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: Oncogenesis

Article Title: Neuron-specific enolase promotes stem cell-like characteristics of small-cell lung cancer by downregulating NBL1 and activating the BMP2/Smad/ID1 pathway.

doi: 10.1038/s41389-022-00396-5

Figure Lengend Snippet: Fig. 5 NSE downregulates and interacts with NBL1 in SCLC cells. A, B Western blot images showed the interaction between NSE and NBL1 by Co-IP experiment. C The double-immunofluorescence labeling experiment detected NSE and NBL1 and displayed co-location in clinical samples (original magnification ×40, scale bar 50 μm). D Western blot images show the interaction of BMP2 and BMPR1A enhanced after overexpression of NSE. E The photo shows a significant and negative correlation between ENO2 (encode NSE) and NBL1 in mRNA level using the CCLE database.

Article Snippet: The membranes were incubated with the following antibodies: NSE (1:2000, Abcam), GFP (1:2000, Proteintech), Flag (1:1000, Proteintech), OCT4 (1:1000, CST), Nanog (1:2000, CST), SOX2 (1:1000, CST), β-actin (1:5000, Proteintech), BMP2 (1:1000, Proteintech), Smad1 (1:2000, Proteintech), pSmad1/5/8 (1:1000, CST), and ID1 (1:500, Proteintech).

Techniques: Western Blot, Co-Immunoprecipitation Assay, Labeling, Over Expression

Journal: Function (Oxford, England)

Article Title: Deletion of Mechanosensory β1-integrin From Bladder Smooth Muscle Results in Voiding Dysfunction and Tissue Remodeling.

doi: 10.1093/function/zqac042

Figure Lengend Snippet: Figure 1. Characterization of the mouse model. (A) RT-PCR of β1-integrin from detrusor extracted mRNA at 6 wk after i.p. injections. Expression of functional Cre in β1-integrin floxed mice is evidenced by a smaller DNA product lacking exon 3 (ex3), which is not present in wild type mice. Myh11β 1-fl/fl mice were induced with TMX or injected with corn oil vehicle (Myh11-TMX and Myh11- Oil, respectively). (B) Western blotting of β1-integrin in detrusor from oil- and TMX-injected mice at 6 wk after injections. β-actin loading controls are shown in the lower panel. (C) Quantitation of densitometry from (B) normalized to the amount of actin in each lane. Data are mean ± SD, ∗P < 0.05, based on t-test with Welch’s correction for unequal variances. (D) Immunofluorescence of β1- integrin in BSM of oil- and TMX-injected Myh11β 1-fl/fl mice (left panels, green). Phalloidin staining shows actin (red) and DAPI highlights nuclei (blue). Scale bar (white) in top left panel = 10 μm.

Article Snippet: Western blotting: Rabbit antibodies to β1-integrin (ab52971), and muscarinic receptor 3 (M3, ab126168) were from Abcam (Waltham, MA, USA); α1-integrin (#71 747), β3-integrin (#13 166), and β-actin (#4697) were from Cell Signaling Technology (Danvers, MA, USA); M2 (G206) was from Assay Biotech (Fremont, CA, USA) and P2X1 (APR001) was from Alomone Laboratories (Jerusalem, Israel).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Functional Assay, Injection, Western Blot, Quantitation Assay, Immunofluorescence, Staining