Journal: bioRxiv

Article Title: The CDK7 inhibitor CT7001 (Samuraciclib) targets proliferation pathways to inhibit advanced prostate cancer

doi: 10.1101/2022.06.29.497030

Figure Lengend Snippet: (A) Thermal stability profiles of putative human CDK targets, using data from the Meltome atlas. (B) Calculated melting temperatures (T m ) of human CDK targets. (C) (i) Validation of the CDK7 melt curve in live LNCaP cells and (ii) immunoblots showing the effect of heat shock (54°C) on soluble levels of CDK1, CDK2, CDK4, CDK7, and CDK9. Numbers underneath blots represent remaining fraction of soluble protein. (D) (i) Cellular thermal shift assay (CeTSA) isothermal dose-response fingerprints at 54°C in intact LNCaP cells treated with the indicated CT7001 concentrations for 3 hours and (ii) quantification of target engagement relative to untreated samples. All immunoblots are representative of 3 biologically independent experiments.

Article Snippet: Immunoblotting was carried out as previously described [ ] using the following primary antibodies: CDK7 (Cell Signaling Technology, 2916), CDK1 (Cell Signaling Technology, 9116), CDK2 (Cell Signaling Technology, 2546), CDK4 (Cell Signaling Technology, 12790), CDK9 (Cell Signaling Technology, 2316), β-actin (Abcam, ab6276), GAPDH (Cell Signaling Technology, 2118), P-S5 PolII (Abcam, ab5401), P-S2 PolII (Abcam, ab5095), PolII (Abcam, ab817), P-S780 Rb (Abcam, ab47763), P-S807/811 Rb (Cell Signaling Technology, 8516), Rb (Abcam, ab6075), cyclin H (Abcam, ab54903), MAT1 (Santa Cruz Biotechnology, sc135981), P-S15 p53 (Cell Signaling Technology, 9284), p53 (Santa Cruz Biotechnology, sc-126), P-T161/T160 CDK1/CDK2 (Abcam, ab201008), P-S10 Histone H3 (Abcam, ab139417), AR (MilliporeSigma, 06-680), P-T1457 MED1 (Abcam, ab60950), MED1 (Bethyl Laboratories, A300-793A), cleaved PARP1 (Abcam, ab4830), p21 (Santa Cruz Biotechnology, sc-817).

Techniques: Biomarker Discovery, Western Blot, Thermal Shift Assay, Drug discovery

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 1 The protective efficacy of the Ad-AURKA/CDK7 vaccine on various tumors in the preventive model. (A) Schematic diagram of subcutaneous tumors inoculation (Renca, RM-1, MC38 or Hepa1-6) after immunization with Ad-Ctrl or Ad-AURKA/ CDK7 vaccine (n=5 mice per group). (B, F, J and N) Average tumor volumes of each group in the Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors were measured twice a week. The tumor volume was statistically analyzed 35 days after tumor inoculation. (C, G, K and O) The tumor volume of an individual mouse in each group of Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors was plotted. (D, H, L and P) Tumor weights were measured at the end of the experiment. (E, I, M and Q) Tumor inhibition rate in the Renca, RM-1, MC38, or Hepa1-6 subcutaneous tumors was, respectively, calculated by (D, H, L and P). The two-tailed independent Student’s t-test was used to analyze two-group comparisons. The data showed as means±SD. The statistical significance levels were set as *p<0.05, **p<0.01 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; i.m, intramuscular; s.c, subcutaneous.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Inhibition, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 2 Antitumor efficacy of Ad-AURKA/CDK7 immunization in the Renca subcutaneous tumor model. (A) Schematic diagram showed the overall design of Renca therapeutic subcutaneous tumor (n=5 mice per group). (B) Tumor growth volume of each group after Renca tumor inoculation was measured twice a week. The tumor volume was statistically analyzed 35 days after tumor inoculation. (C) The final tumor volume of different treatment groups on day 35 after Renca tumor implantation. (D) Tumor weights were measured at the end of the experiment. (E) The tumor inhibition rate in (D) is shown. (F, G) The percentages of CD3+ T cells, CD4+ T cells, CD8+ T cells, DCs, NK, Mφ, MDSC, or regulatory T cells (Treg) were from spleens and tumors in each group. One-way analysis of variance was used for comparisons among multiple groups. Data are means±SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001 and not significant (ns). Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin- dependent kinase 7; DCs, dendritic cells; i.m, intramuscular; MDSC, myeloid-derived suppressor cells; NK, natural killer; s.c, subcutaneous; TIL, tumor-infiltrating leukocyte.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Tumor Implantation, Inhibition, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 3 The maturation and differentiation of DC subgroups induced by Ad-AURKA/CDK7 vaccine in vivo. Different DC subgroups in spleens were analyzed by flow cytometry at the end of the experiments. (A) The percentages of CD8+CD11c+ DC subsets in immunized mice of each group (n=5), a typical flow cytometry data is displayed from each group. (B, C) Statistical analysis of the ratio of CD11c+ DCs or CD8+CD11c+ DCs. (D) The representative flow cytometry data of the percentage of CD80+CD11c+, CD86+CD11c+, MHC-II+CD11c+, or CD40+CD11c+ DC subsets in spleens of each group. (E–H) Statistical analysis of different DC subgroups in (D). One-way analysis of variance was used for comparisons among multiple groups. Data are means±SD. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin- dependent kinase 7; DCs, dendritic cells.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: In Vivo, Flow Cytometry

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 4 Ad-AURKA/CDK7 treatment induced memory CD8+ T-cell immune response. (A, B) The proliferation capability of antigen-specific CD8+ T cells from mice spleens in each group was detected by the EdU assay after the continuous stimulation with AURKA/CDK7 antigens. (C, D) The number of IFN-γ-secreting T lymphocytes was observed using the ELISpot assay. (E, F) The representative flow cytometry data of CD8+ T cells secreting IFN-γ, TNF-α, or IL-2 in each group after antigen stimulation. (G, H) The co-culture experiment was detected to assess CTL-specific killing ability. (I, J) The proportions of effector memory T cells or central memory T cells in spleens were measured, and typical flow cytometry data was selected from each group. (K) The tumor volume of a single mouse in the Renca subcutaneous tumors was measured twice a week after tumor rechallenge (n=5 mice per group). (L) The survival curve of the Ad-AURKA/CDK7 or Ad-Ctrl group was observed after re-implantation of the Renca tumor (n=10 mice per group). One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. Data are means±SD. **p<0.01, ***p<0.001, ***p<0.001 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocytes; ELISpot, enzyme-linked immunosorbent spot; IFN, interferon; IL, interleukin; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: EdU Assay, Enzyme-linked Immunospot, Flow Cytometry, Co-Culture Assay, ELISpot Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 5 Multifunctional CD8+ T cells exerted an indispensable role in the antitumor effects of Ad-AURKA/CDK7 vaccine. (A–D) The percentages of multifunctional CD8+ T cells secreting TNF-α+IFN-γ+, TNF-α+IL-2+, IFN-γ+IL-2+, or TNF-α+IFN-γ+IL-2+ in spleens of each group were detected by flow cytometry after continuous stimulation with AURKA/CDK7 antigens for 96 hours. (E–H) The proportions of tumor-infiltrating multifunctional CD8+ T lymphocytes secreting TNF-α+IFN-γ+, TNF-α+IL-2+, IFN- γ+IL-2+ or TNF-α+IFN-γ+IL-2+ in tumor tissues of each group were analyzed on day 35 after Renca tumor inoculation. (I) The tumor weights of mice in each group were measured at the end of CD8 depletion. (J) Tumor inhibition rates in (I). (K, L) The percentages of CD8+ T cells or CD8+CD11c+ DCs were detected in spleens and tumor tissues of each group in the CD8+ T-cell depletion assay. The two-tailed independent Student’s t-test was used to analyze two-group comparisons. One-way analysis of variance was used for comparisons among multiple groups. The data are shown as means±SD, with n=5 mice per group. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; IFN, interferon; IL, interleukin; TIL, tumor-infiltrating leukocytes; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Flow Cytometry, Inhibition, Depletion Assay, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 6 Ad-AURKA/CDK7 treatment suppressed tumor metastases by activating multifunctional CD8+ T cells. (A) A schematic diagram displayed the design and treatment of lung metastasis. (B) The representative image of lung metastasis nodules in each group. (C) The survival curve of mice in different groups after Renca tumor lung metastasis (n=10 mice per group). (D) The number of metastatic nodules was counted on the lung surface of immunized mice (n=5 mice per group). (E) Typical immunohistochemistry image of the lung-infiltrating CD8+ T cells in each group immunized with different vaccines. The scale was 200 µm. (F, G) The proportion of CD8+ T cells and CD8+CD11c+ DCs in the lungs of treated mice in each group. (H, I) Percentages of multifunctional CD8+ T lymphocytes producing IFN-γ, TNF-α, or IL-2 in spleens and tumor tissues of different groups. (J) The EdU assay was used to detect the proliferation of CD8+ T cells. (K) The number of IFN-γ-secreting T lymphocytes was counted using ELISpot assay. (L) The percentages of tumor-specific killing capability of CTL in each group. One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. The data expressed as means±SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocyte; DC, dendritic cell; ELISpot, enzyme- linked immunosorbent spot; IFN, interferon; IL, interleukin; i.m, intramuscular; i.v, intravenous; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Immunohistochemistry, Vaccines, EdU Assay, Enzyme-linked Immunospot, ELISpot Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 7 Antitumor efficacy of Ad-AURKA/CDK7 vaccine requires for multi-functional CD8+ T cells in the Renca orthotopic model. (A) Schematic diagram illustrating the design of the Renca orthotopic model and vaccination. (B) Typical image of renal orthotopic tumors in the different vaccine-treated groups at the endpoint of the experiment (n=5 mice per group). (C) The survival curve of each group in the orthotopic model (n=10 mice per group). (D) Tumor weights were calculated by the formula: Tumor mass (g)=left kidney mass − right kidney mass. (E) Tumor inhibition rate in (D). (F) Tumor-infiltrating CD8+ T lymphocytes were captured by immunohistochemical staining in the left kidney and the representative images were shown. The scale was 200 µm. (G, H) Statistical analysis of the proportion of CD8+ T cells or CD8+CD11c+ DCs in renal tumor tissues of each group. (I, J) The percentages of multifunctional CD8+ T lymphocytes secreting IFN-γ+, TNF-α+, IL-2+, TNF-α+IFN-γ+, TNF-α+IL-2+, IFN- γ+IL-2+, or TNF-α+IFN-γ+IL-2+ were detected by flow cytometry in spleens and tumors in each group. (K) The antigen-stimulated proliferation of CD8+ T cells in different groups. (L) The tumor-specific killing capability of CTL was detected by the co-culture experiment. One-way analysis of variance was used for comparisons among multiple groups. Survival analysis was performed using the log-rank (Mantel-Cox) test. The data are shown as means±SD. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; CTL, cytotoxic T lymphocyte; DC, dendritic cell; IFN, interferon; IL, interleukin; i.m, intramuscular; s.c, subcutaneous; TNF, tumor necrosis factor.

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Functional Assay, Inhibition, Immunohistochemical staining, Staining, Flow Cytometry, Co-Culture Assay

Journal: Journal for immunotherapy of cancer

Article Title: Adenovirus vaccine targeting kinases induces potent antitumor immunity in solid tumors.

doi: 10.1136/jitc-2024-009869

Figure Lengend Snippet: Figure 8 The therapeutic effect induced by Ad-AURKA/CDK7 vaccine in the humanized mice model. (A) Schematic diagram explaining the establishment of the humanized mice model and experiment design. (B) Final tumor volumes were measured in each group on day 35 after OSRC-2 tumor inoculation. (C) Tumor weights of each group at the end of the experiment. (D) Tumor inhibition rate in (C). (E) The representative flow cytometry data of CD8+CD11c+, CD103+CD11c+, CD80+CD11c+, CD86+CD11c+, and HLA-A2+CD11c+ DC subgroups in spleens of each group. (F–J) Statistical analysis of DC subsets in (E) in the spleens of humanized mice. (K–O) The proportions of tumor-infiltrating DC subsets in different groups were detected. (P) The typical flow cytometry image of multi-functional CD8+ T lymphocytes secreting IFN-γ+, TNF-α+, or IL-2+ in spleens in the Ad-Ctrl or Ad-hAURKA/CDK7 treatment group. (Q, S) The percentages of multifunctional CD8+ T cells in (P) in spleens. (R, T) Statistical analysis of the proportion of IFN-γ+CD8+, TNF-α+CD8+, IL-2+CD8+, TNF-α+IFN-γ+CD8+, TNF-α+IL-2+CD8+, IFN-γ+IL-2+CD8+, or TNF-α+IFN-γ+IL-2+CD8+ in antigen-specific CD8+ T lymphocytes of tumor tissues per group. The two-tailed independent Student’s t-test was used to analyze two-group comparisons. One-way analysis of variance was used for comparisons among multiple groups. The data are shown as means±SD, with n=5 mice per group. **p<0.01, ***p<0.001, and ****p<0.0001. Ad, adenovirus; AURKA, Aurora kinase A; CDK7, cyclin-dependent kinase 7; DC, dendritic cell; IFN, interferon; IL, interleukin; i.m, intramuscular; TIL, tumor-infiltrating leukocytes; TNF, tumor necrosis factor..

Article Snippet: After blocking with 5% skim milk at room temperature for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies targeting AURKA (Proteintech, Cat# 66 757–1- Ig) or CDK7 (Proteintech, Cat# 27 027–1- AP).

Techniques: Inhibition, Flow Cytometry, Functional Assay, Two Tailed Test

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: MNAT1 is upregulated in OS tissues and cell lines. a The mRNA expression of MNAT1 in OS cell lines (MG63, U2OS, Well5 and 143B) and control cells (HOBC and HFOB) was analyzed by qRT-PCR. b The protein expression of MNAT1 in OS cell lines and control cells. c The mRNA expression of MNAT1 in 30-paired OS tissues and adjacent normal tissues from affiliated hospital of qingdao university OS cohort was analyzed by qRT-PCR. d The protein expression of MNAT1 in 6-paired OS tissues (T) and adjacent normal tissues (N) was analyzed by western blot. * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: MNAT1 expression in OS tissues and its prognostic value. a Representative MNAT1 staining patterns via IHC assays. b MNAT1 expression was significantly higher in OS tissues ( n = 78) compared with that in non-tumor tissues ( n = 40). The correlation of MNAT1 expression level with distant metastasis ( c ), vascular invasion ( d ), and TNM stage ( e ). f and g The OS and DFS were analyzed by Kaplan-Meier analysis. * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Expressing, Staining

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: MNAT1 knockdown suppressed OS cell proliferation and migration. a The transfection efficiency of the MNAT1 siRNA was evaluated by qRT-PCR and western blot. b and c The changes in the proliferation of OS cells transfected with si-MNAT1 or NC were determined by the CCK-8 assay. d EdU staining assays (Scale bars, 50 μm) and e colony formation assays (Scale bars, 8 mm) were performed to determine the growth of U2OS and 143B cells. f Cell migration was assessed by wound-healing assay (Scale bars, 500 μm). g Cell invasion was assessed by transwell assay (Scale bars, 50 μm). * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Staining, Wound Healing Assay, Transwell Assay

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: Ectopic overexpression of MNAT1 promotes OS cell proliferation and invasion in vitro . U2OS and 143B cells were transfected with MNAT1 plasmid or negative control (NC). a The mRNA expression of MNAT1 was examined by qRT-PCR. b The transfection efficiency was evaluated by western blot assay and EdU staining assays (Scale bars, 50 μm) ( c ) and colony formation assays (Scale bars, 8 mm) ( d ) were performed to determine the proliferation of U2OS and 143B cells. e Cell migration was assessed by wound-healing assay (Scale bars, 500 μm). f Cell invasion was assessed by transwell assay (Scale bars, 50 μm). * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Over Expression, In Vitro, Transfection, Plasmid Preparation, Negative Control, Expressing, Quantitative RT-PCR, Western Blot, Staining, Migration, Wound Healing Assay, Transwell Assay

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: In vivo functional analysis of MNAT1 in OS. a The strength of the luciferase signal of xenograft tumor was decreased by MNAT1 knockdown. b The relative photon flux of OS tumor in nude mice of NC or sh- MNAT1 group was analyzed by a live imaging system to measure the luciferase signal. Comparison of tumor volume ( c ) and weight ( d ) in NC and sh-MNAT1 infected U2OS cells. e and f Representative IHC staining images and relative expression levels for MNAT1 and ki-67 in tumor sections from sh-MNAT1 group and NC group. * p < 0.05, ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: In Vivo, Functional Assay, Luciferase, Knockdown, Imaging, Comparison, Infection, Immunohistochemistry, Expressing

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: MNAT1 regulated PI3K/Akt/mTOR signaling pathway in OS. a KEGG analysis the different regulated signaling pathways in OS tumor compared with adjacent non-tumor tissues. b GSEA analysis the enrichment of signature genes in MNAT1 high or low group. c PI3K/Akt/mTOR pathway related proteins were analyzed by western blot. d IHC staining of PI3K, AKT and mTOR in xenograft tumor tissues from NC or sh-MNAT1 group. e The expression of MNAT1 was evaluated by western blot assay. * p < 0.05; ** p < 0.01

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Protein-Protein interactions, Western Blot, Immunohistochemistry, Expressing

Journal: BMC Cancer

Article Title: MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

doi: 10.1186/s12885-020-07687-3

Figure Lengend Snippet: MNAT1 regulated OS chemo-sensitivity to DDP-based therapy. U2OS and 143B cells were transfected with si-NC, si-MNAT1, VECTOR or MNAT1 plasmid. After 48 h cells were treated with different concentration of DDP. a and b Cell viability analysis of U2OS and 143B cells treated with si-MNAT1 or MNAT1 plasmid and different concentrations of DDP in comparison with the negative control. c and d Cell viability analysis of U2OS and 143B cells treated with PI3K inhibitor PF-04979064 or MNAT1 plasmid and different concentrations of DDP in comparison with the negative control. e and f Colony formation (Scale bars, 8 mm) and EdU assay (Scale bars, 50 μm) performed on U2OS cells treated with si-MNAT1 or MNAT1 plasmid and different concentration of DDP in comparison with the negative control

Article Snippet: Primary antibodies against MNAT1 (Proteintech, 11,719–1-AP), β-tubulin (Abcam, ab210797), PI3K (Proteintech, 20,584–1-AP), Akt (Proteintech, 10,176–2-AP) and mTOR (Proteintech, 20,657–1-AP).

Techniques: Transfection, Plasmid Preparation, Concentration Assay, Comparison, Negative Control, EdU Assay

Journal: Cells

Article Title: CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

doi: 10.3390/cells9030638

Figure Lengend Snippet: CDK7 RNA expression in breast cancer patient tumor samples. Association of CDK7 RNA expression with Relapse Free Survival (RFS) in microarray data from 3,951 breast cancer patient samples in all breast cancer patients ( A ), luminal-A patients ( B ), luminal-B patients ( C ), basal patients ( D ), and HER2+ patients ( E ), determined using KM-plotter online survival analysis tool .

Article Snippet: Protein (50 μg) was resolved on 4–20% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies: CDK7 (sc-529, Santa Cruz Biotechnology, Dallas, TX), ER (sc-543, Santa Cruz), HER2 (#4290, Cell Signaling Technology, Danvers, MA), RNA-Pol II-S2P (C152000005, Diagenode, Denville, NJ), RNA-Pol II-S5P (C152000007, Diagenode), RNA-Pol II-S7P (04-1570, Millipore Sigma, Burlington, MA) and RNA-Pol II (sc-900X, Santa Cruz), CITED2 (MAB5005, R+D Systems, Minneapolis, MN), Tubulin (T-9026, Millepore Sigma), followed by anti-rabbit (#31460, ThermoFisher Scientific), anti-mouse (31430, ThermoFisher Scientific) or anti-rat (AP136P, Millipore Sigma) secondary antibodies.

Techniques: RNA Expression, Microarray

Journal: Cells

Article Title: CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

doi: 10.3390/cells9030638

Figure Lengend Snippet: ER, HER2 and CDK7 expression in breast cancer cell lines. ( A ) Immunoblotting and ( B ) mRNA analysis of ERα (ESR1), HER2 (ERBB2) and CDK7 expression in a panel of cell lines representing multiple subtypes of breast cancer. mRNA represented as mRNA expression level normalized to GAPDH mRNA expression in sample.

Article Snippet: Protein (50 μg) was resolved on 4–20% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies: CDK7 (sc-529, Santa Cruz Biotechnology, Dallas, TX), ER (sc-543, Santa Cruz), HER2 (#4290, Cell Signaling Technology, Danvers, MA), RNA-Pol II-S2P (C152000005, Diagenode, Denville, NJ), RNA-Pol II-S5P (C152000007, Diagenode), RNA-Pol II-S7P (04-1570, Millipore Sigma, Burlington, MA) and RNA-Pol II (sc-900X, Santa Cruz), CITED2 (MAB5005, R+D Systems, Minneapolis, MN), Tubulin (T-9026, Millepore Sigma), followed by anti-rabbit (#31460, ThermoFisher Scientific), anti-mouse (31430, ThermoFisher Scientific) or anti-rat (AP136P, Millipore Sigma) secondary antibodies.

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

doi: 10.3390/cells9030638

Figure Lengend Snippet: Correlations between CDK7, ER and HER2 expression in breast cancer cell line panel Bi-variant scattergraphs and Spearman rank correlations for CDK7, ER (ESR1) and HER2 (ERBB2) protein expression ( A ) and mRNA gene expression ( B ) levels compared to THZ1 2-day and 7-day IC 50 values across the cell line panel. Protein levels expressed as mean densitometric measurements normalized to α-tubulin protein expression on the same blot. mRNA levels expressed as mean relative expression (determined by RT-qPCR) normalized to GAPDH mRNA expression in the same sample.

Article Snippet: Protein (50 μg) was resolved on 4–20% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies: CDK7 (sc-529, Santa Cruz Biotechnology, Dallas, TX), ER (sc-543, Santa Cruz), HER2 (#4290, Cell Signaling Technology, Danvers, MA), RNA-Pol II-S2P (C152000005, Diagenode, Denville, NJ), RNA-Pol II-S5P (C152000007, Diagenode), RNA-Pol II-S7P (04-1570, Millipore Sigma, Burlington, MA) and RNA-Pol II (sc-900X, Santa Cruz), CITED2 (MAB5005, R+D Systems, Minneapolis, MN), Tubulin (T-9026, Millepore Sigma), followed by anti-rabbit (#31460, ThermoFisher Scientific), anti-mouse (31430, ThermoFisher Scientific) or anti-rat (AP136P, Millipore Sigma) secondary antibodies.

Techniques: Expressing, Variant Assay, Gene Expression, Quantitative RT-PCR

Journal: Cells

Article Title: CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

doi: 10.3390/cells9030638

Figure Lengend Snippet: HER2 and ER are not markers of response to THZ1. ( A ) Bi-variant scattergraphs and Spearman rank correlations between CDK7, ER (ESR1) and HER2 (ERBB2) protein and mRNA expression levels ( B ) Bi-variant scattergraphs and Spearman rank correlations for CDK7 protein compared to protein and mRNA expression of ER (ESR1) or HER2 (ERBB2) across the cell line panel.

Article Snippet: Protein (50 μg) was resolved on 4–20% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies: CDK7 (sc-529, Santa Cruz Biotechnology, Dallas, TX), ER (sc-543, Santa Cruz), HER2 (#4290, Cell Signaling Technology, Danvers, MA), RNA-Pol II-S2P (C152000005, Diagenode, Denville, NJ), RNA-Pol II-S5P (C152000007, Diagenode), RNA-Pol II-S7P (04-1570, Millipore Sigma, Burlington, MA) and RNA-Pol II (sc-900X, Santa Cruz), CITED2 (MAB5005, R+D Systems, Minneapolis, MN), Tubulin (T-9026, Millepore Sigma), followed by anti-rabbit (#31460, ThermoFisher Scientific), anti-mouse (31430, ThermoFisher Scientific) or anti-rat (AP136P, Millipore Sigma) secondary antibodies.

Techniques: Variant Assay, Expressing

Journal: Cells

Article Title: CDK7 Inhibition Is Effective in all the Subtypes of Breast Cancer: Determinants of Response and Synergy with EGFR Inhibition

doi: 10.3390/cells9030638

Figure Lengend Snippet: Dual inhibition of EGFR and CDK7. Cell growth curves for cell lines treated with low-dose THZ1 (0–50 nM) alone and in combination with erlotinib (0–5 µM) in fixed ratio combinations for 7 days.

Article Snippet: Protein (50 μg) was resolved on 4–20% Express-Plus PAGE gels in Tris-MOPS (SDS) running buffer (GenScript, Piscataway, NJ), transferred to PVDF membranes and incubated at 4 °C overnight with primary antibodies: CDK7 (sc-529, Santa Cruz Biotechnology, Dallas, TX), ER (sc-543, Santa Cruz), HER2 (#4290, Cell Signaling Technology, Danvers, MA), RNA-Pol II-S2P (C152000005, Diagenode, Denville, NJ), RNA-Pol II-S5P (C152000007, Diagenode), RNA-Pol II-S7P (04-1570, Millipore Sigma, Burlington, MA) and RNA-Pol II (sc-900X, Santa Cruz), CITED2 (MAB5005, R+D Systems, Minneapolis, MN), Tubulin (T-9026, Millepore Sigma), followed by anti-rabbit (#31460, ThermoFisher Scientific), anti-mouse (31430, ThermoFisher Scientific) or anti-rat (AP136P, Millipore Sigma) secondary antibodies.

Techniques: Inhibition

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: The DEAD-box DDX1 is a novel interactor of CDK7. ( A ) Immunoblot analysis with antibodies raised against different TFIIH subunits (XPB and p62 of the core-TFIIH, the bridging factor XPD and the CDK7 and CycH subunits of the CAK), the chromatin (H3), and cytoplasm (Mek2) markers in whole extracts (WCE), 1% triton-soluble (Sol), and chromatin-enriched (Chr) fractions of primary dermal fibroblasts from healthy donors (C3PV and C8PV), PS-TTD (TTD8PV and TTD23PV), or XP (XP26VI and XP15PV) patients with pathogenic variants in ERCC2/XPD gene. Protein quantifications were obtained by normalizing each protein band on the corresponding MEK2 or H3 protein amounts for the soluble and chromatin-enriched fractions, respectively, in order to even out the loading differences. For each TFIIH subunit, the soluble (white bar) and chromatin-bound (black bar) amounts are expressed as a percentage of their sum, indicated as 100%. The graph (bottom panel) includes the data obtained by the immunoblots shown in . The percentage of chromatin-associated proteins in PS-TTD or XP primary dermal fibroblasts is compared with that observed in CTR cells. Bars indicate standard errors (** P < .01, *** P < .001; Student’s t -test). ( B ) Silver staining of proteins co-immunoprecipitated with CDK7 antibodies in the chromatin-enriched (Chr) fraction of MRC5 cells. ( C ) Table list of the novel chromatin-associated CDK7-interacting proteins in MRC5 cells identified through mass spectrometry analysis. Immunoblot analysis of DDX1 protein, the CAK (CDK7 and CycH) and XPD subunits of TFIIH in 1% triton-soluble (sol) and chromatin-enriched fractions (chr) of MRC5 cells before (Input) and after immunoprecipitation (IP) with anti-CDK7 ( D ), anti-DDX1 ( E ), or IgG (D and E) antibodies.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Silver Staining, Immunoprecipitation, Mass Spectrometry

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: DDX1 helicase binds to holo-TFIIH and Pol II. ( A ) In vitro pull-down assay of XPD or the CAK subunits CDK7 and CycH with DDX1 recombinant protein under permissive (100 mM KCl) or restrictive (300 mM KCl) salt conditions. Proteins are visualized by immunoblot analysis with antibodies raised against DDX1, XPD, CDK7, and CycH. The membranes were first hybridized with anti-XPD (*) and subsequently with anti-DDX1. Due to the similar molecular weight of the two proteins, the XPD protein band is still visible in the DDX1 immunoblotting. The input represents 10% of the total protein amount used in each pull-down reaction. In vitro pull-down assays of XPD WT, XPD fragments ( B ), or mutated forms of XPD ( C ) with DDX1 recombinant protein. XPD fragments contain specific functional domains of the protein, as depicted on the XP schematic representation (top). Mutated forms include the Arg112His and Arg722Trp substitutions causative of PS-TTD and the Arg683Trp amino acid change causative of XP. Proteins are visualized by immunoblot analysis with antibodies specific for XPD and DDX1. The input represents 10% of the total protein amount used in each pull-down reaction. ( D ) Immunoblot analysis with antibodies raised against the DDX1 helicase, various TFIIH subunits (XPB and p62 of core-TFIIH, the bridging factor XPD, CDK7, and CycH of the CAK sub-complex), and the RPB1 subunit of Pol II of two-step (TIP) immunoprecipitations performed first with anti-CDK7 and subsequently anti-DDX1 antibodies in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of control MRC5 cells. As a negative control, the first immunoprecipitation step was performed with IgG antibodies.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: In Vitro, Pull Down Assay, Recombinant, Western Blot, Molecular Weight, Functional Assay, Control, Negative Control, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: Reduced DDX1 protein amount leads to impaired Pol II-mediated transcription. ( A ) NER efficiency by UDS analysis in MRC5 cells upon gene silencing with either scrambled (CTR) or DDX1 siRNA. The number of individual grains per nuclei was counted. The diagram reports the mean values of three independent experiments. ( B ) Repair efficiency by slot blot analysis of CPD or 6–4PP in untreated MRC5 cells or treated with either DDX1 or control siRNA (CTR) at 0, 1.5, 4.5, 6.5 h after 20 J/m 2 UV-C irradiation. The amount of CPD or 6–4PP has been normalized to the amount of loaded single-strand DNA in at least three independent experiments. No significant differences have been recorded by the Student’s t -test when comparing the mean values of DDX1 silenced cells with the corresponding controls (MRC5 or CTR siRNA). ( C ) Global RNA synthesis by in vivo MRC5 cells labelled with 5-ethynyluridine (EU) and EU-click reaction (red). As a negative control, cells were exposed to Actinomycin D (ACTD) before EU treatment. Nuclei were counterstained with DAPI. The diagram on the left reports the intensity of the EU nuclear fluorescent signal measured by ImageJ in cells from three independent experiments (shown by different colours). The mean value and standard error for each experiment are indicated. ( D ) Immunoblot analysis of DDX1, TFIIH subunits (XPB, p62, XPD, CDK7, and CycH), the α subunit of the basal transcription factor TFIIE and the RPB1 subunit of Pol II in MRC5 whole cell extract transfected with either scrambled (CTR) or DDX1 siRNA (left). The amount of DDX1 and Pol II protein levels was normalized to the amount of β-actin. The diagram reports the mean values of three independent experiments (right). ( E ) Immunoblot analysis with antibodies raised against DDX1, the CDK7 and CycH subunits of TFIIH, the RPB1 subunit of Pol II, and the additional novel CDK7-interactors (NONO, SFPQ, and DHX9) in TIP samples performed first with anti-CDK7 and subsequently anti-DDX1 antibodies in the chromatin-enriched fractions of control MRC5 cells. As a negative control, the first immunoprecipitation step was performed with IgG antibodies (IgG). In all the graphs of the figure, when depicted, bars indicate standard errors (** P < .01, **** P < .0001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Dot Blot, Control, Irradiation, In Vivo, Negative Control, Western Blot, Transfection, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: Reduced DDX1 protein level leads to increased R-loop amount. ( A ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or DDX1 siRNA in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control dsDNA (right). The values are the mean of at least three independent experiments (* P < .05; Student’s t -test). ( B ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled (CTR) or DDX1 siRNA for 120 h. The amount of RNA/DNA hybrids at a, b, c, d, and e positions, indicated as horizontal bars within the β-actin locus (schematic representation on the top), was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments (* P < .05, ** P < .01; Student’s t -test). ( C ) Density plot of DDX1 ChIP-seq peak distribution relative to the gene TSS in Mus musculus . The plot shows the distribution of DDX1 occupancy across 8504 genes, each showing at least one DDX1 ChIP-seq peak within −1000 to +1000 bp of TSS. Peaks were mapped separately to both the plus strand and the minus strand. The x -axis indicates the upstream and downstream distance in bp from the TSS corresponding to position 0 bp. The y -axis indicates the density of DDX1 peaks.

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Dot Blot, Transfection, Control, Real-time Polymerase Chain Reaction, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: DDX1 -dependent R-loop accumulation leads to transcriptional stress. ( A ) Global RNA synthesis by in vivo labelling with 5-ethynyluridine (EU) and EU-click reaction in MRC5 cells 48 h after transfection with DDX1 or scrambled (CTR) siRNA as well as with the plasmid expressing the RNase H1 GFP or the empty vector. Nuclei were counterstained with DAPI. The diagram (top right) reports the intensity of the EU nuclear fluorescent signal measured by ImageJ in cells from two independent experiments (shown by different colours). The mean value and standard errors of each experiment are indicated (*** P < 0.001; Student’s t -test). ( B ) Immunoblot analysis with antibodies raised against DDX1, XPD, γH2AX, and ORC2 in the chromatin-enriched fraction of cells transfected with either scrambled (CTR) or DDX1 siRNA (left). The amount of DDX1, XPD, and γH2AX protein levels was first normalized to the amount of the chromatin loading control ORC2 and then expressed as fold increased relative to the corresponding protein amount in CTR siRNA. The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (** P < .01, *** P < .001; Student’s t -test). ( C ) Immunoblot analysis with antibodies raised against DDX1, γH2AX, and γTub in MRC5 cells transfected with DDX1 or scrambled (CTR) siRNA as well as with the plasmid expressing the RNase H1 GFP (+) or the empty vector (−) (left). The amount of DDX1 and γH2AX protein levels was normalized to the amount of the corresponding γTub loading control and reported as arbitrary units (au). The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (* P < .05, *** P < .001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: In Vivo, Transfection, Plasmid Preparation, Expressing, Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: TFIIH plays a role in R-loop processing. ( A ) Immunoblot analysis with antibodies raised against the RPB1 subunit of Pol II and various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK) in MRC5 whole cell extracts transfected with scrambled control (CTR) or XPD siRNA. Protein levels were normalized to the amount of γ-tubulin and reported as arbitrary units (au). The diagram reports the mean values of three independent experiments (right). Bars indicate the standard errors (* P < .05, ** P < .01, *** P < .001; Student’s t -test). ( B ) Slot blot of 0.5 and 1 μg of genomic DNA from MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs in the absence (−) or presence (+) of RNase H1 and hybridized with antibodies recognizing the RNA/DNA hybrids (S9.6 antibody) or dsDNA (left panel). The intensity of the bands was measured with ImageJ. The amount of S9.6 signal was normalized to the amount of the loading control, the dsDNA signal (right panel). The values are the mean of at least three independent experiments. Bars indicate the standard error (* P < .05; Student’s t -test). ( C ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of MRC5 cells treated with scrambled control (CTR) or XPD siRNA for 72 h. The amount of RNA/DNA hybrids at the a, b, c, d, and e positions of the β-actin locus was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least three independent experiments. Bars indicate the standard errors (* P < .05, ** P < .01; Student’s t -test). ( D ) Immunoblot analysis of XPD and the CDK7 subunits of TFIIH, NONO, SFPQ, and DDX1 proteins in the chromatin-enriched fractions of MRC5 cells transfected with scrambled control (CTR) or XPD siRNAs before (Input) and after immunoprecipitation (IP) with anti-CDK7 or IgG antibodies. Two independent IPs are shown (upper and lower left panels). In the cells treated with XPD -siRNA, the amount of co-immunoprecipitated proteins has been normalized to the amount of the corresponding immunoprecipitated CDK7 and expressed as fold increased relative to the sample CTR siRNA (right panel). The values are the mean of at least three independent experiments. When depicted, bars indicate standard errors (* P < .05; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Transfection, Control, Dot Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Trichothiodystrophy-causative pathogenic variants impair a cooperative action of TFIIH and DDX1 in R-loop processing

doi: 10.1093/nar/gkaf745

Figure Lengend Snippet: PS-TTD cells exhibit altered TFIIH-DDX1 interaction and impaired R-loop processing. ( A ) Immunoblot analysis with antibodies raised against various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK), DDX1, NONO, SFPQ, and the RPB1 subunit of Pol II in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of MRC5, TTD2GL, and XP102LO cells before (Input) and after immunoprecipitation with anti-CDK7 antibodies. The amount of co-immunoprecipitated DDX1, NONO, or SFPQ proteins was normalized to the amount of immunoprecipitated CDK7 (right panel). Bars indicate standard errors (* P < .05; ** P < .01; *** P < .001; Student’s t -test). ( B ) Immunoblot analysis with antibodies raised against various TFIIH subunits (XPB and p62 of the core-TFIIH, XPD, CDK7, and CycH of the CAK), DDX1, NONO, SFPQ, and the RPB1 subunit of Pol II in 1% triton-soluble (sol) and chromatin-enriched (chr) fractions of control (C3PV), TTD23PV, and XP26VI primary dermal fibroblasts before (Input) and after immunoprecipitation (IP) with anti-CDK7 or IgG antibodies. The amount of co-immunoprecipitated DDX1, NONO, and SFPQ was normalized to the amount of immunoprecipitated CDK7. The graph (right panel) also includes data collected from the samples shown in . Bars indicate standard errors (** P < .01; Student’s t -test). ( C ) DRIP analysis with the S9.6 antibody at the β-actin locus ( ACTB ) of primary dermal fibroblasts from PS-TTD with pathogenic variants in ERCC2/XPD (TTD8PV, TTD12PV, and TTD23PV) or in ERCC3/XPB (TTD6VI), from XP with pathogenic variants in ERCC2/XPD (XP15PV and XP49PV), or healthy individuals (CTR, C3PV, and C5PV). The amount of RNA/DNA hybrids at the a, b, c, d, and e positions of the ACTB locus was evaluated by real-time PCR. When applied, the RNase H1 treatment is indicated. Data are expressed as fold enrichment over the input. The values are the mean of at least two independent experiments. Bars indicate standard errors (* P < .05, ** P < .01, *** P < .001; Student’s t -test).

Article Snippet: Cell fractions were immunoprecipitated (single IP) overnight at 4°C, as previously described [ ], and using either CDK7 or DDX1 primary antibodies (Santa Cruz Biotechnology) conjugated to agarose beads.

Techniques: Western Blot, Immunoprecipitation, Control, Real-time Polymerase Chain Reaction