Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effects of ActRIIA and its downstream signaling proteins on the survival of LUAD. Cox regression was used to analyze the data (n=530). The Kaplan-Meier survival curves represented the impacts of different proteins on the survival of LUAD: (A) ActRIIA, (B) SMAD3, (C) MAPK1 and (D) MAPK3. (E) Sample distribution differences of LUAD data from GSE116959 were analyzed through principal component analysis. (F) Volcano plot of GSE116959 was analyzed using log 2 FC>1 and adjusted P-value <0.05. Upregulated DEGs were shown in red and downregulated DEGs were shown in blue. (G) Statistical analysis results of microarray-based data analysis from GSE116959. LUAD, lung adenocarcinoma; DEGs, differentially expressed genes.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Microarray

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effect of activin A on viability and proliferation of A549 cells. (A) The viability of A549 cells was examined by Cell Counting Kit-8 assay after treated with activin A. (B and C) The proliferation of A549 cells was determined by real-time cell analysis in the presence or absence of activin A. (D) The proliferation of A549 cells treated with activin A for 24 was examined by BrdU incorporation. *P<0.05 and **P<0.01 compared with control group (n=3).

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Cell Counting, Cell Analysis, BrdU Incorporation Assay, Control

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effect of activin A on the apoptosis of A549 cells. (A) The apoptosis of A549 cells treated with activin A for 24 h was assayed by Hoechst fluorescent staining. Typical cells were marked by white arrows. Scale bar, 100 µm. (B) The apoptotic ratio of A549 cells was examined by flow cytometry with YF ® 488-Annexin V and PI staining after treated with activin A for 24 h. *P<0.05 and **P<0.01 compared with control group.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Staining, Flow Cytometry, Control

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effect of activin A on expression of endoplasmic reticulum stress pathway-related proteins in A549 cells. Levels of proteins were examined by western blotting in A549 cells after treated with activin A for 24 h. The graph represented the relative levels of proteins in three separate experiments. The levels of proteins were normalized against GAPDH expression, and the results were shown as the fold-increase of the control. **P<0.01 compared with control group.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Western Blot, Control

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effects of calcium signaling on apoptosis of A549 cells. (A and B) The calcium levels in A549 cells treated with activin A were measured by Fluo-4 fluorescence signal intensity. F0, fluorescence baseline. F, fluorescence intensity after treated with 20 ng/ml activin A. The graph represented the comparison of the peak value of calcium signal normalized to the baseline (F/F0). Typical cells were marked by white arrows. Scale bar, 1,000 µm. (C) BAPTA-AM and ionomycin affected apoptosis of activin A-induced A549 cells. The apoptosis of cells labeled with YF ® 488-Annexin V and PI was tested by flow cytometry. The graph revealed the percentage of apoptotic cells in three separate experiments. **P<0.01 compared with 0.025% DMSO control group; ## P<0.01 compared with 0.025% DMSO + Activin A group.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Fluorescence, Comparison, Labeling, Flow Cytometry, Control

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effects of activin A on expression of activin receptors, Smad3 and MAPK signaling proteins in A549 cells. (A) Levels of ActRIA, ActRIB, ActRIIA, ActRIIB and Smad3 mRNAs were determined by reverse transcription-quantitative PCR in A549 cells treated with activin A for 4 h. The graph represented the relative levels of mRNA in three separate experiments. The levels of mRNA were normalized against GAPDH expression, and the results were shown as the fold-increase of the control. (B) Level of ActRIIA protein was examined by western blotting in A549 cells treated with activin A for 4 h. The graph represented the relative levels of proteins in three separate experiments. The levels of ActRIIA protein were normalized against GAPDH, and the results were presented as the fold-increase of the control. (C) Levels of Smad3, p-Smad3, ERK1/2, p-ERK1/2, JNK and p-JNK proteins were determined by western blotting in A549 cells subject to 0–40 ng/ml of activin A for 4 h. The graph represented the relative levels of protein in three separate experiments. The levels of protein were normalized against GAPDH expression, and the results were presented as the fold-increase of the control. **P<0.01 compared with control group. p-, phosphorylated.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot

Journal: Oncology Reports

Article Title: Activin A induces apoptosis of human lung adenocarcinoma A549 cells through endoplasmic reticulum stress pathway

doi: 10.3892/or.2023.8688

Figure Lengend Snippet: Effects of ERK inhibitor FR180204 on activin A-induced A549 cell apoptosis. (A) A549 cells were pretreated for 2 h with 1% DMSO or 10 µM FR180204 in 1% DMSO, then treated with 20 ng/ml activin A for 4 h. Levels of p-ERK and ERK protein were examined by western blotting. The graph represented the relative levels of proteins in three separate experiments. The levels of p-ERK and ERK protein were normalized against GAPDH, and the results were shown as the fold-increase of the control. (B) A549 cells were pretreated for 2 h with 1% DMSO or 10 µM FR180204, then treated for 12 h with or without 20 ng/ml activin A. The apoptosis of cells labeled with YF ® 488-Annexin V and PI was assayed by flow cytometry. The graph revealed the percentage of apoptotic cells in three separate experiments. (C) A549 cells were pretreated for 2 h with 1% DMSO or 10 µM FR180204, then treated for 12 h with or without 20 ng/ml activin A. Levels of CHOP and caspase-12 protein were examined by western blotting. The graph represented the relative levels of proteins in three separate experiments. *P<0.05 and **P<0.01 compared with 1% DMSO control group; ## P<0.01 compared with activin A + 1% DMSO control group. p-, phosphorylated.

Article Snippet: The human LUAD cell line A549 ( https://www.cellosaurus.org/CVCL_0023 ; cat. no. CCL-185; American Type Culture Collection) was cultured in RPMI-1640 (cat. no. 11875093; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) (cat. no. C04001; Shanghai VivaCell Biosciences, Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO 2 .

Techniques: Western Blot, Control, Labeling, Flow Cytometry

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Identification of KSR1-regulated phosphoproteome in breast cancer cells. ( A ) Experimental schematic outline of SILAC experiment. ( B ) Scatter plot comparison of phosphosite ratios quantified from control vs KSR1-overexpressed MCF7 cells. ( C ) Gene ontology (GO) Classification of the KSR1-regulated phosphoproteome in MCF7 cells according to molecular functions, biological processes and cellular compartmentalisation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Multiplex sample analysis, Comparison, Phospho-proteomics, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 transcriptional activity in the presence or absence of etoposide by luciferase assays. ( A ) MCF7 cells were transiently co-transfected with either pCMV6 (vector) or pCMV6-KSR1 plasmids in the presence of four individual p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1, p53-CYCLIN G1 and p53-IGFBP3) following dimethylsulphoxide (DMSO) or etoposide (40 μ M ) treatment for 3 h. ( B ) MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 48 h, followed by transfection of three p53-dependent promoter constructs expressing firefly luciferase genes (p53-R2, p53-AIP1 and p53-CYCLIN G1) for additional 24 h. DMSO or etoposide (40 μ M ) were subsequently added as described above. Firefly luciferase activity was measured (renilla luciferase activity was used to normalise transfection efficiency). The normalised luciferase activity of empty vector is set as 1. Results shown are the average of at least three independent experiments and error bars represent s.d. Student's t -test was performed using SPSS 16.0 statistical software (SPSS Inc.). (* P <0.05, ** P <0.01).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Luciferase, Transfection, Plasmid Preparation, Construct, Expressing, Control, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 on p53 mRNA, total protein and neddylation levels and on p53 subcellular localisation. ( A ) Effects on p53 mRNA and total protein levels after KSR1 overexpression. MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, relative mRNA levels of TP53 and p53 total protein were measured by RT-qPCR and western blotting, respectively. Gene expression level from cells transfected with pCMV6 was set as 1. Results shown are the average of at least three independent experiments. Similarly, in MCF7 stably overexpressing KSR1 cells, p53 total protein was evaluated by western blot. Blots shown are representatives of at least three independent experiments. ( B ) Immunofluorescence staining of p53 cells after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. p53 was detected with an anti-p53 antibody while the nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI). Representative pictures of three independent experiments are shown. Subcellular fractionation assays were performed after 24-h transfection with either pCMV6 or pCMV6-KSR1 plasmids in MCF7. Tubulin and histone deacetylase 1 (HDAC1) expression served as positive normalising control for cytoplasmic and nuclear proteins respectively. Blots shown are representatives of at least three independent experiments. ( C ) Neddylation assay on p53 after KSR1 overexpression. MCF7 cells were co-transfected with HA-NEDD8 and pCMV6 or pCMV6-KSR1 plasmids as indicated. p53 was immunoprecipitated using a p53-specific antibody (DO-1) and the neddylated-p53 was detected by immunoblotting using anti-NEDD8 and anti-p53-specific antibodies. Blots shown are representatives of at least three independent experiments. Abbreviations: IgG= immunoglobulin G; IP= immunoprecipitation.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Over Expression, Transfection, Quantitative RT-PCR, Western Blot, Gene Expression, Stable Transfection, Immunofluorescence, Staining, Fractionation, Histone Deacetylase Assay, Expressing, Control, Immunoprecipitation

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Mechanisms of KSR1-regulated p53 transcriptional activity. ( A ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 overexpression followed by etoposide treatment. MCF7 cells were transiently transfected with pCMV6 (vector) or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with various concentrations of etoposide (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( B ) Effects on p53 acetylation and phosphorylation of DBC1 after KSR1 silencing followed by a titration of etoposide treatment. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 for 72 h followed by etoposide treatment (20, 40, 80 μ M , 3 h). p53 acetylation and DBC1 phosphorylation at Thr454 were assessed by immunoblotting with specific antibodies as indicated. ( C ) Effect of KSR1 on p53 acetylation is through DBC1. MCF7 cells were transfected with control siRNA (siCT) or siKSR1 in concordance with siCT or siDBC1 for 72 h followed by etoposide treatment (40 μ M , 3 h). Acetylated p53, DBC1 and KSR1 protein levels were assessed by immunoblotting with specific antibodies as indicated. ( D ) Effect of KSR1 on DBC1 phosphorylation is dependent on its intact kinase domain. MCF7 cells were transiently transfected with vector, wild-type KSR1 or mutant KSR1 (R502M) plasmids for 24 h followed by etoposide treatment (40 μ M , 3 h). DBC1 phosphorylation was measured by immunoblotting with specific antibody. ( E ) Interaction of DBC1 and SIRT1 after KSR1 overexpression with etoposide treatment by immunoprecipitation (IP). MCF7 cells were transiently transfected with pCMV6 or pCMV6-KSR1 plasmids for 24 h. Subsequently, cells were treated with etoposide (40 μ M , 3 h). The interactions between SIRT1 and DBC1 were detected by IP of SIRT1 or DBC1 followed by immunoblotting with DBC1 and SIRT1 antibodies respectively. Blots shown are representatives of at least three independent experiments. Quantification of blots was analysed by ImageJ software (NIH, Bethesda, MD, USA). ( F ) Schematic model illustrating the role of KSR1 on p53 transcriptional activity in breast cancer cells with (i) basal or (ii) up-regulated levels of KSR1. Abbreviation: IgG= immunoglobulin G.

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Activity Assay, Phospho-proteomics, Over Expression, Transfection, Plasmid Preparation, Western Blot, Titration, Control, Mutagenesis, Immunoprecipitation, Software

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: Effects of KSR1 silencing on breast cancer cell proliferation in vitro . SRB assays of MCF7, ZR75-1, SKBR3 and MDA231 cells after transfection with 20 n M of either siKSR1 or ‘non-targeting' siRNA (control siRNA) or vehicle (Hiperfect) for 6 days. Error bars represent s.d. of three experiements each in quintuplicates (* P <0.05, compared with control siRNA at day 6; Student's t- test).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: In Vitro, Transfection, Control

Journal: British Journal of Cancer

Article Title: SILAC-based phosphoproteomics reveals an inhibitory role of KSR1 in p53 transcriptional activity via modulation of DBC1

doi: 10.1038/bjc.2013.628

Figure Lengend Snippet: KSR1 expression is altered in breast cancer tissues. Oncomine analysis was performed to examine KSR1 expression in breast normal and cancer tissues using online TCGA microarray data ( www.oncomine.org ).

Article Snippet: The following antibodies were used: KSR1 rabbit polyclonal from Cell Signaling (Hitchin, UK), anti-Flag mouse monoclonal (Sigma Aldrich), p53 mouse monoclonal DO-1 from Santa Cruz (Wiltshire, UK), acetylated-p53 and phospho-p53 Ser15 rabbit polyclonal (Cell Signaling), SIRT1 rabbit polyclonal (Santa Cruz), DBC1 and phospho-DBC1 Thr454 rabbit polyclonal (Cell Signaling) and β -actin mouse monoclonal from Abcam (Cambridge, UK).

Techniques: Expressing, Microarray

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: Expression levels of ADGRG6 mRNA in PAAD and clinical subgroups (GEPIA and UALCAN). ( A ) Comparison of ADGRG6 mRNA expression between PAAD tissues ( n = 179) and normal tissues ( n = 171) from the GEPIA database (TCGA + GTEx). ( B – I ) UALCAN-based subgroup analysis of ADGRG6 expression levels in PAAD samples stratified by sex ( B ), pancreatitis status ( C ), age ( D ), drinking habits ( E ), diabetes status ( F ), tumor grade ( G ), lymph node metastasis ( H ), and TP53 mutation status ( I ). The “normal” group in UALCAN ( n = 4) includes adjacent non-tumor tissues, without subgroup annotations. The comparison to the “normal” group in the UALCAN analysis should be interpreted with caution due to the small size of the normal cohort ( n = 4). “Drinking status” data is incomplete (missing in 80 samples), and comparisons among subgroups should be interpreted cautiously. Subtype descriptions of “Tumor Grade”: Grade 1-Well differentiated (low grade), Grade 2-Moderately differentiated (intermediate grade), Grade 3-Poorly differentiated (high grade), Grade 4-Undifferentiated (high grade). Pathologic descriptions of “Nodal Metastasis Status”: N0-No regional lymph node metastasis, N1-Metastases in 1 to 3 axillary lymph nodes. Data are presented as Mean ± SD. Statistical analysis was performed using Student’s t -test for two-group comparisons and one-way ANOVA followed by Bonferroni’s post hoc test. “*” indicates comparison with the control group; “#” indicates significance between experimental groups (* p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01).

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Expressing, Comparison, Mutagenesis, Control

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: Protein expression of ADGRG6 in PAAD based on UALCAN and HPA databases. ( A ) Comparison of ADGRG6 protein expression between PAAD tissues ( n = 137) and normal pancreatic tissues ( n = 74) using the UALCAN database (CPTAC dataset). ( B – L ) Subgroup analyses of ADGRG6 protein expression stratified by sex ( B ), chromatin modifier alteration status ( C ), age ( D ), weight ( E ), tumor grade ( F ), tumor stage ( G ), MYC/MYCN alteration ( H ), SWI/SNF complex alteration ( I ), and activity status of the mTOR ( J ), Hippo ( K ), and RTK ( L ) signal pathways. Each subgroup was compared to normal tissues. The “others” group in ( C , H – L ) refers to patients without the specific mutation or alteration listed. ( M ) Representative IHC staining images from the HPA database showing ADGRG6 expression in normal pancreatic tissue and PAAD tissue. Scale Bar: 200 µm. Data are presented as Mean ± SD. Statistical analysis was performed using Student’s t -test for two-group comparisons and one-way ANOVA followed by Bonferroni’s post hoc test. “*” indicates comparison with the control group; “#” indicates significance between experimental groups (* p < 0.05, *** p < 0.001; # p < 0.05).

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Expressing, Comparison, Activity Assay, Mutagenesis, Immunohistochemistry, Control

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: Prognostic value of ADGRG6 expression in PAAD. ( A ) Kaplan–Meier survival curves showing OS relative to ADGRG6 expression. ( B – I ) Subgroup OS analyses, including ( B , C ) gender, ( D , E ) stage 1–2, ( F ) T2 (tumor size > 2 cm but ≤4 cm), ( G ) T3 (tumor size > 4 cm), ( H ) N0 (no regional lymph node metastasis), and ( I ) M0 (no distant metastasis).

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Expressing

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: Single-cell analysis of ADGRG6 in the TME. ( A ) TISCH database analysis of ADGRG6 expression across different cell types in the TME. ( B , C ) The distribution of ADGRG6 expression in various immune and stromal cell types in the PAAD_CRA001160 and PAAD_GSE154778 datasets. The left panels ( B , C ) depict the Uniform manifold approximation and projection (UMAP) of single-cell transcriptome data with cell typing (major lineages) in the two datasets. The right panels ( B , C ) show the expression of the ADGRG6 gene in different cell types (major lineages) within the two datasets.

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Single-cell Analysis, Expressing

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: ADGRG6 silencing suppresses PAAD cell proliferation, migration, and invasion in vitro. ( A ) IHC staining of ADGRG6 in PAAD tissues based on tissue microarray analysis, showing higher expression in advanced TNM stages. Scale Bar: 200 µm. ( B ) ADGRG6 mRNA levels in AsPC-1 and BxPC-3 post si- ADGRG6 . ( C ) Cell proliferation of si- ADGRG6 -transfected AsPC-1 and BxPC-3 measured by CCK-8 assay. ( D ) Wound-healing assay demonstrating reduced migration capacity in si- ADGRG6 -transfected AsPC-1 and BxPC-3 cells. Scale Bar: 50 µm. ( E ) Transwell invasion assays (200×) confirming decreased invasive ability post-knockdown. Scale Bar: 50 µm. ( F ) 3D spheroid culture assays demonstrating impaired spheroid growth in si- ADGRG6 cells, quantified by spheroid diameters across 21 days (4×). Scale Bar: 200 µm. Data are presented as Mean ± SD. Statistical analysis was performed using Student’s t -test for two-group comparisons and one-way ANOVA followed by Bonferroni’s post hoc test. “*” indicates comparison with the control group; “#” indicates significance between experimental groups (** p < 0.01, *** p < 0.001; # p < 0.05).

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Migration, In Vitro, Immunohistochemistry, Microarray, Expressing, Transfection, CCK-8 Assay, Wound Healing Assay, Knockdown, Comparison, Control

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: In vivo evidence of ADGRG6 oncogenic function in zebrafish and murine xenograft models. ( A ) Representative fluorescence microscopy images of zebrafish xenografts injected with CM-DiI-labeled AsPC-1 cells (si-NC vs. si- ADGRG6 ) at 48 h post-injection (hpi), showing reduced tumor fluorescence area in si- ADGRG6 xenografts. Scale Bar: 300 µm. ( B ) Migration distance of tumor cells in zebrafish xenografts at 24 hpi, significantly reduced upon ADGRG6 knockdown. Fluorescence (red) resulting from CM-Dil labeling was used to monitor the behavior of the cells in the zebrafish model. ( C ) Relative ADGRG6 mRNA and ( D ) protein levels in AsPC-1 cells transfected with siRNA. ( E ) Representative images of mice in vivo tumorigenesis assay. ( F ) Representative images of excised tumors from the tumor-bearing mice. ( G ) Body weight of mice across groups was measured every three days for each mouse and the growth curve was plotted ( n = 6). ( H ) Comparison of the tumor weight ( n = 6). Data are presented as Mean ± SD. Statistical analysis was performed using Student’s t -test for two-group comparisons and one-way ANOVA followed by Bonferroni’s post hoc test. * p < 0.05, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: In Vivo, Fluorescence, Microscopy, Injection, Labeling, Migration, Knockdown, Transfection, Comparison

Journal: Current Issues in Molecular Biology

Article Title: ADGRG6 Promotes Pancreatic Adenocarcinoma Progression Through the NF-κB/STAT6 Axis and Modulation of the Tumor Immune Microenvironment

doi: 10.3390/cimb47120991

Figure Lengend Snippet: Regulatory Role of ADGRG6 in the NF-κB→STAT6→GATA3 Signaling Axis. ( A ) KEGG pathway analysis of the gene set co-expressed with ADGRG6 using the LinkInterpreter module; ( B – E ) Pearson correlation analysis between ADGRG6 and key genes of the signaling axis ( NFKB1 , RELA , STAT6 , and GATA3 ) in 178 clinical samples. Values represent Pearson correlation coefficients and corresponding p -values, n = 178; ( F , G ) Relative expression levels of STAT6 and GATA3 genes in the signaling axis in AsPC-1 and BxPC-3 cells after ADGRG6 knockdown detected by RT-qPCR. Data are presented as mean ± standard deviation; ( H , I ) Secretion levels of secretory cytokines IL-6 and IL-8 in AsPC-1 and BxPC-3 cells detected by ELISA; ( J ) Expression and activity of key proteins in the signaling axis after ADGRG6 knockdown detected by Western blot, *** p < 0.001.

Article Snippet: Sections were then incubated overnight at 4 °C with a rabbit polyclonal anti-ADGRG6 antibody (1:500; Proteintech, Rosemont, IL, USA, Cat. no. 17774-1-AP).

Techniques: Expressing, Knockdown, Quantitative RT-PCR, Standard Deviation, Enzyme-linked Immunosorbent Assay, Activity Assay, Western Blot

Journal: Nutrition & Diabetes

Article Title: Human mediastinal adipose tissue displays certain characteristics of brown fat

doi: 10.1038/nutd.2013.6

Figure Lengend Snippet: Comparison of gene expression in human subcutaneous and mediastinal adipose tissue. Real-time PCR validation of genes selected from the microarray analysis. Each dot represents one individual ( n =23). Box plots represent median (thick black lines), first and third quartiles (outlined boxes), the lowest data point still within 1.5 times the interquartile range from the first quartile (lower whiskers) and the highest data point still within 1.5 times the interquartile range from the third quartile (upper whiskers) of the expression levels of UCP1 , PPARGC1A , CIDEA , PRDM16 , S HOX2 and HOXC8 in subcutaneous and mediastinal adipose tissue. Gene expression was normalized to reference gene PPIA . P -values were calculated according to Wilcoxon paired-sample test.

Article Snippet: RNA samples from a separate group of 23 patients (see description in ) were used for complementary DNA synthesis with SuperScript III (Invitrogen) and analysed with TaqMan gene expression assays ( PPIA : Hs99999904_m1, TBP : Hs00427620_m1, UCP1 : Hs00222453_m1, PRDM16 : Hs00223161_m1, COBL: Hs00391205_m1, CIDEA : Hs00154455_m1, PPARGC1A : Hs00222453_m1, SHOX2 : Hs00243203_m1 and HOXC8 : Hs00224073_m1; Applied Biosystems, Foster City, CA, USA).

Techniques: Comparison, Gene Expression, Real-time Polymerase Chain Reaction, Biomarker Discovery, Microarray, Expressing

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet: T cell-depleted tumors and maturation trajectories of human DC vaccines (A) CIBERSORT deconvolution across TCGA cancer types. Population abundances were row normalized (C1, n = 1,313; C2, n = 1,210, C3, n = 688; C4, n = 222, C5, n = 2; C6, n = 111). (B) Overall survival of cancer patients’ transcriptome profiled before ICBs treatment (anti-PD-1/CTLA4/PD-L1 ICBs, or combinations thereof) sub-grouped in T cell-depleted C4/C5 tumors (n = 667) and immunogenic C2/C3/C6 tumors (n = 474). Statistics: log rank test. (C) GISTIC 2.0 analysis with indicated 12 genes. Statistical significance: false discovery rate (FDR) < 0.05 (random permutations to background score distribution, BH adjusted). Bladder cancer, n = 136; breast cancer, n = 880; colorectal adenocarcinomas, n = 585; glioblastoma multiforme, n = 580; head and neck cancer, n = 310; kidney cancer, n = 497; acute myeloid leukemia, n = 200; lung adenocarcinoma, n = 357; lung squamous cell carcinoma, n = 344; ovarian cancer, n = 563; endometrial cancer, n = 496. (D–J) Single-cell trajectory reconstruction exploration and mapping (STREAM) DC vaccine trajectory of 93 DC vaccines from 18 prostate adenocarcinoma patients vaccinated with five to eight vaccines. (D) Overview of STREAM DC vaccine trajectory. (E and F) Pseudo-time inferred from DC vaccines’ transcriptome based on variable genes. Principal graph initiated with epg_alpha = 0.01, epg_mu = 0.2, epg_lambda = 0.03, and epg_n_nodes = 5. Dots depict individual DC vaccines and dot color represents (E) patient number or (F) DC vaccine batch/cycle (chi-squared test of independence of variables). (G and H) Signature scores overlaid on the graph as streamplots. Type I IFN/ISG-response signature (G) or mature regulatory DC signature (H) were used as color intensity. (I and J) Patient outcomes were overlaid on the graph as streamplots. PSA doubling time at week 48 (I) and intensity of IFNγ production of peripheral blood mononuclear cell after antigen restimulation (J) were used as color intensity. Here, “n” represents different patients (biological replicates). See also Figure S1 .

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Vaccines

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet: Optimization of DCvax-IT for T cell-depleted tumors (A) Metagene expression for indicated signatures in different subcutaneous tumors (from GEO: GSE85509 ). (B) Flow cytometry analysis of CD45 + fraction from subcutaneous MC38/TC1 tumors on day 23 after injection (percentage of CD8 + of CD3 + cells, n = 6; two-tailed Student’s t test). (C) Tumor volume of TC1-tumor-bearing mice treated with anti-PD-1/CTLA4 on day 9/16 after injection (n = 6; area under curve; one-way ANOVA, Kruskal-Wallis test). (D) Survival of WT, Ripk3 −/− , and Mlkl −/− TC1 cells 24/48 h after treatment (three or four repeats). (E) Cell death of WT and Mlkl −/− TC1 cells 48 h after treatment. p values depict comparison WT vs. Mlkl −/− TC1 cells (n = 3; two-way ANOVA, Sidak’s multiple comparisons test). (F) Schematic overview of the vaccine formulation process. (G and H) Functional analysis of DCs untreated or stimulated with LPS, IFNβ, or with untreated or dying TC1s (with/without IFNβ). (G) Flow cytometry of DC maturation (MHCII + CD86 + frequency of CD11c + ). p values depict comparison vs. UT DCs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (H) IFN-signature expression (qPCR). p values depict comparison vs. UT DCs (n = 3; one sample t test). (I) Flow cytometry of frequency of PD-L1 + PD-L2 + CD200 + of CD11c + cells (moDCs alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells). p values depict comparison vs. UT moDCs (n = 4, LPS/IFNβ n = 3; one-way ANOVA, Fischer least significant difference [LSD]). (J) Flow cytometry of frequency of CD11b + F4/80 + in moDCs (alone/cocultured with untreated/dying WT/ Mlkl −/− TC1 cells) or bone-marrow-derived macrophages (BMDMs). p values depict comparison vs. BMDMs (n = 3; one-way ANOVA, Dunnett’s multiple comparisons test). (K) Cytokine secretion via cytokine array. From all values, the background was subtracted. Normalization was done using moDCs + untreated cancer cells (n = 3). Here, “n” represents biological replicates and error bars represent SEM. See also Figures S2 and .

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Expressing, Flow Cytometry, Injection, Two Tailed Test, Comparison, Formulation, Functional Assay, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Lymph node and tumor-associated PD-L1 + macrophages antagonize dendritic cell vaccines by suppressing CD8 + T cells

doi: 10.1016/j.xcrm.2023.101377

Figure Lengend Snippet:

Article Snippet: For DC vaccine creation, bone marrow derived DCs were stimulated with dying cancer cells in a 1:1 ratio or with TC1 antigens (i.e., Human Papillomavirus (HPV) e6/e7 epitopes: VYDFAFRDL/DKKQRFHNI, RAHYNIVTF/LCVQSTHVD)), with or without 2.5 ng/mL interferon beta (IFNβ) (R&D systems #8234-MB-010) for 48h.

Techniques: Control, Recombinant, Lysis, Protease Inhibitor, Western Blot, Staining, Stripping, Liposomes, CRISPR, MTS Assay, ATP Assay, Reverse Transcription, Cell Isolation, Enzyme-linked Immunosorbent Assay, Conjugation Assay, Selection, Drug discovery, Vaccines, Single-cell Analysis, RNA Sequencing, Mutagenesis, Microarray, Purification, Software