Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A ) Enrichment of the indicated pathways in Myc-CaP cells treated with YJ1206 at 1 μM for 24 hours. Adj., adjusted. ( B ) Immunoblot of the indicated proteins in Myc-CaP cells treated with YJ1206 at increasing concentrations for 4 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. GAPDH was used as a loading control. ( C ) Top: Analysis of the indicated gene expression by RT-qPCR in Myc-CaP cells treated with YJ1206 at 1 μM for 15 hours, or siRNAs targeting Cdk12 and/or Cdk13 . Nontargeting siRNA was used as control. Bottom left: IFN-β ELISA results in Myc-CaP cells treated as described above. Bottom right: Flow cytometry assessing surface MHC-I expression in Myc-CaP cells treated as described above. ( D ) Immunoblot of the noted proteins in B16-F10 cells treated with YJ1206 at increasing concentrations for 4 hours. ( E and F ) Flow cytometry median fluorescence intensity (MFI) quantifications of surface MHC-I ( E ) or PD-L1 ( F ) in the indicated cells treated with YJ1206 at 1 μM or 3 μM for 15 hours. ( G ) Quantification of immunofluorescence DNA/RNA hybrid (red) staining in Myc-CaP cells treated with 1 μM YJ1206 for 4 hours or siRNA targeting Cdk12 and/or Cdk13 , with/without RNase H. Representative images are in . Nontargeting siRNA was used as control. Forty (siRNA treatment) or 20 (YJ1206 or DMSO treatment) cells were used per data point. Data in C , E , and F are displayed as mean ± SD of triplicate experiments. Data in G are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons in C and E – G .

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Western Blot, Control, Gene Expression, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Fluorescence, Immunofluorescence, Staining, Whisker Assay

Journal: The Journal of Clinical Investigation

Article Title: CDK12/13 inactivation triggers STING-mediated antitumor immunity in preclinical models

doi: 10.1172/JCI193745

Figure Lengend Snippet: ( A and B ) Representative images ( A ) or quantification ( B ) of immunofluorescence assessing PCNA-RNAPII PLA foci in Myc-CaP cells treated with siRNA targeting Cdk12 and/or Cdk13 , or YJ1206 at 3 μM for 4 hours, with or without DRB or triptolide treatment. ( C – E ) Representative images ( C ) or quantification ( D and E ) of dsDNA ( C , left, and D ) and ssDNA ( C , right, and E ) in Myc-CaP cells treated with 3 μM YJ1206 for 4 hours, with or without DRB treatment. Scale bars: 5 μm. ( F ) ELISA measuring cGAMP levels in Myc-CaP cells treated with YJ1206 at the indicated concentrations. ( G ) Immunoblot of the noted proteins in Myc-CaP cells treated with YJ1206 at 1 μM, with or without DRB for 4 hours. Data in D and E are presented as box-and-whisker plots, with the median (center line), 25th–75th percentiles (box), and minimum to maximum values (whiskers). Data are displayed as mean ± SEM in F of triplicate experiments. One hundred cells were used per data point in B , D , and E . **** P < 0.0001 by 2-tailed t test. NS, not significant. Bonferroni’s correction was applied for multiple comparisons.

Article Snippet: IFN-β ELISA (R&D Systems, DY8234-05 and DY008B) and cGAMP ELISA (Cayman Chemical, 501700) kits were used per the manufacturer’s protocols.

Techniques: Immunofluorescence, Enzyme-linked Immunosorbent Assay, Western Blot, Whisker Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

Journal: International journal of molecular medicine

Article Title: Involvement of brain-derived neurotrophic factor in exercise‑induced cardioprotection of post-myocardial infarction rats.

doi: 10.3892/ijmm.2018.3841

Figure Lengend Snippet: Figure 4. Serum BDNF level is associated with myocardium angiogenesis and left ventricle function. (A) ELISA measurements of the serum BDNF con- centration at the (a) beginning and the (b) end of the exercise programme. *P<0.05 vs. Sham and †P<0.05 vs. MIC. (B) The serum BDNF concentration was positively correlated with myocardial microvessel density (r=0.542, P=0.006). (C) The serum BDNF concentration was positively correlated with the EF (r=0.631, P=0.001). (D) A positive correlation was identified between improved serum BDNF levels and the EF (r=0.502, P=0.013). The values are presented as the mean ± standard error of the mean (n=8). MI, myocardial infarction; L‑NAME, NG‑nitro‑L‑arginine methyl ester; MIC, sedentary MI group; MIE, MI + exercise group; MIE+L, MI + exercise + L‑NAME group; BDNF, brain‑derived neurotrophic factor; EF, ejection fraction; HPF, high‑powered fields.

Article Snippet: Serum and media BDNF concentrations were detected using an ELISA kit (cat. no. DBNT00; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: International journal of molecular medicine

Article Title: Involvement of brain-derived neurotrophic factor in exercise‑induced cardioprotection of post-myocardial infarction rats.

doi: 10.3892/ijmm.2018.3841

Figure Lengend Snippet: Figure 5. SS of 12 dyn/cm2 increased BDNF/TrkB‑FL protein levels and sustained TrkB‑FL activation in HUVECs. (A) Representative western blot analysis images for BDNF and the protein level analysis in HUVECs exposed to SS (*P<0.01 vs. Ctrl). (B) ELISA measurement of BDNF levels in the culture medium following exposure of HUVECs for 1, 3, 6 and 12 h of SS (*P<0.01 vs. Ctrl). (C) Representative western blot analysis images for TrkB‑FL and the protein level analysis in HUVECs exposed to SS (*P<0.01 vs. Ctrl). (D) TrkB‑T protein expression and phosphorylation levels were not modified by SS at 12 dyn/cm2. (E) Representative western blot analysis images for phosphorylated TrkB‑FL and the protein level analysis in HUVECs exposed to SS (**P<0.001 vs. Ctrl). (F) TrkB‑T phosphorylation levels were not modified by SS at 12 dyn/cm2. The values are presented as the mean ± standard error of the mean (n=6). SS, shear stress; BDNF, brain‑derived neurotrophic factor; TrkB, BDNF/NT‑3 growth factors receptor; FL, full‑length; T, truncated; p, phosphorylated; ctrl, control; HUVECs, human umbilical vein endothelial cells; t, time.

Article Snippet: Serum and media BDNF concentrations were detected using an ELISA kit (cat. no. DBNT00; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's protocol.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Phospho-proteomics, Modification, Shear, Control