Journal: Bioengineering & Translational Medicine

Article Title: The effect of local non‐thermal plasma therapy on the cancer‐immunity cycle in a melanoma mouse model

doi: 10.1002/btm2.10314

Figure Lengend Snippet: NTP treatment affects tumor immunogenicity. Tumor sections were collected on Days 7, 10, and 14, individually stained for specific cancer proteins, and counterstained with DAPI to identify the nuclei. (a) Quantification of protein expression was performed with our software, which identifies cell nuclei from the DAPI stain (far left panel; blue) within the tumor and analyzes the signal around it (middle left panel; green). A representative mask of the nuclei is shown (middle right panel) along with the analysis area (far right panel; white). Inserts are 500 μm × 500 μm zoomed‐in sections to show the segmentation capability of the software and analysis. All scale bars are 500 μm. Representative images of the (b) CRT staining (green), (d) CD47 staining (red), and (f) PD‐L1 staining (yellow) are shown. Scale bars are 100 μm. The immunofluorescence signal was quantified with our program and graphed for (c) CRT, (e) CD47, and (g) PD‐L1. Data are represented as mean ± SEM and each biologically independent sample is shown ( n = 3–5). Statistical significance of the NTP treatment condition was compared to that of the control for each day, using the generalized linear mixed model. *p ≤ 0.05 (detailed p values are reported in the text)

Article Snippet: Incubation with the primary antibody was performed overnight at 4°C for CRT (1/500 dilution; clone 3EPR3924, Abcam, ab92516), CD47 (1/100 dilution; clone B6H12.2, ThermoFisher Scientific, MA5‐11895), and PD‐L1 (1/200 dilution; clone 10F.9G2, Biolegend, 124301).

Techniques: Immunopeptidomics, Staining, Expressing, Software, Immunofluorescence, Control

Journal: Nature Communications

Article Title: Theranostic near-infrared-IIb emitting nanoprobes for promoting immunogenic radiotherapy and abscopal effects against cancer metastasis

doi: 10.1038/s41467-021-27485-0

Figure Lengend Snippet: 4T1 cells were incubated under hypoxic conditions (37 °C, 1% O 2 , 5% CO 2 ) and irradiated with X-ray doses of 6 Gy in vitro. a Representative photographs of stained colonies of 4T1 cells treated with hypoxia, hypoxia + QD-RGD, hypoxia + QD-Cat-RGD, hypoxia + RT (6 Gy), hypoxia + RT + QD-RGD (6 Gy) or hypoxia + RT + QD-Cat-RGD (6 Gy) after 7 days. The photographs of stained colonies ( a ) were representative of those generated from three independent samples each group. b Histogram plot of the survival fraction of 4T1 cells with different treatments. c Immunofluorescence staining of p-H2AX (Ser139) and quantitative analysis of foci density of foci per cell at 4 h after different treatments. Scale bars = 25 μm. d Apoptosis analysis measured by flow cytometry of 4T1 cells at 24 h after different treatments. e QD-Cat-RGD reinforces the ICD induced by RT and emits several types of DAMPs from dying cancer cells. f Immunofluorescence staining of calreticulin of 4T1 cells with different treatments after 24 h. Scale bars = 25 μm. g Flow cytometry analysis of surface calreticulin expression in 4T1 cells with different treatments after 24 h. h Histogram plot of secreted ATP production by 4T1 cells with different treatments after 24 h. i Histogram plot of HMGB1 released by 4T1 cells with different treatments after 24 h. j Immunofluorescence staining of HMGB1 in 4T1 cells with different treatments after 24 h. Scale bars = 25 μm. The images of immunofluorescence staining ( c , f , j ) were representative of those generated from three independent samples each group. All data are shown as the mean ± s.e.m. of ( n = 3) and n represents the number of independent samples. Statistical significance was calculated via one-way ANOVA with Tukey’s multiple comparisons test. n.s. not significant. Source data are provided as a Source Data file.

Article Snippet: For detection of calreticulin expression on the cell surface, an anti-calreticulin antibody (1:100, Cell Signaling Technology, Alexa Fluor 488, Cat 62304) was used to stain the cells after different treatments.

Techniques: Incubation, Irradiation, In Vitro, Staining, Generated, Immunofluorescence, Flow Cytometry, Expressing

Journal: Toxins

Article Title: Suppression of Hepatic Epithelial-to-Mesenchymal Transition by Melittin via Blocking of TGFβ/Smad and MAPK-JNK Signaling Pathways

doi: 10.3390/toxins9040138

Figure Lengend Snippet: Effects of melittin (MEL) on TGF-β1-induced epithelial-to-mesenchymal transition (EMT) in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 48 h. ( A ) MEL inhibited the TGF-β1-stimulated EMT marker in AML12. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin; ( B ) E-cadherin, ZO-1, fibronectin and vimentin mRNA expression was analyzed by real-time PCR in vitro. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin; ( C ) Immunofluorescence double staining for E-cadherin (green) and vimentin (red) in TGF-β1-stimulated AML12 after treatment of MEL. Cells was counterstained with Hoechst 33342 (blue). Magnifications ×200; ( D ) Real-time PCR of EMT related gene markers. The quantitative ratios are shown as relative optical density that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p < 0.05 versus normal control, † p < 0.05 versus TGF-β1 treatment.

Article Snippet: A non-tumorigenic mouse hepatocyte cell line, AML12 (America Tissue Culture Collection, CRT-2254; ATCC, Manassas, VA, USA), was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium (Gibco, Grand Island, NY, USA) containing 5 μg/mL ITS premix (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL dexamethasone (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco).

Techniques: In Vitro, Incubation, Marker, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Double Staining, Control

Journal: Toxins

Article Title: Suppression of Hepatic Epithelial-to-Mesenchymal Transition by Melittin via Blocking of TGFβ/Smad and MAPK-JNK Signaling Pathways

doi: 10.3390/toxins9040138

Figure Lengend Snippet: MET antagonizes the TGF-β1-stimulated Smad signal pathway in vitro. Cells were pretreated for 1 h with MEL, followed by incubation with TGF-β1 for 24 h. ( A ) Nuclear extracts were subjected to SBE DNA binding and Smad4 antibody (Ab) assay by EMSA supershift assay; ( B ) MEL inhibits TGF-β1-dependent transcriptional activity of the CAGA x12 -Luc reporter in a dose-dependent manner; ( C ) Immunoblot of the effect of MEL on the TGF-β1-stimulated pSmad2, pSamd3 and Smad4; ( D ) AML12 was transfected with control (Con) or specific Smad4 siRNA and then treated with TGF-β1 for 24 h or 48 h. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p < 0.05 versus normal control, † p < 0.05 versus TGF-β1 treatment.

Article Snippet: A non-tumorigenic mouse hepatocyte cell line, AML12 (America Tissue Culture Collection, CRT-2254; ATCC, Manassas, VA, USA), was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium (Gibco, Grand Island, NY, USA) containing 5 μg/mL ITS premix (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL dexamethasone (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco).

Techniques: In Vitro, Incubation, Binding Assay, Activity Assay, Western Blot, Transfection, Control, Expressing

Journal: Toxins

Article Title: Suppression of Hepatic Epithelial-to-Mesenchymal Transition by Melittin via Blocking of TGFβ/Smad and MAPK-JNK Signaling Pathways

doi: 10.3390/toxins9040138

Figure Lengend Snippet: MEL antagonizes the TGF-β1-stimulated non-Smad/MAPK signal pathway. ( A ) Immunoblot of mitogen-activated protein kinase (MAPK). MEL strongly inhibited the TGF-β1-stimulated pJNK1/2; ( B ) AML12 was transfected with control (Con) or specific c-Jun N-terminal kinase (JNK)1/2 siRNA and then treated with TGF-β1 for 24 h or 48 h; ( C ) Smad4 siRNA, Smad3 inhibitor (SIS) and MEL inhibited TGF-β1-stimulated pJNK1/2 expression; ( D ) JNK siRNA, JNK inhibitor (SP, SP600125) and MEL inhibited TGF-β1-stimulated Smad4 expression. The quantitative ratios are shown as relative optical densities of bands that are normalized to the expression of β-actin. The data are representative of three similar experiments and quantified as mean values ± S.E. * p < 0.05 versus normal control, † p < 0.05 versus TGF-β1 treatment.

Article Snippet: A non-tumorigenic mouse hepatocyte cell line, AML12 (America Tissue Culture Collection, CRT-2254; ATCC, Manassas, VA, USA), was cultured in a 1:1 mixture of Dulbecco’s Modified Eagle’s Medium/Ham’s F-12 medium (Gibco, Grand Island, NY, USA) containing 5 μg/mL ITS premix (Sigma-Aldrich, St. Louis, MO, USA), 40 ng/mL dexamethasone (Sigma-Aldrich), and 10% fetal bovine serum (FBS, Gibco).

Techniques: Western Blot, Transfection, Control, Expressing