Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 1. PDL1 expression in cancer and noncancer cell lines. A, relative expression of PDL1 from total RNA isolated from cell lines. B, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by IHC. C, representative PDL1 protein expression levels in corresponding high- and low- expressing cell lines by flow cytometry.

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Isolation, Cytometry

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 2. Relative expression of PDL1 in 38 osteosarcoma specimens. PDL1 expression was evaluated from total RNA by quantitative real-time RT-PCR and showed that the expression levels range over 4 log (5,000-fold).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 3. Overall survival of 37 patients with osteosarcoma in relation to PDL1 gene expression. The median overall survival for PDL1-low patients was 89 months compared with 28 months for PDL1-high patients, which showed a trend but was not statistically significant (P ¼ 0.0544).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 4. Correlation between PDL1 gene expression and TILs by IHC. A, representative TILs in osteosarcoma tissues (400); score 0, no TILs; 1, rare/few TILs; 2, brisk/prominent TILs. B, significant positive correlation was shown between PDL1 gene expression and TILs in patients with osteosarcoma (P ¼ 0.0117).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Gene Expression

Journal: Cancer Immunology Research

Article Title: Programmed Cell Death Ligand 1 Expression in Osteosarcoma

doi: 10.1158/2326-6066.cir-13-0224

Figure Lengend Snippet: Figure 5. Characterization of the origins of metastases. A, PDL1 expression is significantly higher in metastatic osteosarcoma tumors that originate from lung than from other locations (P ¼ 0.0024). B, TILs also exhibit a positive correlation with pulmonary osteosarcoma metastasis compared with nonpulmonary metastasis (P ¼ 0.0443).

Article Snippet: Unfortunately, the ProSci rabbit polyclonal failed to detect PDL1 on the positive control cell line SKOV3, and the XW mouse mAb exhibited unspecific binding on the negative control cell line MCF7 (Supplementary Fig. S1).

Techniques: Expressing

Journal: Biomolecules

Article Title: Apelin-13-Mediated Upregulation of METTL3 Ameliorates Alzheimer’s Disease via Inhibiting Neuroinflammation Through m6A-Dependent Regulation of lncRNA BDNF-AS

doi: 10.3390/biom15081188

Figure Lengend Snippet: Effect of AP on cell proliferation, apoptosis, and neuroinflammation. ( A ) Flow cytometry for Cell apoptosis analysis. ( B ) MTT for evaluating Cell proliferation. ( C ) Protein level of BDNF and METTL3 detected by Western blotting. ( D ) RNA levels of BDNF detected by qRT-PCR. ( E ) RNA levels of lncRNA BDNF-AS , detected by qRT-PCR. ( F ) RNA levels of BDNF-AS m6A detected by MeRIP-qPCR. ( G ) Inflammatory factor TNF-α expression by qRT-PCR. ( H ) Inflammatory factor IL-1β expression by qRT-PCR. Original images can be found in . Data is expressed as mean ± SEM, n = 3, * p < 0.05 vs. Control, # p < 0.05 vs. Aβ25-35, ^ p < 0.05 vs. Aβ25-35+AP-L, & p < 0.05 vs. Aβ25-35+AP-M.

Article Snippet: AP (4029110, Bachem, Bubendorf, Switzerland); Aβ25–35 (A4559, Sigma-Aldrich, St. Louis, MO, USA); thiazolyl blue (MTT) (M2128, Sigma-Aldrich, St. Louis, MO, USA); PC12 cell line (Shanghai Cell Bank, Chinese Academy of Sciences, Shanghai, China); microglia bv2 (CL-0493, Pricella, Wuhan, China); PrimeScriptTM RT reagent Kit (RR037A, Takara, Tokyo, Japan); ELISA kit (IL-1β, ERC007QT and TNF-α, ERC102aQT.96, Neobioscience Technology, Shenzhen, China); ANNEXIN V-FITC/PI Apoptosis Detection Kit (CA1020, Solarbio, Beijing, China); protein secondary antibody (A21020 and A21010, Abbkine, Wuhan, China), β3-tubulin (ab18207, Abcam, Cambridge, MA, USA), NeuN (ab177487, Abcam), anti-β-actin (ab8227, Abcam); BDNF (ab108319, Abcam), METTL3 (ab195352, Abcam), IBA 1 (ab178846 m, Abcam), GFAP (ab7260, Abcam), TrkB (ab187041, Abcam), p-TrkB (ab229908, Abcam), DAA (6736-58-9, MCE, Shanghai, China).

Techniques: Flow Cytometry, Western Blot, Quantitative RT-PCR, Expressing, Control

Journal: Cell Death Discovery

Article Title: FNDC5 inhibits autophagy of bone marrow mesenchymal stem cells and promotes their survival after transplantation by downregulating Sp1

doi: 10.1038/s41420-023-01634-4

Figure Lengend Snippet: A CCK-8 assay detection of cell proliferation after H/SD treatment. B Flow cytometry detection of apoptosis upon H/SD treatment. C Western blot analysis of autophagy representative proteins, Beclin 1 and p62. D Immunofluorescence of LC3 II/B in BMSCs at different times after injury. E Immunofluorescence of LC3 II/B after H/SD and autophagy inhibitor (3-MA, 5 mM) or autophagy inducer (rapa, 1.0 μg/L) treatment. F CCK-8 assay detection of cell proliferation after autophagy inhibition or promotion. G Flow cytometry detection of BMSC apoptosis after autophagy inhibition or promotion. Data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, n = 3, Scale bar = 100 μm. H/SD hypoxia/serum deprivation, LC3 II/B Microtubule-associated protein 1 light chain 3 II/B, DAPI 4'6-diamidino-2-phenylindole, 3-MA 3-methyl-adenine, rapa rapamycin.

Article Snippet: BMSCs were washed with PBS, processed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions and then detected using a flow cytometer (BD Biosciences, USA).

Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Immunofluorescence, Inhibition

Journal: Cell Death Discovery

Article Title: FNDC5 inhibits autophagy of bone marrow mesenchymal stem cells and promotes their survival after transplantation by downregulating Sp1

doi: 10.1038/s41420-023-01634-4

Figure Lengend Snippet: A – C Validation of Sp1 overexpression in BMSCs. A Immunofluorescence confirmed the transfection efficiency of OE-Sp1 lentivirus, which was GFP-labeled. B Western blotting confirmed Sp1 protein overexpression. C qRT-PCR detected overexpression of Sp1 mRNA in transfected BMSCs. D qRT-PCR detection of ULK2 mRNA levels in BMSCs-OE-Sp1 and BMSCs-OE-NC subject to H/SD. E Western blotting of ULK2 protein in BMSCs treated with OE-Sp1 lentivirus or vehicle control. F Immunofluorescence of LC3 II/B in BMSCs after OE-Sp1 lentivirus or vector control transfection. G Western blotting of autophagy representative proteins, Beclin 1 and p62, in BMSCs. H CCK-8 assay detection of cell proliferation after OE-Sp1 or OE-NC treatment and H/SD. I Flow cytometry detection of BMSC apoptosis after OE-Sp1 lentivirus or vector control transfection. Data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, n = 3, Scale bar = 100 μm. OE-Sp1 overexpression of Sp1, OE-NC overexpression negative control.

Article Snippet: BMSCs were washed with PBS, processed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions and then detected using a flow cytometer (BD Biosciences, USA).

Techniques: Biomarker Discovery, Over Expression, Immunofluorescence, Transfection, Labeling, Western Blot, Quantitative RT-PCR, Control, Plasmid Preparation, CCK-8 Assay, Flow Cytometry, Negative Control

Journal: Cell Death Discovery

Article Title: FNDC5 inhibits autophagy of bone marrow mesenchymal stem cells and promotes their survival after transplantation by downregulating Sp1

doi: 10.1038/s41420-023-01634-4

Figure Lengend Snippet: A Immunofluorescence of LC3 II/B in BMSCs subject to H/SD after OE-FNDC5 lentivirus transfection or OE-Sp1 lentivirus transfection. B Western blotting of ULK2, Beclin 1, and p62 proteins in BMSCs. C CCK-8 assay detection of cell proliferation after OE-FNDC5 lentivirus transfection or combined with OE-Sp1 lentivirus transfection. D Flow cytometry detection of BMSC apoptosis after OE-FNDC5 treatment or combined with OE-Sp1 treatment. Data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, n = 3, Scale bar = 100 μm.

Article Snippet: BMSCs were washed with PBS, processed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions and then detected using a flow cytometer (BD Biosciences, USA).

Techniques: Immunofluorescence, Transfection, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Cell Death Discovery

Article Title: FNDC5 inhibits autophagy of bone marrow mesenchymal stem cells and promotes their survival after transplantation by downregulating Sp1

doi: 10.1038/s41420-023-01634-4

Figure Lengend Snippet: A Immunofluorescence of BDNF at the edge of the infarct area on day 14 after BMSCs-OE-FNDC5 or BMSCs-OE-FNDC5 + OE-Sp1 transplantation. B Immunofluorescence of CD31 and α-SMA at the edge of the infarct area on day 14 after BMSC transplantation. C Schematic diagram of the protective effect of FNDC5 on BMSC survival after ischemic injury by regulation of autophagy. FNDC5 down-regulates the transcription factor, Sp1, and reduces expression of the autophagy target protein, ULK2, thereby inhibiting BMSC autophagy, decreasing apoptosis and enhancing cell vitality under hypoxia and nutrient-deprivation. BMSCs-OE-FNDC5 transplantation has a neuroprotective effect in MCAO rats. Data are presented as the mean ± SD, * p < 0.05, ** p < 0.01, n = 6, Scale bar = 100 μm. BDNF Brain-derived neurotrophic factor, CD31 Platelet endothelial cell adhesion molecule 1, α-SMA α-Smooth muscle actin.

Article Snippet: BMSCs were washed with PBS, processed using the Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions and then detected using a flow cytometer (BD Biosciences, USA).

Techniques: Immunofluorescence, Transplantation Assay, Expressing, Derivative Assay

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: Knockdown of UCA1 inhibited cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with shNC, sh-UCA1#1, and sh-UCA1#2. (A) The efficiency of transfection was quantified by RT-qPCR analysis. (B) Cell viability was measured by trypan blue exclusion assay. (C) Cell migration and (D) invasion were detected by Transwell migration assay. (E) Apoptotic cells and (F) the levels of apoptosis-related proteins were measured by flow cytometry and Western blot, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Knockdown, Migration, Transfection, Quantitative RT-PCR, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: miR-301a overexpression blocked the effect of UCA1 on cell growth and migration in MHCC97 cells. MHCC97 cells were transfected with sh-UCA1 or shNC, or cotransfected with sh-UCA1 and miR-301a mimic or mimic control. (A) Cell viability, (B) cell migration, and (C) invasion were assessed by trypan blue exclusion assay and Transwell migration assay, respectively. (D) Apoptotic cells were quantified by flow cytometry assay. (E) Western blot was conducted to measure the expression of apoptosis-related core factors. * p < 0.05, ** p < 0.01.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Over Expression, Migration, Transfection, Control, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry, Western Blot, Expressing

Journal: Oncology Research

Article Title: Knockdown of Urothelial Carcinoma-Associated 1 Suppressed Cell Growth and Migration Through Regulating miR-301a and CXCR4 in Osteosarcoma MHCC97 Cells

doi: 10.3727/096504018X15201143705855

Figure Lengend Snippet: CXCR4 was involved in the regulatory effect of miR-301a on cell growth and migration. MHCC97 cells were transfected with sh-CXCR4 or sh-NC, or cotransfected with sh-CXCR4 and miR-301a mimic, or mimic control. The (A) mRNA and (B) protein expression levels of CXCR4 were tested by RT-qPCR analysis and Western blot analysis, respectively. (C) Cell viability, (D) cell migration, (E) invasion, and (F) apoptotic cells were assessed by trypan blue exclusion assay, Transwell migration assay, and flow cytometry assay, respectively. (G) The protein levels of apoptosis-related factors were measured by Western blot. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The apoptotic cell rates were determined using an Annexin-V-Phycoerythrin (PE) Apoptosis Detection Kit (Beyotime Biotechnology).

Techniques: Migration, Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Trypan Blue Exclusion Assay, Transwell Migration Assay, Flow Cytometry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: SNAI1 promotes epithelial-mesenchymal transition and maintains cancer stem cell-like properties in thymic epithelial tumors through the PIK3R2/p-EphA2 Axis

doi: 10.1186/s13046-024-03243-0

Figure Lengend Snippet: Validation of the oncogenic effect of SNAI1 both in vitro and in vivo. a . qRT-PCR reveals the relative mRNA expression levels in Ty82 cells under various conditions, including those overexpressing SNAI1 (LV-SNAI1), suppressing SNAI1 (LV-shSNAI1), treated with the SNAI1 inhibitor (Rosiglitazone), and the control group. b - c . Western blot analysis depicting the protein expression levels of SNAI1 in TET cells under different experimental conditions. d - e . The impact of SNAI1 overexpression on the proliferation of TET cells was assessed using the CCK-8 assay. Absorbance at 450 nm was measured at various time points. f - i . Transwell assays were employed to evaluate the impact of SNAI1 overexpression or suppression on the in vitro migration and invasion ability of TET cells. Representative micrographs and quantification of cells attached to the lower surface of the chamber are presented (Scale bars: 200 μm). j - m . The clone assay was utilized to assess the influence of SNAI1 on the in vitro proliferation of TET cells. Representative micrographs are presented (Scale bars: 2 mm). The quantifications of cell clone spheres are presented. n . The apoptosis assay was conducted using an Annexin V-APC/PI Apoptosis Kit in Ty82 cells treated with Rosiglitazone or the control group. o - p . Western blot analysis was performed to assess the protein expression levels of EMT markers, including N-cadherin, E-cadherin, and Vimentin, in TET cells transfected with LV-SNAI1 or LV-Ctrl. q . Representative images depict Ty82 cells transfected with LV-SNAI1, LV-shSNAI1, or LV-Ctrl grown as spheres (Scale bars: 200 μm). r . Ty82 cells transfected with LV-SNAI1 or LV-Ctrl were injected into the left ventricle of 5- to 6-week-old male wild-type BALB/c nude mice. Representative bioluminescent images depict TET cell metastases. * denotes p < 0.05, ** denotes p < 0.01, *** denotes p < 0.001, and ns denotes not statistically significant

Article Snippet: For the cell apoptosis assay, the Annexin V-APC/PI Apoptosis Kit (Elabscience) was employed.

Techniques: Biomarker Discovery, In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Control, Western Blot, Over Expression, CCK-8 Assay, Migration, Clone Assay, Apoptosis Assay, Transfection, Injection

Journal: Theranostics

Article Title: Conditional knockout of TGF-βRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.

doi: 10.7150/thno.35686

Figure Lengend Snippet: Figure 7. Knockdown of Smad2 attenuates cisplatin-induced renal injury, inflammation response, and programmed cell death in established nephrotoxic AKI model. A: PAS staining and quantitative analysis show knockdown of Smad2 reduced renal injury in cisplatin nephropathy. B: Creatinine assay. C: BUN assay. Serum creatinine and BUN show knockdown of Smad2 prevented decline of renal function in cisplatin nephropathy. D: Real-time PCR data show knockdown of Smad2 reduced mRNA of TNF-α, IL-1β, MCP-1 and KIM-1. E: Immunohistochemistry and quantitative data show knockdown of Smad2 reduced KIM-1 protein and F4/80+ macrophages in cisplatin-induced nephropathy. F: TUNEL assay. Knockdown of Smad2 reduced apoptosis in injured kidney. G: Western blot analysis of KIM-1, RIPK1, RIPK3, cleaved caspase-3. Data represent mean ± SEM for 6 mice. *P<0.05, **P<0.01, ***P<0.001 versus control; #P<0.05, ##P<0.01, ###P<0.001 versus Smad2 vector control+cisplatin. KD: knockdown. Magnification: 100X.

Article Snippet: Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Beyotime (Shanghai, China).

Techniques: Knockdown, Staining, Real-time Polymerase Chain Reaction, Immunohistochemistry, TUNEL Assay, Western Blot, Control, Plasmid Preparation

Journal: Theranostics

Article Title: Conditional knockout of TGF-βRII /Smad2 signals protects against acute renal injury by alleviating cell necroptosis, apoptosis and inflammation.

doi: 10.7150/thno.35686

Figure Lengend Snippet: Figure 10. Role of TGF-βRII and Smad2 in acute kidney injury. Conditional knockout of TGF-βRII or Smad2 from kidney protects against AKI by alleviating cell necroptosis, apoptosis and inflammation, and Smad2 may serve as a therapeutic target for nephrotoxic and ischemic AKI.

Article Snippet: Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Beyotime (Shanghai, China).

Techniques: Knock-Out

Journal: Molecular Biotechnology

Article Title: miR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway

doi: 10.1007/s12033-024-01078-w

Figure Lengend Snippet: MiR-324-3p suppresses the activation of transforming growth factor (TGF)-β1- induced LX-2 cells in vitro. A Hepatic stellate cells (HSCs) were transfected with miR-324-3p mimic, and expression of the miR-324-3p was determined by real-time-quantitative polymerase chain reaction (RT-qPCR). B RT-qPCR to analyze the miR-324-3p expression in HSCs transfected with the miR-324-3p inhibitor. C Cell counting kit-8 (CCK-8) to analyze the proliferation of transforming growth factor (TGF)-β1-induced LX-2 cells with indicated treatment. D Flow cytometry to detect the cycle and apoptosis of TGF-β1-induced LX-2 cells. E , G Western blot (WB) assay and RT-qPCR to evaluate α-smooth muscle actin (α-SMA) and Vimentin expression in transfected HSC cells. F , H The α-SMA, and Vimentin levels in transfected cells were analyzed by WB assay as well as RT-qPCR. * P < 0.05 and ** P < 0.01 vs . Control group; # P < 0.05 and ## P < 0.01 vs . NC + TGF-β1 group

Article Snippet: Then, the apoptosis was detected using the Annexin V-FITC/PI Apoptosis Analysis Kit (Bestbio, China) and analyzed using a CytoFLEX flow cytometer (Beckman Coulter, USA).

Techniques: Activation Assay, In Vitro, Transfection, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Cell Counting, CCK-8 Assay, Flow Cytometry, Western Blot, Control