Journal: iScience

Article Title: Metallo-protease Peptidase M84 from Bacillus altitudinis induces ROS-dependent apoptosis in ovarian cancer cells by targeting PAR-1

doi: 10.1016/j.isci.2024.109828

Figure Lengend Snippet:

Article Snippet: Scrambled (SCR) siRNA (Cat#sc-37007, Santa Cruz Biotechnology) was used as control for knockdown studies.

Techniques: Virus, Staining, Transfection, Adjuvant, SYBR Green Assay, BIA-KA, Isolation, cDNA Synthesis, Extraction, Sequencing, Software

Journal: iScience

Article Title: Metallo-protease Peptidase M84 from Bacillus altitudinis induces ROS-dependent apoptosis in ovarian cancer cells by targeting PAR-1

doi: 10.1016/j.isci.2024.109828

Figure Lengend Snippet:

Article Snippet: Scrambled (SCR) siRNA , Santa Cruz Biotechnology , Cat#sc-37007.

Techniques: Virus, Staining, Transfection, Adjuvant, SYBR Green Assay, BIA-KA, Isolation, cDNA Synthesis, Extraction, Sequencing, Software

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: TLR2 signaling in MSC is crucial to induction of immunosuppressive monocytes/macrophages. A. Experimental scheme. THP-1 macrophages were cocultured with human MSCs in a Transwell system. Prior to coculturing, MSCs were subjected to pretreatment with Pam2CSK4 (100 ng/mL) or anti-TLR2 Ab (10 μg/mL) or transfected with TLR2 siRNA or SCR siRNA for 24 h. After 18 h of coculturing, THP-1 macrophages were analyzed for PTGS2 mRNA levels and the secreted levels of PGE2, IL-10 and AREG. B. qRT-PCR for PTGS2 in THP-1 macrophages . The mRNA levels are fold changes relative to THP-1 macrophages cultured alone. C. ELISA for PGE2, IL-10 and AREG in supernatants of THP-1 macrophages with or without MSC coculturing. D. Experimental scheme. Murine BM cells were cocultured with human MSCs under GM-CSF stimulation for 5 d. Prior to coculturing, MSCs were pretreated with Pam2CSK4 (100 ng/mL) or anti-TLR2 Ab (10 μg/mL) or transfected with TLR2 siRNA or SCR siRNA for 24 h. After 5 d of coculturing, BM cells were assayed. E. Representative and quantitative flow cytometry results for CD11b hi Ly6C hi Ly6G lo and CD11b mid Ly6C mid Ly6G lo cells in BM cells. F. qRT-PCR for Arg1 , Nos2 and Ptgs2 in BM cells. Shown are the mRNA levels relative to BM cells cultured alone without GM-CSF. G. ELISA for PGE2, IL-10 and active TGF-β1 levels in cell-free supernatants of BM cells with or without MSC coculturing. Mouse-specific ELISA kits were used for measurement of IL-10 and active TGF-β1. The PGE2 assay kit recognizes both human and murine PGE2. H. Experimental scheme. MSCs were isolated from WT C57BL/6 or TLR2 KO mice. BM cells from WT C57BL/6 mice were cocultured with WT or TLR2 KO MSCs under GM-CSF for 5 d, and then subjected to assays. I, J. Representative flow cytometry results for CD11b, Ly6C, Ly6G and MHC class II in BM cells. K. Quantitative flow cytometric analysis for CD11b hi Ly6C hi Ly6G lo cells, CD11b mid Ly6C mid Ly6G lo cells and MHC class II hi Ly6C hi Ly6G lo cells in BM cells. L. ELISA for murine TNF-α and IL-1β in supernatants of cell cultures. Data are means ± SD from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as analyzed by one-way ANOVA and Tukey's test, Kruskal-Wallis test and Dunn's multiple-comparison test (CD11b hi Ly6C hi Ly6G lo and MHC class II hi Ly6C hi Ly6G lo cells in K) or Student's t -test (L).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Transfection, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Isolation, Comparison

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: TLR2 signaling in MSCs is essential for suppression of inflammatory angiogenesis in the cornea following sterile injury. A. Experimental protocol. Immediately after corneal suturing injuries, MSCs, Pam2CSK4-pretreated MSCs, TLR2 siRNA-transfected MSCs, SCR siRNA-transfected MSCs or HBSS (vehicle) were administered via tail vein injection in BALB/c mice. Seven days later, the corneas were subjected to assays. B, C. Representative corneal photographs and microphotographs of CD31/LYVE1-stained corneal whole-mounts. Scale bar: 500 μm for the first and second rows and 200 μm for the third row (magnified images of yellow-outlined insets from the first row). D, E . Clinical scoring of corneal NV and quantification of CD31-stained area in corneal whole-mounts. F, G. qRT-PCR for pro-angiogenic factors and pro-inflammatory cytokines in cornea. The mRNA levels are presented relative to those in BALB/c control corneas that had not received injury or treatment. H. Experimental protocol. Corneal sutures were applied to TLR2 KO mice, and either MSCs or HBSS were intravenously injected. After 7 d, the corneas were subjected to assays. I, J. Representative corneal photographs and microphotographs after CD31/LYVE1 immunostaining. Scale bar: 500 μm for the upper row and 200 μm for the lower row (magnified images of yellow-outlined insets from the upper row). K, L. Clinical scoring of corneal NV and measurement of CD31- and LYVE1-stained areas. Data represent means ± SD, where a circle indicates the data from an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, as analyzed by one-way ANOVA and Tukey's test or by Student's t -test (Injury + MSC vs. Injury + MSC/Pam2CSK4 in F, G).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Sterility, Transfection, Injection, Staining, Quantitative RT-PCR, Control, Immunostaining

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: Single-cell transcriptional profiling of Pam2CSK4-treated and untreated MSCs. A. Experimental scheme. MSCs treated with Pam2CSK4 100 ng/mL for 24 h and untreated MSCs were subjected to scRNA-seq on the 10x Genomics Chromium platform. B. Combined UMAP and tSNE plots of Pam2CSK4-treated MSCs (blue) and untreated MSCs (orange) showing overlapping. C. tSNE plots of Pam2CSK4-treated MSCs and untreated MSCs depicting 7 clusters (C0-C6) in both cell libraries. D, E. Feature and dot plots displaying positive and negative markers for MSCs. F. Heatmap of top 10 DEGs in Pam2CSK4-treated MSCs vs. untreated MSCs as determined by RNA-Seq analysis. AKR1C1 was identified as the top DEG upregulated in Pam2CSK4-treated MSCs relative to untreated MSCs. G. ELISA for secreted levels of AKR1C1 in cell-free supernatants of MSC cultures treated with Pam2CSK4 (0-100 ng/mL). H. qRT-PCR and ELISA for mRNA and protein levels of AKR1C1 in MSCs transfected with TLR2 siRNA or control SCR siRNA. Means ± SD are presented. ** p < 0.01 as analyzed by Kruskal-Wallis test and Dunn's multiple-comparison test (G) or Student's t -test (H).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: RNA Sequencing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, Control, Comparison

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: AKR1C1 inhibition promotes MSC ferroptosis. A. Cell viability assay in MSCs transfected with AKR1C1 siRNA or control SCR siRNA or in MSCs treated with AKR1C1 inhibitor, 5-PBSA (10 or 100 ng/mL). B, C. Quantification of intracellular Fe 2+ fluorescence intensity in MSCs and representative fluorescence images. Red indicates FerroOrange staining, while blue represents DAPI-stained nuclei. Scale bar: 50 μm for the upper row and 200 μm for the lower row. Data are means ± SD from 3-4 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant, as analyzed by one-way ANOVA and Tukey's test.

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Inhibition, Viability Assay, Transfection, Control, Fluorescence, Staining

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: AKR1C1 upregulates production of therapeutic factors in MSCs, whereas AKR1C1 inhibition abrogates it. A. Experimental scheme. MSCs were subjected to AKR1C1 inhibition or replenishment and analyzed for the expression of key therapeutic factors. B. qRT-PCR for key therapeutic factors in MSCs after transfection with AKR1C1 siRNA or SCR siRNA. C. qRT-PCR in MSCs with rhAKR1C1 replenishment (1 or 5 ng/mL). D, E. qRT-PCR and ELISA for mRNA and secreted protein levels of key therapeutic factors in MSCs with 5-PBSA treatment (10 or 100 ng/mL). Data are means ± SD from 2-3 independent experiments. * p < 0.05, *** p < 0.001, **** p < 0.0001, as analyzed by one-way ANOVA and Tukey's test, Kruskal-Wallis test and Dunn's multiple-comparison test ( STC1 , TGFB1 , SDF1 in C and STC-1 in E) or Student's t -test (B).

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Theranostics

Article Title: Activation of Toll-like receptor 2 promotes mesenchymal stem/stromal cell-mediated immunoregulation and angiostasis through AKR1C1

doi: 10.7150/thno.100327

Figure Lengend Snippet: AKR1C1 in MSCs is required for amelioration of inflammatory angiogenesis in the cornea following sterile injury. A. Experimental protocol. Immediately after corneal suturing injury, AKR1C1 siRNA-transfected MSCs, SCR siRNA-transfected MSCs or HBSS (vehicle) were intravenously administered into BALB/c mice. Seven days later, the corneas were assayed. B, C. Representative corneal photographs and microphotographs of corneal flat mounts with CD31/LYVE1 immunostaining. Scale bar: 500 μm or 200 μm (magnified views of yellow-outlined insets). D, E. Clinical scoring of corneal NV and quantification of LYVE1-stained area in corneal whole-mounts. F, G. Representative flow cytometry cytograms of CD11b hi Ly6C hi cells in the spleen (F) and quantitation of CD11b hi Ly6C hi cells in spleen and blood (G). H, I. qRT-PCR assays for pro-inflammatory cytokines in ocular draining cervical lymph nodes (DLNs) and blood cells. The mRNA levels are presented as fold changes relative to naïve BALB/c mice without injury or treatment. Data represent means ± SD, where a circle indicates the data from an individual animal. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant, as analyzed by one-way ANOVA and Tukey's test.

Article Snippet: For TLR2 activation or blocking, MSCs were treated with 10-1000 ng/mL Pam2CSK4 (tlrl-pm2s-1, InvivoGen, San Diego, CA) or 10 μg/mL anti-human TLR2 Ab (pab-hstlr2, InvivoGen) for 24 h. For TLR2 or AKR1C1 gene knockdown, MSCs were transfected with 10 μM TLR2 siRNA (sc-40256, Santa Cruz Biotechnology, Dallas, TX), the corresponding SCR siRNA (sc-37007, Santa Cruz Biotechnology), 10 μM AKR1C1 siRNA (AM16708, Ambion, Calsbad, CA) or the corresponding SCR siRNAs (AM4611, Ambion) using Lipofectamine TM RNAiMAX transfection reagent (Invitrogen/Thermo Fisher Scientific, Waltham, MA).

Techniques: Sterility, Transfection, Immunostaining, Staining, Flow Cytometry, Quantitation Assay, Quantitative RT-PCR