Journal: Cell reports

Article Title: A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

doi: 10.1016/j.celrep.2019.01.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: IRDye 680LT Goat anti-Rat IgG , LI-COR , Cat# 925-68076.

Techniques: Recombinant, In Vitro, Transfection, DNA Extraction, RNA Extraction, Purification, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Bradford Assay, Software

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: Immunity

Article Title: T cell-derived interleukin-22 drives the expression of CD155 by cancer cells to suppress NK cell function and promote metastasis

doi: 10.1016/j.immuni.2022.12.010

Figure Lengend Snippet:

Article Snippet: InVivoMAb rat IgG2a (RG7/1.30) , BioXcell , Cat# BE0251; RRID: AB_2687732.

Techniques: Purification, Control, Virus, Recombinant, Red Blood Cell Lysis, Transfection, Flow Cytometry, DNA Extraction, Gel Extraction, Plasmid Preparation, cDNA Synthesis, Cell Differentiation, Staining, Cell Isolation, Conjugation Assay, Sequencing, CRISPR, Retroviral, Software

Journal: STAR Protocols

Article Title: Protocol for detecting macrophage-mediated cancer cell phagocytosis in vitro and in vivo

doi: 10.1016/j.xpro.2022.101940

Figure Lengend Snippet: Gating strategy for in vivo phagocytosis assay A549 cells are used to construct a mouse subcutaneous xenograft model. The phagocytosis efficiency is represented by the percentage of GFP + F4/80 + CD11b + cells in total F4/80 + CD11b + cells.

Article Snippet: APC Rat Anti-Mouse CD11b (1:100) , SouthernBiotech , 1561-11.

Techniques: In Vivo, Phagocytosis Assay, Construct

Journal: STAR Protocols

Article Title: Protocol for detecting macrophage-mediated cancer cell phagocytosis in vitro and in vivo

doi: 10.1016/j.xpro.2022.101940

Figure Lengend Snippet:

Article Snippet: APC Rat Anti-Mouse CD11b (1:100) , SouthernBiotech , 1561-11.

Techniques: Recombinant, DNA Extraction, Red Blood Cell Lysis, Modification, Cell Culture, Software, Sterility, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Frontiers in molecular biosciences

Article Title: Exosomes derived from M2 macrophages induce angiogenesis to promote wound healing.

doi: 10.3389/fmolb.2022.1008802

Figure Lengend Snippet: FIGURE 1 The characterization of M2 and exosome derived from M2. (A) Schematic of PEG precipitation exosome extraction flow. A detailed description was shown in the Methods part. Created with BioRender.com. (B) Flow cytometry scatter plots of M2 representative surface biomarkers CD200R and CD206 expression level. With a 58.9% CD200R+/CD206+ expression level, these cells were verified as M2 cells. (C) qRT-PCR was used to test the expression levels of M2 markers, MRC1, and TGM2. (D) ELISA kit was used to test the concentration of IL-10 in the M0 and M2 cell culture medium. (E) TEM image of extracted exosomes. Exosomes were labeled by white arrows. (F) Nanoparticle Tracking Analysis (NTA) of exosomes. (G) Western blot was used to test exosome biomarkers expression. With a higher expression of TSG-101, CD63, CD9, and lower expression of Calnexin compared with normal cells, the extracted vesicles are verified as exosomes. (H) Representative immunofluorescence images of PKH26- labeled M2-EXO (red) internalized into HUVECs. Scale bar was shown in the images. Data was expressed as mean ± 3σ (n = 3); * = significant, *p < 0. 05; ** p < 0.01,***p < 0.005; ****p < 0.001, Student t-test.

Article Snippet: The VEGF ELISA Kits used in this project were all purchased from the R&D system (Human VEGF Quantikine ELISA Kit, DVE00; Mouse VEGF Quantikine ELISA Kit MMV00, Mouse IL-10 Quantikine ELISA Kit M1000B, Mouse IL-1 beta Quantikine ELISA Kit MLB00C, Mouse TNF-alpha Quantikine ELISA Kit MTA00B, and Mouse TGF-beta 1 Quantikine ELISA DB100C).

Techniques: Derivative Assay, Extraction, Flow Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Concentration Assay, Cell Culture, Labeling, Western Blot

Journal: Frontiers in molecular biosciences

Article Title: Exosomes derived from M2 macrophages induce angiogenesis to promote wound healing.

doi: 10.3389/fmolb.2022.1008802

Figure Lengend Snippet: FIGURE 2 M2-EXO promotes Angiogenesis. (A) ELISA assay used to test VEGF concentration in gradient exosomes treated HUVEC culture medium. The VEGF concentration increased with M2-EXO concentration from 0 μg/ml to 1.0 μg/ml and got the optimal concentration in the 0.5 μg/ml group. (B) CCK-8 assay was used to estimate HUVEC viability. (C) Flow cytometry was used to evaluate the percentage of EdU-positive cells. With a higher EdU positive cell count (46.6 ± 9.2%) than the control group (30.0 ± 5.7%), M2-EXO can increase DNA synthesis, indicating a proliferation improvement. (D) Wound healing assay used to test migration ability. Scratch distance and width closure was collected at 0 h, 12 h, and 24 h. The wound area collected at 0 h was qualified and standardized as 100%. After 12 h, the control cells migrated and covered 29.3 ± 3.7% of the wound area, while the exosome incubation cells covered 56.4 ± 3.6% of the wound area. 24 h later, exosomes co-incubated cells migrated 94.5 ± 8.1% of the wound area, significantly higher than the migration area of the control cells. (E) Tube formation assay was used to demonstrate the influence of M2-EXO on angiogenesis. ImageJ analyzed the number of branch points and the total tube length. M2-EXO incubated HUVECs had 9992.3 ± 745.8 sizes of entire tube and 76 ± 13.1 branch points, which proved the angiogenesis improvement ability of M2 exosomes. Exosomes derived from M2 was presented as EXO in figures. Data was expressed as mean ± 3σ (n = 3); * = significant, *p < 0.05; ** p < 0.01,***p < 0.005; ****p < 0.001, Student t-test.

Article Snippet: The VEGF ELISA Kits used in this project were all purchased from the R&D system (Human VEGF Quantikine ELISA Kit, DVE00; Mouse VEGF Quantikine ELISA Kit MMV00, Mouse IL-10 Quantikine ELISA Kit M1000B, Mouse IL-1 beta Quantikine ELISA Kit MLB00C, Mouse TNF-alpha Quantikine ELISA Kit MTA00B, and Mouse TGF-beta 1 Quantikine ELISA DB100C).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, CCK-8 Assay, Flow Cytometry, Cell Counting, Control, DNA Synthesis, Wound Healing Assay, Migration, Incubation, Tube Formation Assay, Derivative Assay

Journal: Frontiers in molecular biosciences

Article Title: Exosomes derived from M2 macrophages induce angiogenesis to promote wound healing.

doi: 10.3389/fmolb.2022.1008802

Figure Lengend Snippet: FIGURE 3 mTOR pathway participates in angiogenesis promotion induced by M2-EXO. (A) Western blot was used to test critical markers in the mTOR pathway. Treatment of M2-EXO increased phosphorylation of mTOR and p70S6K. (B) Western blot was used to test p-p70S6K expression. Rapamycin treatment counteracted the expression promotion of p-p70S6K induced by M2-EXO. (C) ELISA was used to test VEGF expression in Rapamycin treatment HUVEC culture medium. Treatment of Rapamycin counteracted the secretion promotion of VEGF induced by M2-EXO. (D) CCK-8 assay was used to demonstrate the promotion role of M2-EXO and the counteraction role of Rapamycin. After M2-EXO treatment, the viability of the cells was sharply increased, while after co-treated with Rapamycin, the viability of the cells was barely equal to the control group. (E) Flow cytometry (Continued)

Article Snippet: The VEGF ELISA Kits used in this project were all purchased from the R&D system (Human VEGF Quantikine ELISA Kit, DVE00; Mouse VEGF Quantikine ELISA Kit MMV00, Mouse IL-10 Quantikine ELISA Kit M1000B, Mouse IL-1 beta Quantikine ELISA Kit MLB00C, Mouse TNF-alpha Quantikine ELISA Kit MTA00B, and Mouse TGF-beta 1 Quantikine ELISA DB100C).

Techniques: Western Blot, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Control, Flow Cytometry

Journal: Frontiers in molecular biosciences

Article Title: Exosomes derived from M2 macrophages induce angiogenesis to promote wound healing.

doi: 10.3389/fmolb.2022.1008802

Figure Lengend Snippet: FIGURE 7 M2-EXO treatment promotes angiogenesis and tissue regeneration in the wound. (A–E) ELISA assay used to test TNF-α, IL-1β, IL-10, TGF-β, and VEGF concentration in on the 7th day. (n = 6). (F) Immune-fluorescence images were collected from tissue homogenization on the 14th day from the control group, and the M2-EXO treated group, respectively. Red dye was used to label CD31; Green dye was used to label α-SMA; DAPI was used to label the nucleus; Merge figure was presented by integrating all the figures. Scale bar was labeled in the figures. (G) Representative immunofluorescence figures selected from (F) followed by being enlarged. (H) Quantification analysis of the relative thickness of granulation tissue (n = 3). (I) Quantification analysis of α-SMA-positive staining area (n = 3) (J) Quantification analysis of vessel density in sarcomeres (n = 3) (K) Quantification analysis of the number of mature blood vessels in sarcomeres (n = 3). (L–M) Western blot was used to test the expression level of MMP-2, MMP-9, PTEN, p-AKT, AKT, mTOR, and p-mTOR in tissue homogenization collected on the 7th day. Data was expressed as mean ± 3σ; * = significant, *p < 0.05; ** p < 0.01,***p < 0.005; ****p < 0.001, Student t-test.

Article Snippet: The VEGF ELISA Kits used in this project were all purchased from the R&D system (Human VEGF Quantikine ELISA Kit, DVE00; Mouse VEGF Quantikine ELISA Kit MMV00, Mouse IL-10 Quantikine ELISA Kit M1000B, Mouse IL-1 beta Quantikine ELISA Kit MLB00C, Mouse TNF-alpha Quantikine ELISA Kit MTA00B, and Mouse TGF-beta 1 Quantikine ELISA DB100C).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Tissue Homogenization, Control, Labeling, Staining, Western Blot, Expressing

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 1 Ahnak regulates induction of adipocyte differentiation. (A) Epididymal fat pads of wild-type and Ahnak2/2 mice were stained with hematoxylin and eosin. Scale bar, 50 lm. Histological analysis is described in Supporting Information Methods. (B) Adipocyte sizes were quanti- fied using ImageJ software. (C) RT-PCR analysis of the mRNA expression of Ahnak during differentiation of 3T3-L1 (upper) and ADSCs (lower). Day (day 0 to day 8) means time course of adipocyte differentiation, and cells were harvested at each time point. Expression levels of each gene were normalized to the levels of GAPDH. Data are presented as mean 6 SD. (D) Ectopic expression of 4CRUs of Ahnak was confirmed by immunoblot analysis with antibody against Ahnak. Immunoblotting is described in Supporting Information Methods. (E) 3T3-L1 preadipocyte cell line overexpressing 4CRUs of Ahnak were induced to adipocyte after BMP2 (3 ng/mL) treatment for 24 h. For adipocyte differentiation, cells were treated with DMI containing 50 lM methyl-isobutylxanthine, 0.1 lM dexamethasone, and 0.1 lg/mL insulin in DMEM with 10% FBS at 2 days post-confluence. After 2 days, cells were cultured in the DMEM containing 10% FBS and insulin. Subsequently, medium was changed every second day. On day 8, the differentiated adipocytes were assessed by Oil Red O staining. (F) The number of adipocyte-differentiated 3T3-L1. Fold represents ratio of the number of differentiated 3T3-L1 with BMP2 to differentiated control without BMP2 (*P value<0.001).

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Staining, Software, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Cell Culture, Control

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 2 Reduction of Ahnak expression decreased adipogenesis. (A) C3H10T1/2 pluripotent cell lines were transfected with non-targeting or Ahnak-targeting siRNA (25 nM). Information for siRNA is described in Supporting Information Methods. (B) Cells were induced to adipocyte after BMP2 (10 ng/mL) pre- treatment for 24 h. Control or Ahnak siRNA were additionally treated every second day during adipocyte differentiation in order to maintain the effect of Ahnak knockdown in the cells. (C) The number of adipocyte-differentiated C3H10T1/2 cells. Fold represents ratio of the number of differentiated C3H10T1/2 with BMP2 to differentiated control without BMP2. (D) ADSCs from Ahnak wild-type or Ahnak2/2 mice were subjected to the adipocyte differentiation protocol after BMP2 (10 ng/mL) treatment. Rate of adipocyte differentiation was confirmed through Oil Red O which stains lipid droplet in differentiated, mature adipo- cytes. (E) The number of adipocyte-differentiated ADSCs. Fold represents ratio of the number of differentiated ADSCs with BMP2 to differentiated control without BMP2.

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Expressing, Transfection, Control, Knockdown

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 3 Ahnak interaction with Smad1 is increased by BMP2. (A) HEK293T cells were co-transfected with HA or HA-4CRUs as well as Flag-Smad1 and stimulated with BMP2 (10 ng/mL) for 30 min. Cell lysates were immunoprecipitated with anti- bodies against HA. Co-immunoprecipitation is described in Supporting Information Methods. Co-immunoprecipitation was analyzed through Western blot with indi- cated antibodies. (B) C3H10T1/2 pluripotent stem cells were co-transfected with HA or HA-4CRUs as well as Flag-Smad1 and stimulated with BMP2 for 30 min. Cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-HA antibody to detect 4CRUs of Ahnak.

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 4 Ahnak mediates the activation of Smad1 by BMP2. (A,B) 3T3-L1 preadipocyte cells overexpressing 4CRUs of Ahnak were stimulated with BMP2 (5 ng/mL) for 0, 0.5, 1, 2, and 3 h after serum starvation. Cell lysates were subjected to immunoblot analysis with antibodies against phospho-Smad1, Smad1, and actin, respectively. Establishment of 3T3-L1 stably expressing Ahnak and immunofluorescence staining are described in Supporting Information Methods. (C,D) C3H10T1/2 cells were transfected with indicated siRNA and stimulated with BMP2 (1 ng/ mL) for 0, 0.5, 1, 3, and 5 h after serum starvation. Cells were then fixed and stained with antibodies against Smad1. (E,F) Wild-type and Ahnak2/2 ADSCs were serum-starved and stimulated with BMP2 (1 ng/mL) for the indicated time. Then cells were fixed and stained with Smad1 antibody. Relative Smad1 nuclear translocation intensity in panels B, D, and F is obtained from Supporting Information Figure S2 confo- cal microscope images. Red fluorescent intensity of Smad1 was quantified with ImageJ software by selecting nuclear region (*P value<0.01, **P value<0.001).

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Immunofluorescence, Staining, Transfection, Translocation Assay, Microscopy, Software

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 5 Ahnak promotes Smad1 binding to promoter of PPARc. (A) Expression level of PPARc key transcription factor for adipocyte differentia- tion. The levels of PPARc mRNA during adipocyte differentiation of wild-type and Ahnak2/2 ADSCs were measured by RT-PCR. The levels (mean 6 SEM, n 5 9) are shown relative to preinduction levels of wild-type ADSCs (*P value<0.05). (B) C3H10T1/2 cells transfected with con- trol siRNA and Ahnak siRNA were serum-starved for 5 h. Then the cells were stimulated with BMP2 for 0, 1, 3, and 6 h. Soluble chromatin was prepared and immunoprecipitated with anti-Smad1 antibody or control IgG. The final DNA extraction was amplified by RT-PCR using pri- mers that cover the PPARc promoter region containing Smad-binding sites (*P value<0.05, **P value<0.01). (C–E) Expression levels of aP2, adiponectin, and CD36, which are PPARc target genes, were measured by RT-PCR during adipocyte differentiation of wild-type and Ahnak2/2

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Binding Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunoprecipitation, Control, DNA Extraction, Amplification

Journal: Obesity (Silver Spring, Md.)

Article Title: Ahnak stimulates BMP2-mediated adipocyte differentiation through Smad1 activation.

doi: 10.1002/oby.21367

Figure Lengend Snippet: Figure 6 Proposed model for the function of Ahnak protein in BMP2-mediated adipogenesis. BMP2- induced Smad1 signaling activation is regulated by Ahnak. Ahnak stimulates phosphorylation of Smad1 and promotes Smad1 nuclear translocalization. Smad1 binds SBE of the promotor region of PPARc and functions as a transcriptional factor. As a result, downstream signaling promoted by PPARc is positively regulated and adipocyte differentiation is increased.

Article Snippet: Recombinant Human BMP2 was purchased from R&D systems (355-BM).

Techniques: Activation Assay, Phospho-proteomics