Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were transfected with either control mimic (con) or miR-30b mimic (30b) and levels of TGFβ1 and TGFβ2 mRNA were assessed by qRT-PCR. Expression levels relative to control mimic transfected cells and normalized to β-actin expression are presented as the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 expression. * P < 0.05, ** P < 0.01 as determined by unpaired Student’s t -test. (B) Cells were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of TGFβ2 protein levels by western blot. β-actin was used as endogenous control. (C) ELISAs for TGFβ1 and TGFβ2 were performed with 24 hour conditioned supernates from HUVECs transfected with 20 nM of either control or miR-30b mimic. Data represents the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 secretion into cell culture supernate. * P = 0.044 as determined by unpaired Student’s t -test. (D) HUVECs were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of Smad2 phosphorylation by western blot.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Cell Culture, Phospho-proteomics

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) JDP2 mRNA expression was assessed in HUVECs transfected with miR-30b mimic (20 nM) as compared to control by qRT-PCR. Data represents the mean ± SEM (n = 3) normalized to β-actin as endogenous control. * P = 0.016 as determined by unpaired Student’s t -test. (B) HUVEC were transfected with 50 nM of either control siRNA or ATF2 siRNA 1 or 2 and RNA was isolated at 48 hours post transfection. Levels of ATF2 and TGFβ2 mRNA were assessed by qRT-PCR with β-actin as endogenous control. Data presented is mean ± SEM (n = 2). Statistically significant decreases in ATF2 and TGFβ2 expression were seen in ATF2 siRNA treated cells as compared to control siRNA treated cells. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by unpaired Student’s t -tests for each ATF2 siRNA compared to control siRNA. (C) Cells transfected with 5 nM of either control siRNA or ATF2 siRNA 1 were seeded onto growth factor reduced BME and the formation of capillary-like cord structures and number of loops was assessed after 24 hours. (D) A statistically significant increase in cord formation was observed in cells depleted of ATF2 through siRNA. Data represents the mean ± SEM (n = 2). * P = 0.041 as determined by unpaired Student’s t -test. (E) HUVECs were co-transfected with miRNA mimic (20 nM) and ATF2 siRNA 1 or 2 (50 nM) in the combinations displayed and cell lysates were collected at 48 hours post transfection and assessed for TGFβ2 mRNA expression. Data presented is mean ± SEM (n = 2). * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA with post hoc analysis. (F) Cells were transfected as in (E) using miRNA mimic (20 nM) and ATF2 siRNA 1 (5 nM) and serum starved overnight in MCDB 131 with 0.5% FBS prior to protein expression analysis by western blot. Data is representative of expression levels observed in two independently performed experiments.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

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

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were serum starved overnight in MCDB 131 with 0.5% FBS and stimulated with VEGF (50 ng/ml) in the presence or absence of Avastin (1 μg/ml) for 24 hours. Data represents the mean ± SEM (n = 2) for expression of TGFβ1 and TGFβ2 assessed by qRT-PCR relative to β-actin endogenous control. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA. (B) HUVECs were treated with 5 ng/ml of TGFβ2 for 3 days prior to seeding onto growth factor reduced BME for assessment of capillary-like cord formation after 24 hours. (C) A significant decrease in cord formation is observed in the TGFβ2 treated group. Data represents the mean ± SEM (n = 2). ** P = 0.0072 as determined by unpaired Student’s t -test. (D) HUVECs transfected with 1 nM control or miR-30b mimic were treated 4 hours post transfection with 0.8 μg/ml anti-TGFβ2 neutralizing antibody or rabbit IgG. Media was refreshed after 24 hours, again with rabbit IgG or anti-TGFβ2 antibody and cells were seeded onto growth factor reduced BME 24 hours later (ie. 48 hours post transfection) in media containing rabbit IgG or anti-TGFβ2 antibody. (E) Data represents the mean ± SEM (n = 3) of the number of capillary-like cord structures or number of loops formed after 24 hours on BME. * P < 0.05, ns denotes not significant as determined by ANOVA with post hoc analysis.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection

Journal: Brain

Article Title: Distinct transcriptional changes distinguish efficient and poor remyelination in multiple sclerosis

doi: 10.1093/brain/awae414

Figure Lengend Snippet: Cellular expression of genes of interest in human multiple sclerosis brain tissue. ( A – D ) Immunofluorescent double-stained images of TGFβ1 ( A ), TGFβ2 ( B ), EGF ( C ) and BTC ( D ) with GFAP (astrocytes), HLA (microglia/macrophages) and SOX10 or Nogo-A (oligodendrocytes) in an active non-foamy lesion. Scale bars: 15 μm. Arrows indicate target + /cell marker + double-staining, arrowhead indicates target + /cell marker − double-staining, and asterisks indicate target − /cell marker + double-staining. ( E ) Percentage of cells per cell type expressing selected ligand and receptor pairs and average expression of each gene in each cell type (adapted from Absinta et al. ). OPC = oligodendrocyte precursor cell.

Article Snippet: Antibodies included TGFβ1 ( Ab215715 , 1:100; Abcam), TGFβ2 (116020, 1:100; NovoPro, Shanghai, China), EGF (Ab9695, 1:100; Abcam), BTC (AF-261, 1:100; R&D Systems) and BCAS1/NaBC1 (Sc-136342, 1:15 000; Santa Cruz).

Techniques: Expressing, Staining, Marker, Double Staining

Journal: Brain

Article Title: Distinct transcriptional changes distinguish efficient and poor remyelination in multiple sclerosis

doi: 10.1093/brain/awae414

Figure Lengend Snippet: Association between factors of interest with remyelinating multiple sclerosis tissue. ( A and B ) Histological quantification of targets of interest, TGFβ1, TGFβ2, EGF and BTC, in remyelinated lesions (RLs), active non-foamy lesions (ALs non-foamy), active foamy lesions (ALs foamy) and normal-appearing white matter (NAWM). Scale bars: 100 μm. Arrows indicate TGFβ1 + , TGFβ2 + , EGF + and BTC + cells, respectively. ( C ) Immunohistochemical staining of BCAS1. ( D – I ) Heat map of quantified proteins of interest and BCAS1 in RLs, ALs non-foamy, ALs foamy and NAWM shows a positive correlation between TGFβ1 and BCAS1 and between EGF and BTC. Statistics were performed using negative binomial generalized linear model or restricted maximum likelihood with Tukey’s post hoc test to compare values between groups. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Antibodies included TGFβ1 ( Ab215715 , 1:100; Abcam), TGFβ2 (116020, 1:100; NovoPro, Shanghai, China), EGF (Ab9695, 1:100; Abcam), BTC (AF-261, 1:100; R&D Systems) and BCAS1/NaBC1 (Sc-136342, 1:15 000; Santa Cruz).

Techniques: Immunohistochemical staining, Staining

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Effect of RBE-Exos on the adhesion of NK-92 cells. ( A ) Growth status of NK-92 cells. NK cells treated with RBE-Exos lost the normal cluster growing. ( B ) CD11a protein levels. ( C ) CD18 protein levels. ( D ) CD54 protein levels. Western blot showed that the protein levels of CD11a, CD18 and, CD54 were obviously decreased in NK-92 cells treated with RBE-Exos. ( E ) Relative transcriptional levels of CD11a , CD18 , CD54 and, CD2 in NK-92 cells. qRT-PCR displayed that RBE-Exos downregulated the transcriptional levels of CD11a , CD18 , CD54 , but not CD2 . Data were presented as mean ± SEM for three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Cholangiocarcinoma derived exosomes attenuate the anti-tumor functions of NK cells

doi: 10.1038/s41598-026-36706-9

Figure Lengend Snippet: Cytotoxicity and expression of adhesion molecules in primary NK cells. ( A ) Cytotoxicity of primary NK cells from CCA patients and healthy people against target cells. LDH detection displayed the cytotoxicity of NK cells from CCA patients was significantly lower than that of NK cells healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( B ) Protein levels of CD18 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ( C ) Protein levels of CD54 in primary NK cells from CCA patients and healthy people. Data are presented as the mean ± SEM from n = 3 independent experiments. ** P < 0.01.

Article Snippet: The primary antibodies were as follows: rabbit anti-human CD63, BCL-2, BCL-XL, CD11a, β-actin (Abcam, USA), mouse anti-human HSP70 (Santa-Cruz, USA), rabbit anti-human CD18 (Proteintech, China), mouse anti-human CD54 (Proteintech, China).

Techniques: Expressing

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: TGFβ2 antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500 dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108) was used at a 1:250 dilution.

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: TGFβ2 antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500 dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108) was used at a 1:250 dilution.

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: TGFβ2 antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500 dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108) was used at a 1:250 dilution.

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: TGFβ2 antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500 dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108) was used at a 1:250 dilution.

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: TGFβ2 antibody (Santa Cruz Biotechnology; sc-90) was used at a 1:500 dilution, p-CREB1 (Cell Signaling Technology; 9198) was used at a 1:100 dilution, and p-SMAD2 (Cell Signaling Technology; 3108) was used at a 1:250 dilution.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control