Journal: Stem cell reviews and reports

Article Title: MSC secreted extracellular vesicles carrying TGF-beta upregulate Smad 6 expression and promote the regrowth of neurons in spinal cord injured rats.

doi: 10.1007/s12015-021-10219-6

Figure Lengend Snippet: Fig. 2 BMSC-EVs upregulated Smad 6 expression in NSCs via the secretion of TGF-β. A. To confirm whether IL-6, BMP4, and TGF-β existed in the BMSC-EVs, the expression of these factors was determined by PCR (n = 3, Student’s t-test). B. ELISA from five individual samples confirmed the presence of TGF-β and IL-6 in BMSC- EVs. C, D. To evaluate whether TGF-β or IL-6 played a key role in mediating the expression of Smad 6 in NSCs, we added SB431542 and JSH-23, respec- tively, to NSCs in the presence of BMSC-EVs. This revealed that the BMSC-EVs-induced upregulation of Smad 6 could be abolished by the addition of SB431542 (C, n = 5; the data were revealed as fold changes to control NSCs, Student’s t-test). Conversely, the addition of JSH- 23 did not alter the expression of Smad 6 in NSCs (D, n = 5; the data were presented as fold changes to control BMSC-EV treated NSCs, Student’s t-test) E. We added TGF-β to NSCs to assess the effect of TGF-β on mediating Smad 6 expression; this resulted in an increase of Smad 6 expression in NSCs. This TGF-β-induced upregula- tion of Smad 6 expression could be significantly abolished by the addition of SB431542 (n = 5; the data were presented as fold changes to control NSCs, Stu- dent’s t-test). (Smad 6 mRNA expression was normalized to GAPDH mRNA; the data were presented as mean ± S.D; *p < 0.05, **p < 0.01, ***p < 0.001, #p > 0.05.)

Article Snippet: Passage 2 NSCs or the Smad 6-knockdown NSCs were dissociated and reseeded on glass coverslips in 5% FBSDMEM/F12 for 24 h. The medium was then switched to DMEM/F12 supplemented with one of the following: BMSC- EVs or 10 ng/mL TGF-β (R&D Systems); BMSCEVs + 10 μM SB431542 [the TGF-β type I receptor kinase inhibitor (Sigma)]; BMSC-EVs + 20 ng BMP4; 10 ng/ mL TGF-β + 10 μM SB431542; 10 ng/mL TGF-β + 20 ng BMP4 (R&D Systems); 20 ng/mL IL-6 (Sigma), with or without 30 μM JSH-23 (NF-κB inhibitor, MCE); 20 ng/mL IL-6 + BMSC-EVs, with or without SB 431,542; 20 ng/mL BMP4, with or without 200 ng/mL Noggin [BMP- antagonist (Sigma)]; 20 ng/mL BMP4 + BMSC-EVs, with or without SB 431,542; 40 ng/mL IL-6 and 40 ng/mL BMP4, with 1 3 or without BMSC-EVs.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 1. DDX4 forms cytoplasmic granules in cancer cells (A) Of the 88 known components of the CB, 22 have also been identified as CGAs (the dotted rectangular box, DDX4 highlighted in red). (B) Immunostaining of different human epithelial tissues with an anti-DDX4 antibody. DDX4 granules are absent from the normal epithelial tissues, but present (black arrows) in the cytoplasm of cancer cells in breast, colon, and lung adenocarcinoma; scale bar: 20 mm. Selected cancer cells are highlighted in the insets; scale bar: 10 mm. (C) Immunostaining of fibrosarcoma and leiomyosarcoma tissues as examples of DDX4+ cancers that are not of epithelial origin. Selected DDX4 granules are indicated with black arrows. Scale bar: 20 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Immunostaining

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 2. DDX4 granules are found in cancer-cell-line-derived xenograft tumors (A) Immunohistochemistry of UT-SCC-14-derived xenograft tumors with an anti-DDX4 antibody. Selected DDX4 granules are indicated with black arrows. Scale bar: 20 mm. Smaller rectangular box: no primary antibody control. (B) Immunofluorescence of UT-SCC-14 cultured cells and tumors with an anti-DDX4 antibody (red), nuclei are stained with DAPI (gray). DDX4 granules (white arrows) appear in xenograft tumor cells. Neg. Ctrl: no primary antibody. Scale bar: 10 mm. (C) Immunofluorescence of cultured PC3 cells and PC3-derived tumors with an anti-DDX4 antibody (red). DDX4 granules (white arrows) can be detected in tumors. Scale bar: 10 mm. (D) DDX4 IP from PC3 tumors and cells (3 biological replicates each) followed by western blotting. Rabbit IgG was used as a negative control. Graph: DDX4 signal was quantified, and the signal intensities were normalized to the IgG light-chain signal (asterisk) (p = 0.0274, Mann-Whitney U test, 2-tailed). (E) Immunofluorescence of PC3 spheroids with an anti-DDX4 antibody (red). Anti-a-tubulin antibody (green) visualizes cytoplasmic protrusions. White arrows point to selected DDX4 granules. Scale bar: 20 mm. (F) Immunofluorescence of DDX4 (red) in non-treated and puromycin-treated PC3 cells. DAPI (gray) stains the nuclei. Scale bar: 10 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Derivative Assay, Immunohistochemistry, Control, Cell Culture, Staining, Western Blot, Negative Control, MANN-WHITNEY

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 3. DDX4 deletion compromises PC3 spheroid formation (A) The sequence of DDX4 gene after CRISPR-Cas9 in PC3 cells to visualize the deleted 103 nt. (B) Agarose gel of genomic PCR of WT and 2 DDX4-null PC3 cell clones (null1 and null2). (C) Proliferation curve of DDX4-null and WT PC3 cells. (D) Bar charts show the percentage of live cells, early apoptotic cells, and late apoptotic cells in WT and DDX4-null PC3 cells (3 replicates each). The chart at right shows only the early and late apoptotic cells. (E) Calcein AM-stained (green) WT and DDX4-null PC3 spheroids at days 5 and 10. Scale bar: 20 mm. (F) Area of spheroids formed by DDX4-null vs. WT PC3 spheroids (day 5: p = 0.000043, day 10: p = 0.000011, Mann-Whitney U test, 2-tailed). (G) Invasive processes (MaxApp in AMIDA) of DDX4-null vs. WT PC3 spheroids (day 5: p = 0.000011, day 10: p = 0.000022, Mann-Whitney U test, 2-tailed). (H) Immunofluorescence on WT and DDX4-null (number of replicates is 10 for each) PC3 spheroids with vimentin antibody (green). Nuclei were stained with DAPI (gray). Scale bar: 20 mm. (I) Calcein AM-stained (green) spheroids derived from WT and DDX4-null PC3 cells overexpressing either GFP or DDX4-GFP. Scale bar: 20 mm.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Sequencing, CRISPR, Agarose Gel Electrophoresis, Clone Assay, Staining, MANN-WHITNEY, Derivative Assay

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 4. DDX4 deletion compromises PC3 tumor formation and growth (A) The volume of subcutaneous DDX4-null vs. WT PC3 xenograft tumors; 10 biological replicates each. The tumor progression was followed weekly for 4 weeks (Wk1: p = 0.000411, Wk2: p = 0.000119, Wk3: p = 0.00105, Wk4: p = 0.006798, Mann-Whitney U test, 2-tailed). (B) The weights of dissected DDX4-null vs. WT PC3 tumors (p = 0.0232, Mann-Whitney U test, 2-tailed). (C) Immunofluorescence with an anti-DDX4 antibody (red) validated the absence of DDX4 in DDX4-null PC3 tumor cells. Nuclei were stained with DAPI (gray). Scale bar: 20 mm. (D) Immunofluorescence on DDX4-null and WT PC3 tumors with an anti-vimentin antibody (green). Scale bar: 10 mm. (E) Western blotting of 3 biological replicates of WT and DDX4-null PC3 tumors with an anti-vimentin antibody. b-Actin was used as the loading control. The graph shows the quantification of vimentin signal (normalized to b-actin) (p = 0.0309, Mann-Whitney U test, 2-tailed). Data are represented as mean ± SEM. See also Figures S1 and S2.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: MANN-WHITNEY, Staining, Western Blot, Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 5. Transcriptome is misregulated in DDX4-null PC3 cells and DDX4-null PC3 xenograft tumors (A) Hierarchical heatmaps show differentially expressed (|log2FC| R 1, adjusted p value %0.05) genes in DDX4-null vs. WT PC3 cells (left) and xenograft tumors (right). (B) Venn diagram shows the overlap between the genes upregulated in DDX4-null vs. WT PC3 xenograft tumors and cells. (C) Volcano plot shows the differential expression of tumor suppressor genes in DDX4-null vs. WT PC3 tumors, with significantly upregulated (green) and downregulated (orange) genes labeled. (D) Bar chart shows the log2FC (DDX4-null vs. WT tumors) of individually selected genes. (E) Bar chart shows the validation of the selected differentially expressed genes in DDX4-null vs. WT PC3 tumors (number of biological replicates is 3) by qRT-PCR (ADAM12: p = 0.0112, CDH6: p = 0.0161, CDH7: p = 0.0298, CEACAM1: p = 0.0468, SFRP1: p = 0.0293, and CLEC2B: p = 0.0105, Mann-Whitney U test, 2-tailed). (F) Western blotting image of CDH6, CDH1, and CDH2 protein expression in DDX4-null and WT PC3 tumors (3 biological replicates each). b-Actin was used as the loading control. Bar chart shows the quantification of the CDH1, CDH2, and CDH6 protein levels normalized by b-actin signal. CDH1: p = 0.0055, CDH6: p = 0.0004 (Mann-Whitney U test, 2-tailed, 3 biological replicates each). Data are represented as mean ± SEM. See also Figures S3 and S4 and Table S1.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Quantitative Proteomics, Labeling, Biomarker Discovery, Quantitative RT-PCR, MANN-WHITNEY, Western Blot, Expressing, Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 6. DDX4 deletion affects the splicing landscape of cancer cells (A) IP of PC3 tumors (2 biological replicates) with an anti-DDX4 antibody, followed by western blotting with the same antibody. IgG IP was used as the negative control. IgG light and heavy chains are indicated with asterisks. (B) GO term enrichment analysis of the proteins interacting with DDX4 in PC3 xenograft tumors. Bubble plot visualizes the most enriched biological processes selected based on the adjusted p values. Gene ratio: The number of proteins that are associated with the GO term divided by the total number of proteins. Count: represents the number of proteins annotated to the GO term; the size of a bubble dot reflects the number of proteins. (C) Venn diagram illustrating 17 shared components (listed in the rectangular box) between the CB and the DDX4-interacting proteins.

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Western Blot, Negative Control

Journal: Cell reports

Article Title: Germline-specific RNA helicase DDX4 forms cytoplasmic granules in cancer cells and promotes tumor growth.

doi: 10.1016/j.celrep.2024.114430

Figure Lengend Snippet: Figure 7. DDX4 has prognostic significance in human cancers (A) Immunohistochemistry of normal head and neck squamous epithelium (HNSE, 3 representative examples) and HNSCC (bottom) with an anti-DDX4 antibody. The cells were counterstained with hematoxylin. HNSCC samples without granular DDX4 staining were scored as negative, while HNSCC samples with DDX4 granules (black arrows) were scored as positive. Scale bar: 20 mm. Survival curve shows 5-year survival (days) for DDX4 (n = 12/46) and DDX4+ (n = 34/46) patients (p = 0.008, log rank test). (B) Immunofluorescence on PC samples with an anti-DDX4 antibody. PC samples without granular DDX4 staining were scored as negative, while the PC samples with prominent DDX4 granules (white arrows) were scored as positive. Scale bar: 20 mm. Survival curve shows the progression-free survival (months) for DDX4

Article Snippet: The supernatant fraction of the lysed tumor sample was first precleared with 15 mL of washed Dynabeads Protein G (10446293, Invitrogen), then the precleared lysate sample was subjected to immunoprecipitation using beads coupled with 4 mg of anti-DDX4 rabbit polyclonal antibody (51042-1-AP, ProteinTech) and negative control rabbit IgG.

Techniques: Immunohistochemistry, Staining

Journal: Stem cells international

Article Title: Developing a Novel and Convenient Model for Investigating Sweat Gland Morphogenesis from Epidermal Stem Cells.

doi: 10.1155/2019/4254759

Figure Lengend Snippet: Figure 4: Detection of key soluble morphogens in the system of sweat gland morphogenesis and the impact of BMP receptor inhibitor and EGF receptor inhibitor on sweat gland development in vitro. BMP4 and EGF demonstrated sharped difference in the medium of the sweat gland organogenesis system. BMP receptor inhibitor could block the formation of sweat gland in this system, while EGF receptor inhibitor significantly reduced the expression of K18. (a) The variation tendency of BMP4 and EGF in the medium of system. The error bar meant the standard error of BMP4 and EGF concentrations in different systems at different time points. (b) The impact of BMP receptor inhibitor and EFG receptor inhibitor on sweat gland morphogenesis. In comparison with the control group, EGF receptor inhibitor significantly reduced the expression of K18, but glandular structure was still observed. BMP receptor inhibitor completely blocks the expression of K18, and no glandular structure was observed. All the nuclei were counterstained with DAPI (DAPI: blue; K18: red; bars = 200 μm and 50 μm; K18: cytokeratin 18; IM: light microscope; H&E: hematoxylin-eosin staining; IF: immunofluorescence staining).

Article Snippet: BMP4 and EGF concentrations in supernatant were measured using a mouse BMP4 (CSB-E04512m, Cusabio, Wuhan, Hubei, China) and EGF ELISA kit (EM014-96, ExCell Bio, Shanghai, China).

Techniques: In Vitro, Blocking Assay, Expressing, Comparison, Control, Light Microscopy, Staining