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

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 epitope in ALK2 overlaps with BMP6 binding site, and the presence of RKER‐216 competitively inhibits BMP6 from binding to ALK2. (A, E) Binding affinity ( K D ) between ALK2 and RKER‐216 or ALK2 and BMP6 was determined by SPR. RKER‐216 diluted in HBS‐EP+ buffer was injected through a CM4 chip immobilized with ALK2‐Fc at 25 RU (A) while BMP6 diluted in HBS‐EP+ buffer with 30 nM arginine was injected through a CM4 chip immobilized with ALK2‐Fc at 350 RU (E). The results were analyzed by Biacore Insight Evaluation software using 1:1 binding model for RKER‐216 and steady‐state affinity for BMP6. The mean K D ± SEM from 3 separate experiments is reported and a representative sensogram is shown. (B) RKER‐216 binds to regions in ALK2 covering the F2 loop and β4 sheet (blue) as determined by HDX‐MS. The H/D exchange protected regions (blue) were mapped onto a model structure of ALK2 (PDB ID: 7YRU) shown in ribbon (left) and in surface (right) representation in complex with BMP6 structure (PBD ID: 2R52). The model of ALK2:BMP6 complex was generated by aligning individual structure of ALK2 and BMP6 into the structure of ALK1:BMP9 (PDB ID: 4FAO). The sequence alignment between ALK2 and ALK3 are shown (bottom), highlighting the binding region (in blue) of RKER‐216 in ALK2. (C‐D) BLI competition assays were performed in two ways. (C) ALK2 binding site was saturated with 5 nM of RKER‐216 prior to the competition with BMP6 at 0 to 100 nM. In addition to the vehicle control, BMP6 at 100 nM was added to unsaturated ALK2 to confirm binding ability of BMP6 in the competition. No binding activity was observed in the competition, indicating that BMP6 at 100 nM was unable to complete RKER‐216 away for binding ALK2. (D) ALK2 binding site was saturated with 100 nM of BMP6 prior to the competition with RKER‐216 at 0 to 50 nM. RKER‐216 at 50 nM was added to unsaturated ALK2 to confirm binding ability of RKER‐216. This group also served as a control for examining whether RKER‐216 binds to ALK2 or the ALK2‐BMP6 complex, as changes in the wavelength reflects both the size and affinity of the interactor. In the zoomed plot of RKER‐216 competition, data were aligned to zero on the Y ‐axis prior to the competition step for visual comparison, and 50 nM RKER‐216 without saturation had the greatest wavelength shift, indicating that RKER‐216 dislodged BMP6 from ALK2 instead of binding to the ALK2‐BMP6 complex. Experiments were repeated 3 times and a representative sensogram is shown.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: Binding Assay, Injection, Software, Generated, Sequencing, Control, Activity Assay, Comparison

Journal: American Journal of Hematology

Article Title: A Recombinant Antibody Against ALK2 Promotes Tissue Iron Redistribution and Contributes to Anemia Resolution in a Mouse Model of Anemia of Inflammation

doi: 10.1002/ajh.27578

Figure Lengend Snippet: RKER‐216 decreases hepcidin transcription in vitro and controls iron availability by lowering hepcidin secretion in vivo. (A) Hep3B cells were serum starved with 1% FBS overnight and incubated with an ascending dose of RKER‐216 (0.02–1 μg/mL) in the absence or presence of 5 ng/mL of BMP6, BMP2/6, or BMP2 for 6 h ( n = 4–5 per group). (B) Characterization of human ACVR1 knockout in HepG2 and Huh7 cells by quantifying ACVR1 and BMPR1A transcript copy number ( n = 3 per group). (C) ACVR1 KO and the negative control cells were treated with RKER‐216 at 1–30 μg/mL for 6 h ( n = 3 per group). Relative HAMP mRNA levels were determined by qRT‐PCR and transcripts were normalized to an internal control RPL19 . The average of PBS control without ligand stimulation or ACVR1 wildtype without RKER‐216 was set to 1. For ligand preference, hepcidin stimulated by each ligand was set to 100%. Values represent mean ± SEM. Results were compared across RKER‐216 and BMP ligands by one‐ or two‐way ANOVA with Tukey's post hoc test. Means without a common superscript differ significantly ( p < 0.05) or * p < 0.05, *** p < 0.001 relative to untreated cells of the same genotype. (D‐G) B6N male mice at 8 weeks were treated with a single SC dose of isotype control or RKER‐216 at 3 mg/kg for times as indicated ( n = 5 per group). Serum was collected to quantify (D) RKER‐216 exposure and (E) serum hepcidin by ELISA, and (F) serum iron and (G) serum transferrin saturation (TSAT) by colorimetric assays. Values represent mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 relative to the isotype control mice of the same time point by Student's t ‐test.

Article Snippet: Briefly, the library was panned for antibodies recognizing the extracellular domain of human ALK2 (637‐AR; R&D Systems) with three rounds of enrichment and counter‐selected against the Fc control for depletion.

Techniques: In Vitro, In Vivo, Incubation, Knock-Out, Negative Control, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay