Journal: ACS Nano

Article Title: Immune Modulatory Oxysterols Produced from Cholesterol-Containing Lipid Nanoparticles Regulate Tumor Growth

doi: 10.1021/acsnano.5c22020

Figure Lengend Snippet: LNP-oxysterols reduce tumor cell viability by inducing late apoptosis and necrosis in vitro. A) TC-1 tumor cell viability at 24, 48, and 72 h by MTT assay. B–E) Apoptosis and necrosis assessed at 24 h by propidium iodide (PI)/annexin V staining. Each data point represents a biological replicate. Statistical tests were performed by one-way ANOVA compared to vehicle without correction for multiple comparisons, where * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. F) Representative flow cytometry plots, where PI – /annexin V – are viable cells, PI + /annexin V + are cells in late apoptosis, PI – /annexin V + are cells in early apoptosis, and PI + /annexin V – are cells in necrosis. G) Pie charts of the percentage of viable, early apoptotic, late apoptotic, and necrotic cells.

Article Snippet: For proliferation studies, TC-1 tumor cells or BMDMs were plated in triplicate in 96-well plates, incubated at 37 °C with 5% CO 2 overnight for acclimation, and then treated with 55.7 μM LNP-cholesterol or LNP-oxysterols for 24, 48, and 72 h. Cytotoxicity and cell proliferation were assessed using the MTT assay kit (Cat. #4890050K, R&D Systems), and absorbance at 570 nm was measured using a Cytation 5 plate reader.

Techniques: In Vitro, MTT Assay, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Long Non-Coding RNA 74687 Regulates Meiotic Progression and Gonadal Development in Rainbow Trout ( Oncorhynchus mykiss ) via the miR-15a-5p– ccne1 Regulatory Axis

doi: 10.3390/ijms26168036

Figure Lengend Snippet: 5-ethynyl-2′-deoxyuridine (EdU) assay of effect of ccne1 on proliferative capacity of rainbow trout gonadal (RTG)-2 cells. ( A ) EdU results for RTG-2 cells under different transfection conditions. ( B ) Quantification of lnc74687.3 ’s effects on proliferation by ImageJ (ImageJ, 1.53q). ( C ) Quantification of ccne1 ’s effects on proliferation by ImageJ. Data are expressed as mean ± SD ( n = 3), with differences indicated by asterisks (** p < 0.01, * p < 0.05).

Article Snippet: Cell proliferation was assessed using an EdU Cell Proliferation Kit with Alexa Fluor 555 (Beyotime, Shanghai, China) following the manufacturer’s instructions with minor optimisation.

Techniques: EdU Assay, Transfection

Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A The expression of USP39 mRNA was analyzed by RT-PCR in SK-hep-1 and HepG2 HCC cells infected with shRNAs. B Western blotting analysis of USP39 protein level in SK-hep-1 and HepG2 cells infected with shRNAs. C – E The effect of USP39 on HCC cells (SK-hep-1) proliferation was determined by MTT assays ( C ) at different time points and colony formation ( D ). The colony counts were normalized to the control and expressed as a percentage, and results are represented in the bar graph ( E ). F – I Representative images of HCC cell migration ability as shown by wound-healing assays ( F – G ) and migration assay ( H – I ). Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Control, Migration

Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A – D RT-PCR and western blotting indicated that the mRNA expression levels of USP39 and TRIM26 ( A , C ) and the protein level of ZEB1 ( B , D ) in SK-hep-1 cells co-translated with USP39 and TRIM26 expressing plasmids. ( E , G ) The mRNA levels of USP39 and TRIM26 were analyzed by RT-PCR in SK-hep-1 HCC cells. F , H Effect of USP39-knockdown and TRIM26 silencing on the protein level of ZEB1 in SK-hep-1 HCC cell determined by western blotting. I Effect of overexpression of USP39 and TRIM26 on the ZEB1 ubiquitination in SK-hep-1 cells. J Effect of USP39-knockdown and TRIM26 silencing on the cell proliferation of SK-hep-1 assessed by MTT assay. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Knockdown, Over Expression, Ubiquitin Proteomics, MTT Assay

Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A The effect of USP39 and TRIM26 in HCC cell proliferation in vivo was determined by xenograft assays. USP39 and TRIM26 knockdown SK-hep-1 cells were respectively injected into flanks of BALB/c nude mice. After 30 days, tumors were isolated and photographed. B Tumor volumes were calculated. C Tumor weight. D , E Levels of USP39, TRIM26, and ZEB1 were analyzed by RT-PCR ( D ) and western blotting ( E ). F The expression levels of USP39, TRIM26, ZEB1 and Ki67 in tumors of different groups by IHC (original magnification, ×40; inlet, ×10). G , H Representative images showed the tumors metastasis of different groups by whole-body bioluminescence imaging ( G ) and lung metastases ( H ). The number of nodules in the lung was counted and statistically analyzed. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: In Vivo, Knockdown, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Imaging

Journal: Oncogenesis

Article Title: Altering MYC phosphorylation in the epidermis increases the stem cell population and contributes to the development, progression, and metastasis of squamous cell carcinoma

doi: 10.1038/s41389-020-00261-3

Figure Lengend Snippet: a , b TUNEL staining ( a ) and quantitation ( b ) to assess apoptosis in the epidermis and hair follicle of mice 72 h post DMBA/TPA one-time treatment. Scale bar = 50 µm. Shown is mean and SD of 5 mice per genotype. p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, *** p < 0.001. c , d BrdU staining ( c ) and quantitation ( d ) to assess proliferation in the epidermis and hair follicle of mice 48 h post DMBA/TPA one-time treatment. Scale bar = 100 µm. Shown is mean and SD of 3 mice per genotype. p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, ns = not significant. e , f BrdU staining ( e ) and quantitation ( f ) in the epidermis adjacent to the skin lesions after 8 weeks of DMBA/TPA treatment of mice from each genotype. Scale bar = 50 µm. Shown is mean and SD of 5 mice per genotype. p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001. g , h TUNEL staining ( g ) and quantitation ( h ) to assess apoptosis in the epidermis and hair follicle of mice 8 weeks post DMBA/TPA treatment. Scale bar = 100 µm. Shown is mean and SD of 3 mice per genotype. p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, ns = not significant.

Article Snippet: Briefly, the paraffin slides were de-paraffinized, incubated with TdT enzyme at 37 °C for 1 h, washed in TACS 2 TdT stop buffer for 10 min, incubated with streptavidin-HRP for 30 min, and counterstained with methanol green for 10 min. For proliferation analysis, a BrdU cell proliferation Assay Kit (BioVision, Cat. K306), according to the manufacturer’s protocol.

Techniques: TUNEL Assay, Staining, Quantitation Assay, Comparison, BrdU Staining

Journal: Oncogenesis

Article Title: Altering MYC phosphorylation in the epidermis increases the stem cell population and contributes to the development, progression, and metastasis of squamous cell carcinoma

doi: 10.1038/s41389-020-00261-3

Figure Lengend Snippet: a , b Long-term label-retaining cell analysis in control, MYC WT , or MYC T58A mice. Retention of BrdU was assessed 75 days after injection through immunofluorescent staining for BrdU (red) shown in ( a ) and quantification of percent of cells positive for BrdU shown in ( b ). p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, *** p < 0.001. c Immunofluorescence images of skin from the long-term label-retaining analysis stained with anti-CD34 (red). White arrows indicate positive cells in hair follicles. DAPI (blue) is nuclear counterstain. d Quantitation of CD34 staining from (c). Significance was determined by one-way ANOVA, followed by Tukey’s multiple comparison test, ** p < 0.01, *** p < 0.001. e qRT-PCR for Lin28B expression in skin of mice used in long-term label-retaining analysis (normal skin). p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, * p < 0.05, ** p < 0.01. f Immunohistochemistry for CD34 staining in hair follicles from the hyperplastic skin of control, MYC WT , or MYC T58A mice 4 months after DMBA/TPA treatment. Scale bar = 50 µm. Insets are shown below. Red arrowheads indicate CD34 positive cells. Nonspecific staining in the sebaceous gland is indicated by asterisks. g Quantification of CD34 staining from mice as in ( f ). Shown is mean and SD of 5 mice per genotype. p -value is from a one-way ANOVA, followed by Tukey’s multiple comparison test, * p < 0.05, *** p < 0.001. h qRT-PCR for expression of stem cell marker or signaling pathway genes, as indicated, in hyperplastic epidermis from DMBA/TPA-treated control, MYC WT , or MYC T58A mice. p -value is from a two-tailed Welch’s t -test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Briefly, the paraffin slides were de-paraffinized, incubated with TdT enzyme at 37 °C for 1 h, washed in TACS 2 TdT stop buffer for 10 min, incubated with streptavidin-HRP for 30 min, and counterstained with methanol green for 10 min. For proliferation analysis, a BrdU cell proliferation Assay Kit (BioVision, Cat. K306), according to the manufacturer’s protocol.

Techniques: Cell Analysis, Control, Injection, Staining, Comparison, Immunofluorescence, Quantitation Assay, Quantitative RT-PCR, Expressing, Immunohistochemistry, Marker, Two Tailed Test

Journal: Military Medical Research

Article Title: Glucocorticoids trigger muscle-liver crosstalk to attenuate acute liver injury and promote liver regeneration via the FGF6-FGFBP1 axis

doi: 10.1186/s40779-025-00618-y

Figure Lengend Snippet: FGF5 and FGF5s compete for binding to FGFBP1 via LLPS and regulate liver regeneration. a BrdU cell proliferation assay results to detect cell proliferation ability in AML12 cells overexpressing the 18 FGFs and treated with rFGFBP1 (10 ng/ml) or rFGFBP1 and FGFBP1Ab (2 μg/ml) ( n = 3). b Hepatic protein levels of FGF5 and FGF5s at the indicated times after partial (2/3) hepatectomy (PHx), with the corresponding quantification ( n = 2). c Representative liver FGFBP1 and FGF5 immunofluorescence results 24 h after acetaminophen (APAP) or saline administration. d Domain structure and the intrinsically disordered tendency of mouse FGF5 and FGF5s. IUPred assigned scores of the disordered tendencies of the sequences between 0 and 1, with scores higher than 0.5 indicating disorder. The amino acid sequence of FGF5 contains one IDR. e Confocal microscopy images of the assembly status of 5 μmol/L purified rFGF5-mCherry, rFGF5s-mCherry, or rΔIDR-mCherry with 0 or 10% PEG. Scale bar = 5 µm. f Images of purified protein colocalization assay on the cell surface via confocal microscopy and representative curves describing the distribution of the relative fluorescence intensities of FGFBP1 (green) and FGF5 or FGF5s (red). Top two panels: rFGFBP1-EGFP (5 nmol/L) with rFGF5-mCherry (20 nmol/L) or rFGF5s-mCherry (10 nmol/L). Bottom three panels: rFGFBP1-EGFP (5 nmol/L) with rFGF5-mCherry (20 nmol/L) and different amounts of rFGF5s (10, 20, or 40 nmol/L). Scale bar = 5 µm. g Representative albumin (ALB, a hepatocellular function marker) immunofluorescence of human liver organoids (HLOs) in the indicated groups. h Measurement of ALB secretion in HLOs ( n = 3). i Size calculation of HLOs in the indicated groups (CON, n = 146; rFGFBP1, n = 157; rFGFBP1 + rFGF5, n = 184). * P < 0.05, *** P < 0.001. FGF5 fibroblast growth factor 5, FGF5s short form of FGF5, FGFBP1 fibroblast growth factor binding protein 1, LLPS liquid–liquid phase separation, IDR intrinsically disordered region, PEG polyethylene glycol, EGFP enhanced green fluorescent protein, DIC differential interference contrast, DAPI 4’,6-diamidino-2-phenylindole, SP signal peptide

Article Snippet: Cell proliferation was detected using a BrdU Cell Proliferation Assay Kit (6813S, Cell Signaling Technology, MA, USA) according to the manufacturer’s instructions.

Techniques: Binding Assay, BrdU Cell Proliferation Assay, Immunofluorescence, Saline, Sequencing, Confocal Microscopy, Purification, Fluorescence, Marker