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: 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

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: 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: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

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: DCvax-IT fails against T cell-depleted tumors in a curative setup (A–C) Tumor-free survival of mice vaccinated with two prophylactic DC vaccines (day 0/7), followed by subcutaneous TC1 challenge. p values depict comparison vs. PBS-treated mice. (A) Comparison of indicated DC vaccines to PBS-treated mice (PBS, n = 5; all vaccines; n = 5, log rank, Mantel-Cox test). (B) Comparison of indicated DC vaccines to PBS-treated mice (PBS, n = 9; necroptotic/apoptotic DCvax-IT, n = 6; pro-inflammatory cytokine/hyper-inflammatory DC vaccines, n = 5, log rank [Mantel-Cox] test). (C) Comparison of indicated DC vaccines to PBS-treated mice (PBS, n = 6; apoptosis/necroptosis DCvax-IT, n = 5, log rank [Mantel-Cox] test). (D) TC1-tumor-bearing mice treated with DCvax-IT (day 9/16 after injection). Comparison to PBS-treated mice (n = 12, area under curve; Kruskal-Wallis test). (E and F) Flow cytometry analysis of CD45 + fraction from untreated/DCvax-IT-treated TC1 tumors (day 23 after tumor injection). Frequency of (E) CD8 + T cells or (F) CD8 + T cells to TAM ratio. Comparison to PBS-treated mice (UT, n = 3; necroptosis DCvax-IT, n = 4; apoptosis DCvax-IT, n = 3, one-way ANOVA, Dunnett’s multiple comparisons test). (G) Frequency of Celltracker CM-Dil + CD11c + cells in LNs of vaccinated mice. p values depict comparison vs. PBS-treated mice (UT, n = 4, necroptosis/apoptosis DCvax-IT, n = 6, one-way ANOVA, Kruskal-Wallis test). (H) TC1-tumor-bearing mice treated with cisplatin (day 9/16) alone or in combination with DCvax-IT (day 11/18) and after TC1-injection. p values depict comparison vs. cisplatin-treated mice (n = 8; area under curve, one-way ANOVA, Dunnett’s multiple comparisons test). (I) MC38-tumor-bearing mice treated with DCvax-IT (day 9/16) after MC38-injection. p values vs. PBS-treated mice (PBS, n = 8; apoptosis DCvax-IT, n = 10, area under curve, Mann-Whitney test). Here, “n” represents biological replicates and error bars represent SEM. See also Figure S3 .

Article Snippet: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

Techniques: Vaccines, Comparison, Injection, Flow Cytometry, MANN-WHITNEY

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: TC1 tumors enrich CD8 + T cell-suppressive PD-L1 + macrophages (A) Volcano plot of gene expression between MC38 and TC1 tumors (GEO: GSE85509 ). (B) Uniform manifold approximation and projection (UMAP) of untreated TC-1 tumor scRNA-seq data (GSM7103827). (C) Inferred cell-cell communication by CellChat from dataset in (B) (bandwidth indicates intensity of cell-to-cell communication). (D) Macrophages as density over expression of indicate gene levels from dataset in (B). (E) CD45 + cell fraction from TC1 tumors (day 23 after injection). Frequency of TAMs (n = 6; two-tailed paired t test). (F) Flow cytometry analysis of PD-L1 + , CSF1R + , CD206 + (gating on unstained samples) on TAM from TC1 tumors isolated on day 23 post injection. (G) UMAP of TC1-tumor scRNA-seq data indicating normalized Cd274 expression (log1p-transformed reads per 10,000). (H–J) Flow cytometry of T cell recovery after cocultures with TAMs from TC1 tumors (day 23 post injection), pre-incubated with/without anti-PD-L1 for 48 h, together with paired spleen-derived T cells. (H) TAM/T cell coculture experimental setup. (I and J) Frequency of (I) CD8 + T cells and (J) CD4 + T cells (n = 3; two-tailed paired t test). (K) Relative information flow (CellChat) of Cd274 + and Cd274 − macrophages (1,006 cells) from dataset in (B). (L) TNF, TRAIL, FASLG expression of indicated TAMs from TC1 tumors (isolated on day 23 post injection) (n = 5; two-way ANOVA; Sidak’s multiple comparisons test). (M) Flow cytometry analysis of live T cell recovery, as per experimental setup in (H) and (J) but pre-incubated with/without anti-TNF/anti-TRAIL for 48 h, together with paired spleen-derived T cells (n = 5; two-way ANOVA). (N) Flow cytometry analysis of Efluor 780 + dead/dying cell in untreated/anti-PD-L1-treated TC1-derived TAMs (isolated on day 23 post injection) (n = 4; two-tailed paired t test). (O) DGE of Cd274 + macrophages (blue) vs. Cd274 − macrophages (red) from dataset in (B). The x axis: log2 fold change of PD-L1 + to PD-L1 − . Size of circles: −log10-transformed p values. (P) Percentage TAM survival from TC1 tumors (day 23 post injection) treated with different inhibitors (n = 4; one-way ANOVA; Dunnett’s multiple comparisons test). Here, “n” represents biological replicates. See also Figure S4 .

Article Snippet: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

Techniques: Gene Expression, Over Expression, Injection, Two Tailed Test, Flow Cytometry, Isolation, Expressing, Transformation Assay, Cell Recovery, Incubation, 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: DCvax-IT-mobilized PD-L1 + macrophages in tumors are blunted by DCvax-IT and anti-PD-L1 ICB (A–C) Tumor infiltrating leukocyte (TIL) analysis of CD45 + fraction from TC1 tumors (isolated on day 23 post injection) treated with DCvax-IT (day 9/16) and/or anti-PD-L1 (day 10/17). (A) Percentage of TAMs (CD11b + F4/80 + ), (B) percentage of PD-L1 + TAMs, (C) CD8 + -to-TAM ratio. (A–C) p values depict comparison vs. PBS-treated mice unless otherwise specified (n = 3–9; Mann-Whitney test). (D–K) TIL analysis of CD45 + fraction from TC1 tumor (day 23 post injection) treated with DCvax-IT (day 9/16) with/without anti-PD-L1 (day 10/17). Normalized by tumor-weight. (D) CD8 + T cells-to-TAM ratio. (E) Th1-to-Th2 ratio. (F) KI67 + CD8 + -to-dead CD8 + ratio. (G) IFNγ + CD8 + T cells. (H) IL2 + CD8 + T cells. (I) CD127 + CD62 − CD8 + T cells. (J) CD107a + CD8 + T cells. (K) TCF + CD8 + cells. (D, E, F, and I–K) p values depict comparison vs. PBS-treated mice unless otherwise specified (n = 3–5; Mann-Whitney test). (G and H) p values depict comparison vs. PBS-treated mice unless otherwise specified (n = 3–4; one-way ANOVA, Kruskal-Wallis test). (L–N) TC1-tumor-bearing mice treated with DCvax-IT (day 9/11), with anti-PD-L1 (day 10/11) and with anti-CD8 1 day pre-injection and every other day until 500 mm 3 . p values depict comparison vs. PBS-treated mice unless otherwise specified. (M) %MHCII low CD206 high of CD11b + F4/80 + in CD45 + fraction from TC1 tumor (day 23 post injection) (n = 3; two-tailed Student’s test). (N) Tumor volume curve (n = 7; area under curve; one-way ANOVA, Kruskal-Wallis test). (O) TC1-tumor-bearing mice treated with DCvax-IT (day 9/16) and/or anti-PD-L1 ICB (day 10/17) in combination with anti-IFNγ antibody (day 8, 12, 15, 19, 22). p values depict comparison vs. PBS-treated mice (n = 4, area under curve, one-way ANOVA). Here, “n” represents biological replicates and error bars represent SEM. See also Figure S6 .

Article Snippet: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

Techniques: Isolation, Injection, Comparison, MANN-WHITNEY, Two Tailed Test

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: PD-L1 + macrophages are mobilized by DC vaccines in GBM patients (A) Expression of CD274 / cd274 (PD-L1) across indicated datasets (n = 287 patients). (B) Correlation between CD274 vs. M1/M2 macrophage fraction in TCGA cancer types (C1, n = 1,313; C2, n = 1,210, C3, n = 688; C4, n = 222, C5, n = 2; C6, n = 111). (C) Z scores of CoxPH regression of CD274 HIGH macrophages HIGH subgroups, correcting for age, gender, tumor-stage (bladder cancer/BLCA, n = 408; breast cancer/BRCA, n = 1,100; colon adenocarcinoma/COAD, n = 458; GBM, n = 153; head and neck cancer human papillomavirus − /HNSC-HPV − , n = 422; head and neck cancer human papillomavirus + /HNSC-HPV + , n = 98; kidney chromophobe/KICH, n = 66; kidney renal clear cell carcinoma/KIRC, n = 533; kidney renal papillary cell carcinoma/KIRP, n = 290; low-grade glioma/LGG, n = 516; liver cancer/LIHC, n = 371; lung adenocarcinoma/LUAD, n = 515; lung squamous cell carcinoma/LUSC, n = 501; ovarian cancer/OV, n = 303; pancreatic adenocarcinoma/PAAD, n = 179; pheochromocytoma/PCPG, n = 181; prostate adenocarcinoma/PRAD, n = 498; rectum adenocarcinoma/READ, n = 166; sarcoma/SARC, n = 260; melanoma/SKCM, n = 471; stomach adenocarcinoma/STAD, n = 415; thyroid carcinoma/THCA, n = 509; uveal melanoma/UVM, n = 80, Mantel-Cox test). (D and E) log2(metagene expression) of CD274, CD163, CD14, and CD68. (D) Responders vs. non-responders to anti-PD-L1 (atezolizumab/durvalumab) (responders, n = 185 and non-responders, n = 269, where ureter/renal pelvis cancer n = 4, urothelial cancer n = 345, bladder cancer n = 31, esophageal cancer n = 72, renal cell carcinoma n = 2; Mann-Whitney U test). (E) Responders vs. non-responders to anti-PD-1 (nivolumab/pembrolizumab) (responders, n = 183 and non-responders, n = 323, where lung cancer n = 19, GBM n = 19, ureter/renal pelvis cancer n = 7, gastric cancer n = 45, colorectal cancer n = 5, melanoma n = 415, bladder cancer n = 59, hepatocellular carcinoma n = 22, breast cancer n = 14, renal cell carcinoma n = 31, head and neck cancer n = 110; Mann-Whitney U test). (F–J) Analysis of CD45 + fraction of primary and DC vaccinated GBM patients (NCT03395587). Tumor material from day of resection at first diagnosis (primary) or at recurrence after vaccination. (F) Overview of NCT03395587. (G) Frequency of CD4 + /CD8 + of CD3 + cells. (H) Frequency of IFNγ + of CD4 + /CD8 + CD3 + T cells. (G and H) Primary, n = 6; progress vaccine, n = 5, two-way ANOVA, Bonferroni’s multiple comparison. (I) Mean fluorescent intensity of CD163 on CD14 + cells. Primary, n = 15; recurrent DC vaccine, n = 5; two-tailed Student’s t test. (J and K) Immunohistochemistry of tumor slide from unvaccinated and DC vaccinated GBM patients (NCT03395587). (J) Representative images. (K) Correlation between TAM and T cell counts (n = 37 tumor regions from eight unvaccinated/vaccinated, Spearman’s correlation). (L) Mean fluorescent intensity of PD-L1 on CD14 + cells. Primary, n = 15; recurrent DC vaccine, n = 5; two-tailed Student’s t test. (M) Bromodeoxyuridine incorporation in cocultures of PBMC-derived lymphocytes (CD14 depleted PBMC) and TAMs obtained from primary GBM samples with/without anti-PD-L1 blocking (n = 3; area-under-curve-driven two-tailed paired t test). Here, “n” represents different patients (biological replicates) and error bars represent SEM. See also Figure S7 .

Article Snippet: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

Techniques: Vaccines, Expressing, MANN-WHITNEY, Biomarker Discovery, Comparison, Two Tailed Test, Immunohistochemistry, Derivative Assay, Blocking 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: anti-human CD45 microbeads , Miltenyi Biotec , Cat#130-045-801.

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

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: T cells and THP1 cells show differential responses to arginine starvation (A) Left: growth of stimulated CD4 + human T cells in complete (+Arg) or arginine-free medium with (−Arg +Citr) or without (−Arg) citrulline. Data are represented as mean ± SD; n = 4. Right: naive, central memory (Tcm), and effector memory (Tem) T cells (see Figure S1 D for sort strategy) were stimulated and then incubated in the indicated media for 96 h and counted. Data are fold increase over cell number at 0 h; mean ± SD; n = 2. (B) Growth of THP1 cells in the indicated media. Data are represented as mean ± SD; n = 4. (C) Concentration of citrulline in the blood plasma of healthy (control) and plasma or bone marrow of AML patients. Center bar shows mean ± SD. ∗∗∗∗ p < 0.0001 (unpaired t test). (D) Microarray analysis of mRNA in THP1 or stimulated T cells incubated in +Arg or −Arg medium for 72 h. Each column represents a replicate. Class assignments (I–VI) for genes are indicated. (E) Overlap of differentially expressed genes in T cells and THP1 cells, with class assignments (I–VI) indicated. (F) Analysis of KEGG pathway enrichment within each class of differentially expressed genes following arginine starvation, shown in (D). Dot size is proportional to significance (Wallenius method). See also Figure S1 and and .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Incubation, Concentration Assay, Clinical Proteomics, Control, Microarray

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: ATF4-induced ASS1 upregulation facilitates citrulline-dependent growth of THP1 cells (A) Key proteins in arginine uptake and biosynthesis. (B) Forest plot showing changes in gene expression, based on microarray analysis (see Figure 1 D). Horizontal bars show interquartile range. (C) ASS1 and ATF4 expression in primary AML blasts or non-transformed monocytic and myelocytic cells from healthy donors ( Quek et al., 2016 ). Samples are colored by donor. Bars show mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001 (Mann-Whitney test). (D) Western blot for ASS1 and ATF4 in stimulated T cells and THP1 cells incubated for 72 h in complete medium (+), medium containing 20 μM arginine (low), or lacking arginine (−). Two exposures of the ASS1 blot are shown for clarity. Representative of five replicates. (E) Quantification of (D), normalized to GAPDH, relative to +Arg T cells. Expression in T cells is shown on a smaller scale for clarity. Data are represented as mean ± SEM; n = 5. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (Dunnett’s multiple comparison test). ns, not significant. (F) Growth of control (NT) THP1 cells or following KD of ASS1 or ATF4 in the indicated media. Data are represented as mean ± SD; n = 4. (G) Representative western blot for ASS1 and ATF4 in control (NT) THP1 cells or following KD of ASS1 or ATF4 in the presence (+) and absence (−) of arginine for 72 h. Right: quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. ∗ p < 0.05 (Dunnett’s multiple comparison test). (H) CTV-labeled CD8 + T cells were transduced with a GFP - ASS1 coexpression plasmid. Cells were analyzed for GFP and CTV levels after 96 h in the indicated media. Three technical replicates are shown. (I) Proportion of GFP-positive cells from the analysis in (H), normalized to the −Arg ratio. Data are represented as mean ± SD; n = 5 from two donors. ∗∗ p < 0.01 (paired t test). See also Figure S2 .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Gene Expression, Microarray, Expressing, Transformation Assay, MANN-WHITNEY, Western Blot, Incubation, Comparison, Control, Labeling, Transduction, Plasmid Preparation

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: ATF4 activates ASS1 transcription via an intronic enhancer (A) Reference-normalized ChIP-seq for ATF4 and CEBPβ at ASS1 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h, and ChIP-seq for H3K4me3, H3K27ac, and H3K4me1 in THP1 cells in +Arg medium ( Godfrey et al., 2019 ). Gray bars show qPCR primer locations. (B) Reference-normalized ChIP-seq for ATF4 and CEBPβ at SLC7A1 , as in (A). (C) Sequences of the enhancer region in parental (wild type [WT]) and mutant THP1 cells. PAM sequences are underlined. (D) ChIP-qPCR for ATF4 and H3K27ac in WT and mutant THP1 cells, incubated in +Arg or −Arg medium for 72 h. Data are represented as mean ± SEM; n = 3. (E) Western blot for ASS1 and ATF4 in WT and mutant THP1 cells, incubated in +Arg or −Arg medium for 72 h. Representative of three replicates. (F) Growth of WT and mutant THP1 cells, incubated in the indicated media. Data are represented as mean ± SD; n = 3. See also Figure S3 .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: ChIP-sequencing, Incubation, Mutagenesis, ChIP-qPCR, Western Blot

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: ASS1 is repressed in T cells (A) ATAC-seq at ASS1 in THP1 cells incubated in the indicated media for 72 h. ATF4 ChIP-seq from −Arg cells is shown for comparison. Bottom: overlay of ATAC-seq traces at the highlighted region of ASS1 , mean of three replicates. (B) ATAC-seq at ASS1 in stimulated T cells, as in (A). (C) ChIP-qPCR for H3K9me3, H3K27me3, and H3K4me3 in stimulated T cells and THP1 cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 4. (D) ChIP-qPCR for H3K9me3, H3K27me3, and ATF4 in stimulated T cells incubated for 72 h in complete medium (+Arg), medium containing 20 μM arginine, without (low Arg) or with (low Arg + 2HG) addition of 500 μM 2HG, or lacking arginine (−Arg). Data are represented as mean ± SEM; n = 4. (E) Representative western blot for ASS1 and ATF4 in stimulated T cells incubated in the indicated media for 72 h. Non-specific bands are indicated by an asterisk. Right: quantification, normalized to GAPDH, relative to +Arg. Data are represented as mean ± SEM; n = 5. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001 (Dunnett’s multiple comparison test). (F) Model for ASS1 regulation in T cells and THP1 cells in response to arginine depletion. In THP1 cells accessibility at ASS1 allows ATF4 binding under low and −Arg conditions, inducing ASS1 expression. In T cells, ATF4 binding and ASS1 expression are regulated by two competing processes: ATF4 is active under low or −Arg conditions, but the ASS1 promoter is repressed. Under −Arg, elevated H3K9me3/H3K27me3 and reduced accessibility at ASS1 block ATF4 binding. See also Figure S4 .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Incubation, ChIP-sequencing, Comparison, ChIP-qPCR, Western Blot, Binding Assay, Expressing, Blocking Assay

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: ASS1 upregulation is a common tumor response to arginine starvation (A) Growth of tumor cell lines in the indicated media. AML, acute myeloid leukemia; APL, acute promyelocytic leukemia; ALL, acute lymphoblastic leukemia. Data are represented as mean ± SD; n = 4. (B) qRT-PCR for ASS1 in the indicated cell lines, cultured in +Arg and −Arg media. Data are normalized to GAPDH , relative to +Arg in each cell line, represented as mean ± SD; n = 4 or 6 (HeLa). ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 (Šidák’s multiple comparison test). (C) Representative western blot for ASS1 and ATF4 in control (NT) HeLa cells or following KD of ASS1 or ATF4 in the indicated media for 72 h. (Right) Quantification, normalized to GAPDH, relative to +Arg NT cells. Data are represented as mean ± SEM; n = 3. ∗ p < 0.05 (Dunnett’s multiple comparison test). (D) Growth of control (NT) HeLa cells or following KD of ASS1 or ATF4 , incubated in the indicated media. Data are represented as mean ± SD; n = 3. (E) ChIP-qPCR for ATF4 and CEBPβ levels in THP1 and HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3. (F) ChIP-qPCR for H3K9me3 and H3K27me3 in HeLa cells incubated in the indicated media for 72 h. Data are represented as mean ± SEM; n = 3. See also Figure S5 .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Quantitative RT-PCR, Cell Culture, Comparison, Western Blot, Control, Incubation, ChIP-qPCR

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet: Arginine-starved T cells show reduced ATF4/CEBPβ binding and chromatin accessibility (A) Left: number of ATF4 and CEBPβ peaks identified in ChIP-seq from stimulated T cells and THP1 cells incubated in the indicated media for 72 h. Right: overlap of ATF4 ChIP-seq peaks identified in T cells in low Arg or −Arg conditions. (B) Reference-normalized ATF4 (left) and CEBPβ (right) ChIP-seq levels at ATF4 peaks from T cells and THP1 cells incubated in the indicated media (colored lines). Mean level is displayed for T cell ATF4 peaks found only under low Arg conditions, only under arginine starvation, or under both conditions (common), as in (A). (C) Differential chromatin accessibility between stimulated T cells incubated in +Arg and −Arg medium. Red and blue dots indicate significantly increased and decreased ATAC peaks under −Arg; false discovery rate (FDR) < 0.05. (D) Chromatin accessibility (ATAC-seq) at T cell ATF4 peaks, as in (B). (E) Reference-normalized ATF4 and CEBPβ ChIP-seq levels at ATAC peaks from T cells incubated in the indicated media. Mean level is displayed for peaks that show reduced accessibility (more closed), increased accessibility (more open), or no change (unaffected) in arginine-starved T cells, as in (C). (F) Reference-normalized H3K27me3 ChIP-seq levels at T cell ATAC peaks, as in (E). (G) Reference-normalized H3K27me3 ChIP-seq levels at T cell ATF4 peaks, as in (B). See also Figure S6 .

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Binding Assay, ChIP-sequencing, Incubation

Journal: Cell Reports

Article Title: Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation

doi: 10.1016/j.celrep.2021.109101

Figure Lengend Snippet:

Article Snippet: Human tumor cell lines NB4 (female; Cancer Research UK; RRID CVCL_0005), MOLM13 (male; DSMZ, ACC-554; RRID CVCL_2119), RT112 (female; CRUK; RRID CVCL_1670), LNCaP (male; CRUK; RRID CVCL_0395), OCI-AML3 (male; DSMZ, ACC-582; RRID CVCL_1844), THP1 (male; ATCC, TIB-202; RRID CVCL_0006), HL60 (female; ATCC, CCL-240; RRID CVCL_0002) and RS4;11 (female; ATCC, CRL-1873; RRID CVCL_0093) cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum and GlutaMAX (ThermoFisher Scientific).

Techniques: Recombinant, Multiplex sample analysis, Cell Isolation, Activation Assay, Staining, Flow Cytometry, Expressing, Reverse Transcription, Transfection, TA Cloning, Plasmid Preparation, Methylation, Immunoprecipitation, Purification, DNA Library Preparation, Library Quantification, Control, Sequencing, Methylation Sequencing, Amplification, Software