Journal: Cell Death and Differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: A Ad-Seam3A plasmids were transfected into NHEK cells for 48 h. Then, recombinant EGF protein was added to the transfected cells for 15 min. Sema3A, p-EGFR and p-ERK were analysed by western blot. B Wound healing and Transwell assays in transfected Sema3A plasmids in the absence or presence of EGF. C Recombinant EGF protein was incubated in the Ad-Sema3A- or NC-transfected cells for 2 days, and immunoblotting analysis is displayed. D Transfection of short peptides interfering with Sema3A function in keratinocytes, treatment with the EGFR signal inhibitor erlotinib, and testing of the protein expression of EMT markers. E Cells treated with si-Sema3A ± erlotinib were plated in the chamber, and the migration capacity was assessed. Bars indicate the fold changes ± SEM relative to the negative control. F The ERK-specific inhibitor U0126 was introduced into NHEKs. Western blot analysis showed that U0126 attenuated the EMT process mediated by TGF-β1 and that Sema3A deficiency enhanced the protein expression of mesenchymal markers triggered by U0126. G qRT-PCR analysis was performed to confirm the expression level of transcriptional factors including Sema3A, NRP1, GATA-1, CEBPA, XBP1, TP53, CEBPB and TCF4 in Hacat cells. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The blot was incubated with appropriate primary antibodies against Sema3A (ab23393, Abcam), NRP1 (ab81321, Abcam), EGFR (#3777, CST), p-EGFR (#4267, CST), ERK (#4695, CST), p-ERK (#4370, CST), p-Smad2 (ab18338, Abcam), E-cad (#3195, CST), N-cad (ab18203, Abcam), Vimentin (#5741, CST), ZEB2 (#14026, ProteinTech), Snail (#3879, CST), Slug (#9585, CST), VEGFR2 (#9698, CST) or p-VEGFR2 (#2478, CST) for 12 h at 4 °C.

Techniques: Transfection, Recombinant, Western Blot, Incubation, Expressing, Migration, Negative Control, Quantitative RT-PCR

Journal: Cell Death and Differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: A Detection of the EGFR-ERK pathway and EMT inducers by western blotting in Hacat cells incubated with recombinant Sema3A and EGF for 48 h. B Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK in cells transfected with si-NRP1. C Localization of NRP1 and EGFR proteins in Hacat cells. Scale bar = 20 µm. D Co-IP experiment between NRP1 and EGFR. IP: NRP1. WB: EGFR. E EGFR- and NRP1-overexpressing Hacat cells were treated with cycloheximide (CHX) for the indicated time periods to inhibit de novo protein synthesis. As a control, MG132 was added to block the catalytic activity. F si-NRP1 and EGFR plasmids were cotransfected into NHEK cells for 48 h. Protein levels of NRP1, EGFR, p-EGFR, ERK and p-ERK were determined by western blot. G NHEK cells were transfected with si-NRP1 plasmids for 2 days before EGF (50 ng/ml) stimulation. Then the IF analysis of EGFR or NRP1 was showed. Scale bar = 20 µm. H NHEKs were stimulated with EGF (100 ng/ml) for the indicated periods of time. IFs were subsequently conducted in the resulting cells to monitor EGFR and NRP1 localization/expression. Scale bar = 20 µm.

Article Snippet: The blot was incubated with appropriate primary antibodies against Sema3A (ab23393, Abcam), NRP1 (ab81321, Abcam), EGFR (#3777, CST), p-EGFR (#4267, CST), ERK (#4695, CST), p-ERK (#4370, CST), p-Smad2 (ab18338, Abcam), E-cad (#3195, CST), N-cad (ab18203, Abcam), Vimentin (#5741, CST), ZEB2 (#14026, ProteinTech), Snail (#3879, CST), Slug (#9585, CST), VEGFR2 (#9698, CST) or p-VEGFR2 (#2478, CST) for 12 h at 4 °C.

Techniques: Western Blot, Incubation, Recombinant, Transfection, Co-Immunoprecipitation Assay, Control, Blocking Assay, Activity Assay, Expressing

Journal: Cell Death and Differentiation

Article Title: Regulation of Semaphorin3A in the process of cutaneous wound healing

doi: 10.1038/s41418-022-00981-6

Figure Lengend Snippet: A NHEKs were serum starved and subsequently stimulated with Rb-Sema3A for 5 min to 1 h and subjected to IF analyses. B Combined treatment with Rb-EGF and Rb-Sema3A was utilized in keratinocytes at the indicated time points. EGFR and NRP1 localization/expression was shown by IF staining. Wound healing assays ( C ) and Transwell assays ( D ) were performed in Rb-Sema3A-,Rb-EGF and EGFR plasmids incubated in Hacat cells after transfected with si-NRP1 or negative control. The percentage of wound closure is displayed as the mean ± SEM; n = 3. Bars indicate the mean fold changes ± SEM relative to the corresponding control; n = 3. E Western blot analysis of EGFR-ERK pathway after treatment at the indicated time points. F Immunofluorescence in Hacat cells. si-NRP1 or si-NC plasmids were transfected in EGFR-overexpressing cells. Recombinant EGF and Sema3A cytokines were added in cells to test the process of NRP1 and EGFR activation and degradation. Scale bar = 20 µm. G A schematic of the proposed mechanism. ** P < 0.01; *** P < 0.001.

Article Snippet: The blot was incubated with appropriate primary antibodies against Sema3A (ab23393, Abcam), NRP1 (ab81321, Abcam), EGFR (#3777, CST), p-EGFR (#4267, CST), ERK (#4695, CST), p-ERK (#4370, CST), p-Smad2 (ab18338, Abcam), E-cad (#3195, CST), N-cad (ab18203, Abcam), Vimentin (#5741, CST), ZEB2 (#14026, ProteinTech), Snail (#3879, CST), Slug (#9585, CST), VEGFR2 (#9698, CST) or p-VEGFR2 (#2478, CST) for 12 h at 4 °C.

Techniques: Expressing, Staining, Incubation, Transfection, Negative Control, Control, Western Blot, Immunofluorescence, Recombinant, Activation Assay

Journal: iScience

Article Title: Smurf1 silencing restores PTEN expression that ameliorates progression of human glioblastoma and sensitizes tumor cells to mTORC1/C2 inhibitor Torin1

doi: 10.1016/j.isci.2021.103528

Figure Lengend Snippet: Smurf1 is elevated in GB cells (A) Immunohistochemistry (IHC) was performed for Smurf1 protein expression in GB patient tissues and in the normal temporal lobe. Tissues were first sectioned, and then sections were probed with primary antibodies against Smurf1. Target protein expression was evaluated via indirect detection using a labeled secondary antibody. After staining with hematoxylin, the antigen-antibody complex was visualized under a bright-field microscope. In IHC stained images brown tint shows positive immunoreactivity for Smurf1 antigen. Scale bars, 50 μm. (B) Different tumor cells, including PTEN-wt (LN229, U343), and PTEN-mut (U251, LNZ308, U87, U118, and #19005) GB cells were grown under standard culture conditions described in methods. For expression analysis, cells were lysed and whole-cell lysates were examined through Western blotting for the expression of EGFR, p-Akt S473 , Akt, PTEN, Smurf1, and β-actin proteins. Results shown here represent three independent experiments.

Article Snippet: Primary antibodies were used as per manufacturer’s instructions and standard dilutions: Smurf1 (ab57573, Abcam); Akt and p-Akt Ser473 (#9272 and #4060 respectively, CST); β-actin (A1978-200, Sigma); PTEN (sc-7974, Santa); PTEN (10047-1-AP, Proteintech); PTEN (ABM-2052, Cascade BioScience); EGFR (#4267, CST); Phosphotyrosine (p-4G10) (05-321X, EMD Millipore); HA (M180-3, MBL International); Ki67 (ab15580, Abcam); p-p70S6K Thr389 (#9205, CST); p70S6K (sc-8418, Santa); 4EBP1 (#9644, CST); p-4EBP1(#2855, CST).

Techniques: Immunohistochemistry, Expressing, Labeling, Staining, Microscopy, Western Blot

Journal: iScience

Article Title: Smurf1 silencing restores PTEN expression that ameliorates progression of human glioblastoma and sensitizes tumor cells to mTORC1/C2 inhibitor Torin1

doi: 10.1016/j.isci.2021.103528

Figure Lengend Snippet:

Article Snippet: Primary antibodies were used as per manufacturer’s instructions and standard dilutions: Smurf1 (ab57573, Abcam); Akt and p-Akt Ser473 (#9272 and #4060 respectively, CST); β-actin (A1978-200, Sigma); PTEN (sc-7974, Santa); PTEN (10047-1-AP, Proteintech); PTEN (ABM-2052, Cascade BioScience); EGFR (#4267, CST); Phosphotyrosine (p-4G10) (05-321X, EMD Millipore); HA (M180-3, MBL International); Ki67 (ab15580, Abcam); p-p70S6K Thr389 (#9205, CST); p70S6K (sc-8418, Santa); 4EBP1 (#9644, CST); p-4EBP1(#2855, CST).

Techniques: Recombinant, Sequencing, Software