranded breaks in genomic DNA preparations. The intensity of labeling after gel electrophoresis was measured. ( B , C ) Relative ribonucleotide loads are reported as fold-increase of radiolabel incorporation of RNase H2-treated over untreated sample. At least three independent replicates were performed and the error bars indicate SEM. DOI: http://dx.doi.org/10.7554/eLife.08007.005

Article Snippet: Whole-cell protein was extracted and Western blot was performed using antibodies against RNase H2A (1:1000, ProSci) or GFP (1:500, UC Davis/NIH NeuroMab Facility, Davis, CA), and tubulin (1:1000, Sigma–Aldrich, St. Louis, MO).

Techniques: Control, Labeling, Nucleic Acid Electrophoresis

Journal: eLife

Article Title: Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi–Goutières syndrome

doi: 10.7554/eLife.08007

Figure Lengend Snippet: ( A ) Western blot using an anti-RNase H2A antibody shows successful gene knockout in HeLa-GFP cells. Scramble cells were generated with a CRISPR vector carrying a scrambled guide RNA sequence. Tubulin was used as a loading control. ( B ) Immunocytochemistry confirms elevated DNA damage response in RNASEH2A KO cells; DAPI (blue), gamma H2AX antibody (green). ( C ) Bright field and green fluorescent protein (GFP) microscopy images of RNASEH2A KO and scramble control cells. Knockout of the RNASEH2A gene triggers the reactivation of the silent GFP reporter in HeLa-GFP cells. ( D ) (Top) Western blot showing GFP expression in HeLa-GFP cells (RNASEH2A KO and scramble); (bottom) bar graph quantification of Western blot. ( E ) Bisulfite methylation sequencing for two different LINE-1 loci in RNASEH2A KO and scramble control cells. Black and white circles represent methylated and unmethylated CpG sites, respectively. Missing bubbles indicate that a CpG site is absent from the sequence of that particular molecule. The coordinates for each fragment analyzed are indicated at top. ( F ) Quantification of percent methylation at each CpG sites surveyed in panel E . p-value was calculated using a paired Wilcoxon test with the alternative hypothesis that RNASEH2A KO is less methylated than scramble. DOI: http://dx.doi.org/10.7554/eLife.08007.012

Article Snippet: Whole-cell protein was extracted and Western blot was performed using antibodies against RNase H2A (1:1000, ProSci) or GFP (1:500, UC Davis/NIH NeuroMab Facility, Davis, CA), and tubulin (1:1000, Sigma–Aldrich, St. Louis, MO).

Techniques: Western Blot, Gene Knockout, Generated, CRISPR, Plasmid Preparation, Sequencing, Control, Immunocytochemistry, Microscopy, Knock-Out, Expressing, Methylation

Journal: Nature Communications

Article Title: Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

doi: 10.1038/s41467-019-14083-4

Figure Lengend Snippet: a Western blotting was performed to detect the protein levels of RSK3 in MDA-MB-231 and BT549 cells treated with DMSO or JQ1 (1 μM) for 0, 12 and 24 h. b The vector controls and RSK3-overexpressing BLBC cell clones were treated with DMSO or JQ1 (1 μM) for 48 h, and luminescent cell viability assays were performed to measure the killing effects. Statistical data (mean ± SD) are shown (*** P < 0.001, one-way ANOVA). c Tumoursphere formation in RSK3-overexpressing BLBC cells and their vector controls was observed with or without JQ1 (1 μM) treatment. Statistical data of tumoursphere numbers are shown (* P < 0.05, ** P < 0.01; one-way ANOVA). d Measurement of apoptosis in RSK3-overexpressing MDA-MB-231 cells and vector control with or without JQ1 (1 μM) treatment by Annexin V-FITC flow cytometry assay. Statistical data (mean ± SD) are shown based on three independent experiments. A representative experiment is shown (** P < 0.01, one-way ANOVA). e RPS6KA2 -knockdown clones and shRNA control BLBC cells were treated with JQ1 (1 μM) for 48 h, the killing effects were detected by luminescent cell viability assay (** P < 0.01, *** P < 0.001; one-way ANOVA). f Annexin V-FITC flow cytometry assay was used to measure the synergistic pro-apoptotic effects of JQ1 on the silencing of RPS6KA2 . Statistical data of three independent experiments are shown. A representative experiment is shown (** P < 0.01, one-way ANOVA). g Tumoursphere was counted in RPS6KA2 -knockdown BLBC cells and their shRNA controls in the absence or presence of JQ1 (1 μM) treatment. Statistical data (mean ± SD) were shown (** P < 0.01, one-way ANOVA). h – i BALB/c nude mice were injected with RPS6KA2 -knockdown or shRNA control MDA-MB-231 cells ( n = 7), following treatment with vehicle control or JQ1 (35 mg/kg). After 25 days, tumours were weighed and images were taken. h Growth curves of xenograft tumours are shown (* P < 0.05, *** P < 0.001); i Photographs of tumours are shown. Source data are provided as a Source Data file.

Article Snippet: Antibodies against RSK3 (GTX111071) and JunB (GTX79258) were from GeneTex (Irvine, CA).

Techniques: Western Blot, Plasmid Preparation, Clone Assay, Control, Flow Cytometry, Knockdown, shRNA, Cell Viability Assay, Injection

Journal: Nature Communications

Article Title: Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

doi: 10.1038/s41467-019-14083-4

Figure Lengend Snippet: a Western blotting was performed to detect JunD protein levels, MDA-MB-231 and BT549 cells were treated with DMSO or JQ1 (1 μM) for 0, 12 and 24 h. b Photograph depicted the potential JunD binding site in the enhancer region of RPS6KA2 gene. Wild-type and mutant RPS6KA2 gene enhancer luciferase plasmids are shown. c Luciferase assays were performed in MDA-MB-231 and BT549 cells transfected with shRNA for JunD and control, in the presence of DMSO or JQ1 (1 μM) for 6 h. Data are reported as mean ± SD. d Luciferase assays were performed in HEK293T cells transfected with wild-type or mutant RPS6KA2 enhancer luciferase construct with or without JUND co-transfection. HEK293T cells were treated with DMSO or JQ1 (1 μM) for 6 h. Data are reported as mean ± SD. e Chromatin immunoprecipitation (ChIP)-qPCR assay was executed in JUND -knockdown BLBC cells and their vector controls treated with DMSO or JQ1 (1 μM) for 6 h. ‘SP’ indicates specific primers of ChIP for RPS6KA2 gene enhancer and ‘NSP’ indicates non-specific primers that recognize the region downstream of the 3’ end of the gene. f Western blotting was performed to detect the expression levels of JunD and RSK3 in control and JUND -knockdown clones of MDA-MB-231 and BT549. g JUND or RPS6KA2-knockdown MDA-MB-231 and BT549 cells as well as their vector control cells were treated with DMSO or JQ1 (1 μM) for 48 h, and luminescent cell viability assays were done to detect the killing effects (** P < 0.01, one-way ANOVA). h Measured tumoursphere formation in JUND or RPS6KA2-knockdown MDA-MB-231 and BT549 cells as well as their vector controls. The cells were also treated with DMSO or JQ1 (1 μM). Statistical data of numbers of tumoursphere were shown (* P < 0.05, ** P < 0.01; one-way ANOVA). i Rescued expression of RSK3 in control or JUND -knockdown BLBC cells which were treated with DMSO or JQ1 (1 μM) for 48 h. Cell viability was measured by CellTiter-Glo ® luminescent viability assay (** P < 0.01, *** P < 0.001; one-way ANOVA). j Western blotting was done to examine the expression levels of JunD and RSK3 in control or JUND -overexpressing clones of MDA-MB-231 and BT549. k JUND -overexpressing MDA-MB-231 and BT549 cells as well as their vector control cells were treated with JQ1 (1 μM) for 48 h, cell viability was detected by CellTiter-Glo ® luminescent viability assay. Statistical data (mean ± SD) are shown (** P < 0.01, *** P < 0.001; one-way ANOVA). Source data are provided as a Source Data file.

Article Snippet: Antibodies against RSK3 (GTX111071) and JunB (GTX79258) were from GeneTex (Irvine, CA).

Techniques: Western Blot, Binding Assay, Mutagenesis, Luciferase, Transfection, shRNA, Control, Construct, Cotransfection, Chromatin Immunoprecipitation, ChIP-qPCR, Knockdown, Plasmid Preparation, Expressing, Clone Assay, Viability Assay

Journal: Nature Communications

Article Title: Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

doi: 10.1038/s41467-019-14083-4

Figure Lengend Snippet: a Correlation analyses of JUND and RPS6KA2 in four datasets of breast cancer patients from GEO. GSE76275 was separated into two sub-groups: triple-negative (TN) and non-triple-negative (non-TN); GSE43358 was separated into two sub-groups: triple-negative (TN) and non-triple-negative (non-TN); GSE42568 was separated into two sub-groups: ER-positive (ER+) and ER-negative (ER−); GSE31448 was separated into two sub-groups: luminal A (lumA) and luminal B (lumB). Pearson Coefficients of correlation and p values are shown. b Gene differential expression analyses of the mRNA levels of JUND and RPS6KA2 were conducted in two datasets that contained four subtypes of breast cancer patients, including BLBC, luminal A, luminal B and HER2 + . c The mRNA expression statuses of JUND and RPS6KA2 were detected in a panel of breast cancer cell lines by quantitative PCR assay. d Protein expression levels of JunD and RSK3 were detected in a panel of breast cancer cell lines by western blotting. e Luminal and HER2 + breast cancer cell lines as well as BLBC cell lines were treated with DMSO or JQ1 (1 μM) for 48 h, cell growth was detected by CCK8 assay. Statistical data (mean ± SD) are shown. f RPS6KA2 and JUND genes were knocked down in two JQ1-resistant breast cancer cell lines, cell growth was measured by CCK8 assay. Statistical data (mean ± SD) are shown (*** P < 0.001, one-way ANOVA). Source data are provided as a Source Data file.

Article Snippet: Antibodies against RSK3 (GTX111071) and JunB (GTX79258) were from GeneTex (Irvine, CA).

Techniques: Quantitative Proteomics, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay

Journal: Nature Communications

Article Title: Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

doi: 10.1038/s41467-019-14083-4

Figure Lengend Snippet: a Measurement of cell viability of MDA-MB231 and BT549 cells by CellTiter-Glo ® luminescent viability assay. Control and RPS6KA2 -knockdown cells were treated with JQ1 in the absence or presence of seven EGFR inhibitors for 48 h. 1: Ctr; 2: osimertinib; 3: AZ5104; 4: erlotinib; 5: icotinib; 6: gefitinib; 7: lapatinib; 8: EAI045; 9: RPS6KA2 -shRNA; 10: JQ1; 11: JQ1 + osimertinib; 12: JQ1 + AZ5104; 13: JQ1 + erlotinib; 14: JQ1 + icotinib; 15: JQ1 + gefitinib; 16: JQ1 + lapatinib; 17: JQ1 + EAI045; 18: JQ1 + RPS6KA2 -shRNA. Statistical data (Mean ± SD) are shown. P values were calculated between single JQ1-treated samples and samples treated with JQ1 plus EGFR inhibitors. Symbol ‘§’ indicates statistical significance ( P < 0.05, one-way ANOVA). b MDA-MB-231 cells were treated with JQ1 (1 μM) and/or osimertinib, GDC0994, XMD8-92 (1 μM) or GDC0994/XMD8-92 for 12 h, western blotting was performed to detect JunD protein expression, and phosphorylated and total levels of MEK1/2, ERK1/2, ERK5 and RSK3. c CCK8 assays were done to measure the killing effects. Control and RPS6KA2 -knockdown MDA-MB-231 and BT549 cells were treated with indicated the inhibitors for 48 h. 1: Control; 2: osimertinib; 3: BIX-02188; 4: U0126; 5: U0126 + BIX-02188; 6: GDC0994; 7: XMD8-92; 8: GDC0994 + XMD8-92; 9: RPS6KA2 -shRNA; 10: JQ1; 11: JQ1 + osimertinib; 12: JQ1 + BIX-02188; 13: JQ1 + U0126; 14: JQ1 + BIX-02188 + U0126; 15: JQ1 + GDC0994; 16: JQ1 + XMD8-92; 17: JQ1 + GDC0994 + XMD8-92; 18: JQ1 + RPS6KA2 -shRNA; 19: JQ1 + RPS6KA2 -shRNA + BIX-02188 + U0126; 20: JQ1 + RPS6KA2 -shRNA + GDC0994 + XMD8-92; 21: JQ1 + RPS6KA2 -shRNA + osimertinib. Statistical data (mean±SD) are shown. P values were calculated when compared between single JQ1-treated samples and samples that were treated with JQ1 plus kinase inhibitors. Symbol ‘§’ indicates statistical significance ( P < 0.05, one-way ANOVA). d CCK8 assay was performed to observe inhibition effects. Vector control and RSK3 ( upper ) or JunD-overexpressing ( bottom ) MDA-MB-231 cells were treated with the indicated inhibitors for 48 h. 1: DMSO; 2: JQ1; 3: JQ1 + osimertinib; 4: JQ1 + U0126; 5: JQ1 + BIX-02188; 6: JQ1 + BIX-02188 + U0126; 7: JQ1 + GDC0994; 8: JQ1 + XMD8-92; 9: JQ1 + GDC0994 + XMD8-92. Statistical data (mean±SD) are shown. P values were calculated when compared between JunD or RSK3-overexpression clones and vector controls in the presence of the same inhibitors. Symbol ‘§’ indicates statistical significance ( P < 0.05, one-way ANOVA), and symbol ‘#’ indicates no significance ( P > 0.05). Source data are provided as a Source Data file.

Article Snippet: Antibodies against RSK3 (GTX111071) and JunB (GTX79258) were from GeneTex (Irvine, CA).

Techniques: Viability Assay, Control, Knockdown, shRNA, Western Blot, Expressing, CCK-8 Assay, Inhibition, Plasmid Preparation, Over Expression, Clone Assay

Journal: Nature Communications

Article Title: Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance

doi: 10.1038/s41467-019-14083-4

Figure Lengend Snippet: a RPS6KA2 gene enhancer luciferase reporter activity was detected in two resistant clones as well as their parental cells. The data are reported as mean ± SD. ‘P’ indicates parental BLBC cells, and ‘R’ indicates resistant clones. b Measurement of the protein levels of JunD and RSK3 as well as the phosphorylated forms of RSK3 in resistant clones and parental BLBC cells. c MiR-548d-3p levels were measured by RT-PCR in resistant clones and parental BLBC cells. d Sequential chromatin immunoprecipitation was performed in parental and JQ1-resistant BLBC cells. e MDA-MB-231 and BT549 JQ1-resistant clones were treated with the indicated inhibitors for 48 h, and the effects were detected by CCK8 assay. 1: DMSO; 2: osimertinib; 3: GDC0994; 4: XMD8-92; 5: GDC0994 + XMD8-92; 6: U0126; 7: BIX-02188; 8: BIX-02188 + U0126; 9: JQ1; 10: JQ1 + osimertinib; 11: JQ1 + GDC0994; 12: JQ1 + XMD8-92; 13: JQ1 + GDC0994 + XMD8-92; 14: JQ1 + U0126; 15: JQ1 + BIX-02188; 16: JQ1 + BIX-02188 + U0126. Statistical data (mean ± SD) are shown. P values were calculated compared between single JQ1-treated samples and samples that treated with JQ1 plus kinase inhibitors. Symbol ‘§’ indicates statistical significance ( P < 0.05, one-way ANOVA). f Cell growth was detected by CCK8 assay in vector control and RPS6KA2 -knockdown JQ1-resistant clones. Cells were treated as following, 1: DMSO; 2: JQ1; 3: JQ1 + osimertinib; 4: JQ1 + GDC0994 + XMD8-92; 5: JQ1 + BIX-02188 + U0126. Statistical data (mean ± SD) are shown. P values were calculated when compared between RPS6KA2 -knockdown clones and vector controls in the presence of the same inhibitors. Symbol ‘§’ indicates statistical significance ( P < 0.05, one-way ANOVA), and symbol ‘#’ indicates no significance ( P > 0.05). g A proposed model illustrating the underlying mechanism of BET inhibition resistance and potential combination therapies. Source data are provided as a Source Data file.

Article Snippet: Antibodies against RSK3 (GTX111071) and JunB (GTX79258) were from GeneTex (Irvine, CA).

Techniques: Luciferase, Activity Assay, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, CCK-8 Assay, Plasmid Preparation, Control, Knockdown, Inhibition