Journal: Journal of Clinical Investigation

Article Title: STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

doi: 10.1172/jci60083

Figure Lengend Snippet: Figure 3 CD14+HLA-DR–/lo MDSC from blood, LNs, and tumor suppress autologous T cell proliferation and express high levels of pSTAT3, ARG1, and ROS. (A) Circulating CD14+HLA-DR–/lo cells suppress autologous T cell proliferation at a 2:1 T cell/MDSC ratio. Tumor and LN MDSC showed a greater level of suppressive activity, particularly at 2:1 and 1:1 ratios (T cell/MDSC) (*P < 0.05). (B) CD14+HLA-DR–/lo MDSC from blood, LNs, and tumor express high levels of pSTAT3, ARG1, and ROS (DHE-stained cells). Original magnification, ×200. (C) MDSC from PB, LNs, and tumor have differential expression of pSTAT3 and ARG1, with higher pSTAT3 and ARG1 expression levels noted in the tumor in comparison with PB. The percentages are calculated by relative abundance with respect to total CD14+HLA-DR–/lo cells (**P < 0.01).

Article Snippet: For ARG1 rescue assay, CD14+HLA-DR–/lo cells untreated or treated with Stattic were incubated with recombinant human ARG1 (R&D systems) at varying doses (25–100 nM) overnight and then assayed for suppressor activity as described.

Techniques: Activity Assay, Staining, Quantitative Proteomics, Expressing, Comparison

Journal: Journal of Clinical Investigation

Article Title: STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

doi: 10.1172/jci60083

Figure Lengend Snippet: Figure 5 Inhibition of pSTAT3 decreases the expression and the activity of ARG1 on CD14+HLA-DR–/lo MDSC. (A) Inhibition of STAT3 signaling on CD14+HLA-DR–/lo MDSC by Stattic (10 μM) or siSTAT3 appropriately decreased the level of pSTAT3 (*P < 0.05). (B) Intracellular level of ARG1 is decreased with 2 independent methods of STAT3 signaling inhibition. y axis shows MFI (*P < 0.05). (C) ARG1 activity of circulating and tumor-infiltrating CD14+HLA-DR–/lo MDSC after pSTAT3 inhibition with Stattic (**P < 0.01).

Article Snippet: For ARG1 rescue assay, CD14+HLA-DR–/lo cells untreated or treated with Stattic were incubated with recombinant human ARG1 (R&D systems) at varying doses (25–100 nM) overnight and then assayed for suppressor activity as described.

Techniques: Inhibition, Expressing, Activity Assay

Journal: Journal of Clinical Investigation

Article Title: STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

doi: 10.1172/jci60083

Figure Lengend Snippet: Figure 6 STAT3 binds to the promoter region of ARG1 of MDSC. ChIP assay demonstrated pSTAT3 binding to ARG1 promoter regions at 3 of the 6 potential binding sites (from a total of 12 sites matching the consensus sequences generated by Vista genomic program). The sequence of the human ARG1 promoter region with the 6 potential pSTAT3-binding sites is shown in Supplemental Table 2.

Article Snippet: For ARG1 rescue assay, CD14+HLA-DR–/lo cells untreated or treated with Stattic were incubated with recombinant human ARG1 (R&D systems) at varying doses (25–100 nM) overnight and then assayed for suppressor activity as described.

Techniques: Binding Assay, Generated, Sequencing

Journal: Journal of Clinical Investigation

Article Title: STAT3 regulates arginase-I in myeloid-derived suppressor cells from cancer patients

doi: 10.1172/jci60083

Figure Lengend Snippet: Figure 7 Suppressive function of MDSC can be rescued by adding back ARG1 to STAT3-blocked MDSC. (A) Addition of vary- ing concentrations of human recombinant ARG1 to MDSC treated with Stattic rescued the suppressive function of the CD14+HLA-DR–/lo MDSC (**P < 0.01). Addition of ARG1 without STAT3 inhibition did not affect the suppressive func- tion of intact MDSC. T cell/MDSC ratio was 2:1. (B) Both

Article Snippet: For ARG1 rescue assay, CD14+HLA-DR–/lo cells untreated or treated with Stattic were incubated with recombinant human ARG1 (R&D systems) at varying doses (25–100 nM) overnight and then assayed for suppressor activity as described.

Techniques: Recombinant, Inhibition

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 2. Artemin Is Preferentially Expressed by Ter-Cells (A) cDNA microarray assay was performed to compare transcripts of Ter-119+CD45 Ter-cells and CD45+ splenocytes from the spleen of Hepa-bearing mice 4 weeks post inoculation (left). Ten most increased genes in Ter-cells are shown (right). (B) Artemin expression was confirmed by qRT-PCR. (C) Purified Ter-cells were cultured in vitro for 24 hr and artemin in the supernatants was determined by ELISA. (D) Artemin expression in Ter-cells was examined by western blot. (E) Immunofluorescence analysis of Ter-119 or artemin staining in splenocytes from Hepa HCC-bearing mice and DEN-induced HCC mice. Scale bars, 5 mm.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: Microarray, Expressing, Quantitative RT-PCR, Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 3. Serum Artemin Is Increased and Correlated with Poor Prognosis in HCC Patients (A) Serum level of artemin in healthy donors, patients with chronic hepatitis B (CHB), liver cirrhosis (LC), and HCC patients from four independent cohorts (HCC1– HCC4) were determined by ELISA. The horizontal lines in the boxplots represent the median, the boxes represent the interquartile range, and the whiskers represent the 2.5th and 97.5th percentiles. The p values were calculated using Student’s t test as shown. (B) The serum level of artemin derived from HCC patients in cohorts 3 and 4 before surgery and 7 days after curative resection was detected by ELISA. The p values were calculated using Student’s t test as shown. (C) The correlation of serum artemin and serum TGF-b in HCC patients of cohorts 3 and 4 was analyzed by Pearson’s correlation coefficient assay with R and p values indicated. (D) The serum level of TGF-b derived from HCC patients in cohorts 3 and 4 before surgery and 7 days after curative resection was detected by ELISA. The p values were calculated using Student’s t test as shown.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 4. Increased Artemin Receptor GFRa3 Expression and Its Signaling in HCC Tissue Is Correlated with Poor Prognosis in HCC Patients (A) The protein levels of GFRa3 and phosphorylated RET in human normal liver tissues, para-HCC tissues, and HCC tissues were detected by IHC and measured by defining regions of interest (ROI) using automated cell acquisition and quantification software for IHC. (B) The correlation between serum level of artemin in HCC patients and the expression of its receptor GFRa3 in HCC tissues derived from cohorts 1 and 2 was analyzed by Pearson’s correlation coefficient assay with R and p values indicated. (C) The correlation between serum level of artemin in HCC patients and RET phosphorylation in HCC tissues derived from cohorts 1 and 2 was analyzed by Pearson’s correlation coefficient assay with R and p values indicated. (D) The higher GFRa3 mRNA expression in HCC tissues is correlated with the reduced disease-free survival of HCC patients. Shown are Kaplan-Meier survival curves of disease-free survival in cohorts 1 and 2. The median value of GFRa3 mRNA expression in each cohort was chosen as the cutoff point, with log-rank test for significance. (E) The higher RET phosphorylation in HCC tissues is correlated with the reduced disease-free survival of HCC patients. Shown are Kaplan-Meier survival curves of disease-free survival in cohorts 1 and 2. The median value of RET phosphorylation in each cohort was chosen as the cutoff point, with log-rank test for significance. See also Tables S4 and S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: Expressing, Software, Derivative Assay, Phospho-proteomics

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 5. Artemin Promotes HCC Progression Both In Vitro and In Vivo (A) HCC SMMC-7721 and HepG2 cells were stimulated with recombinant human artemin at indicated concentrations in rapamycin (10 ng/mL) for 24 hr, then cells were stained using propidium iodide (PI) and annexin V and analyzed by fluorescence-activated cell sorting (FACS). The annexin V-positive cells were regarded as apoptotic cells. (B and C) SMMC-7721 or HepG2 cells were pretreated with recombinant human artemin at indicated concentrations for 2 hr, then added to the upper compartment of the chamber and incubated for 12 hr (for migration assay) or 24 hr (for invasion assay). Cells adhered to the lower surface were fixed and stained, and the cell number was counted. (D and E) Hepa cells were treated with Ter-cells, Ter-cells plus neutralizing antibody against mouse artemin, or recombinant mouse artemin as indicated. Cells were treated with rapamycin, and apoptosis was determined using annexin V/PI staining (D). Cells invaded to the lower surface were fixed, stained, and counted as indicated (E). (F and G) After subcutaneous inoculation of HepG2 cells (F) or transplantation of SMMC-LTNM tumor tissues (G) into nude mice, recombinant human artemin was delivered by tail vein injection once every 2 days for 14 days, then tumor growth (upper graphs) and survival (lower graphs) of HCC-bearing nude mice were detected as indicated.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: In Vitro, In Vivo, Recombinant, Staining, FACS, Incubation, Migration, Invasion Assay, Transplantation Assay, Injection

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 6. Artemin Induces Phosphorylation of Caspase-9 at Thr125 and Upregulates Expression of TRIOBP, ITGB5 (A–E) Artemin increased GFRa3 expression and RET phosphorylation in SMMC-7721 cells as detected by qRT-PCR (A), western blot (B and C), immunofluo- rescence (D), and in SMMC-LTNM by IHC (E). IHC relative intensity: PBS-GFRa3, 12.4; PBS-p-RET, 38.8; artemin-GFRa3, 49.1; artemin-p-RET, 95.9. (F) Artemin activates MAPK and PI-3K pathways. The expression of ERK, p38, JNK, Akt, and their phosphorylation in SMMC-7721 cells stimulated with re- combinant human artemin were detected by western blot. (G) Artemin-induced phosphorylation of caspase-9 at Thr125 and phosphorylation of ERK in SMMC-7721 cells treated with DMSO or ERK inhibitor PD98059 as indicated. (H) Cell apoptosis of caspase-9 T125A mutant overexpressed SMMC-7721 cells with artemin stimulation and/or rapamycin treatment as examined by annexin V/PI staining. (I) qRT-PCR analysis of artemin-stimulated genes in SMMC-7721 cells treated with artemin (100 ng/mL) for 12 hr. (J) Cell invasion of SMMC-7721 cells with knockdown of the indicated genes, respectively. (K) qRT-PCR analysis of the indicated gene expression in SMMC-7721 cells treated with the indicated inhibitors respectively and artemin. Data are shown as mean ± SD (n = 4) or presented directly. Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figure S4 and Table S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: Phospho-proteomics, Expressing, Quantitative RT-PCR, Western Blot, Mutagenesis, Staining, Knockdown, Gene Expression

Journal: Cell

Article Title: Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression.

doi: 10.1016/j.cell.2018.02.061

Figure Lengend Snippet: Figure 7. Blockade of Artemin or Its Recep- tor GFRa3 Signaling Inhibits HCC Growth In Vivo (A and B) Blockade of artemin inhibited tumor growth and prolonged survival of tumor-bearing mice. After subcutaneous inoculation with HepG2 cells (A) or SMMC-LTNM tumor tissue (B) into nude mice, neutralizing antibody against mouse artemin or normal goat IgG were delivered by tail vein injection once every 2 days for 14 days. The tumor growth and survival of HCC-bearing mice are indicated. (C) Wild-type or artemin-knockout mice were administrated with DEN, and recombinant arte- min or neutralizing antibody against artemin were injected into the wild-type mice between 6–8 months post DEN administration as indicated. Tumor size and splenic Ter-cells on the last day of 8 months post DEN administration were shown. (D) Tumor growth was analyzed in Ctrl Hepa or artemin-knockout Hepa-bearing in artemin- knockout mice as indicated. (E) Tumor growth (left) and survival (right) of HCC- Hepa bearing mice with or without splenectomy as indicated. (F) Hepa tumor growth in splenectomized mice after in vivo administration of artemin, or adoptive transferring of artemin+/+ or artemin/ Ter-cells as indicated. (G) Silence of GFRa3 expression in HepG2 cells inhibited tumor growth in vivo. After subcutaneous inoculation with GFRa3 stably silenced HepG2 cells (shGFRa3 HepG2) or Ctrl silenced cells (Ctrl HepG2) into nude mice, the tumor growth and survival of HCC-bearing mice were observed as indicated. Data are shown as mean ± SD (n = 10). Similar results were obtained in three independent experiments. *p < 0.05; **p < 0.01. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER anti-Phospho-p38 MAPK (Thr180/Tyr182) antibody Cell Signaling Technology Cat #9215 anti-p38 antibody Cell Signaling Technology Cat #9212 anti-JNK antibody Cell Signaling Technology Cat #9252 anti-Phospho-SAPK/JNK (Thr183/Tyr185) antibody Cell Signaling Technology Cat #9251 anti-Phospho-Akt (Ser473) antibody Cell Signaling Technology Cat #4060 anti-Akt antibody Cell Signaling Technology Cat #9272 anti-Smad3 (pSer425) antibody Cell Signaling Technology Cat #9520 anti-Smad2 (pSer250) antibody Cell Signaling Technology Cat #3104 anti-b-actin antibody Cell Signaling Technology Cat #4967 anti-GAPDH antibody Cell Signaling Technology Cat #5174 anti-ITGB5 antibody Cell Signaling Technology Cat #3629 Bacterial and Virus Strains DH5a Transgen Biotech Cat #CD201 Rosetta Tiangen Biotech Cat #CB108 Chemicals, Peptides, and Recombinant Proteins N-Nitrosodiethylamine (DEN) Sigma-Aldrich Cat #N0258 collagenase IV Sigma-Aldrich Cat #C5138 Percoll Sigma-Aldrich Cat #P4937 DMSO Sigma-Aldrich Cat #D2650 PD98059 Calbiochem Cat #513000 SB203580 Calbiochem Cat #559389 SP600125 Calbiochem Cat #420119 Wortmannin Calbiochem Cat #681675 Trizol reagent Invitrogen Cat #15596-018 Rapamycin Sigma-Aldrich Cat #V900930 Recombinant Human artemin R&D Systems Cat #2589-AR/CF Recombinant Mouse artemin R&D Systems Cat #1085-AR/CF Mouse artemin neutralizing antibody R&D Systems Cat #AF1085 TGF-b neutralizing antibody Bio X Cell Cat #BE0057 Critical Commercial Assays Human artemin ELISA kit BlueGene Biotechnology Cat #E01A0032 Mouse artemin ELISA kit BlueGene Biotechnology Cat #E03A0032 the First Strand cDNA Synthesis kit Toyobo Cat #fsq-101 Vybrant Apoptosis assay kit Invitrogen Cat #V13242 Transwell permeable support CORNING Cat #3422 Corning matrigel invasion chamber CORNING Cat #354480 Deposited Data cDNA microarray data This paper GEO: GSE58468 cDNA microarray data This paper GEO: GSE72411 RNA-Seq data This paper GEO: GSE109429 Experimental Models: Cell Lines HepG2 Cell Bank of the Chinese Academy of Sciences N/A Hepa Cell Bank of the Chinese Academy of Sciences N/A (Continued on next page) Cell 173, 1–15.e1–e6, April 19, 2018 e2 Please cite this article in press as: Han et al., Tumor-Induced Generation of Splenic Erythroblast-like Ter-Cells Promotes Tumor Progression, Cell (2018), https://doi.org/10.1016/j.cell.2018.02.061

Techniques: In Vivo, Injection, Knock-Out, Recombinant, Transferring, Expressing, Stable Transfection