Journal:

Article Title: Abnormal integrin-mediated regulation of chronic myelogenous leukemia CD34 + cell proliferation: BCR/ABL up-regulates the cyclin-dependent kinase inhibitor, p27 Kip , which is relocated to the cell cytoplasm and incapable of regulating cdk2 activity

doi:

Figure Lengend Snippet: p27Kip mRNA levels are not significantly different in CML and NL CD34+ cells. High-specific-activity biotin-labeled, anti-sense RNA probes that hybridize with target human mRNAs encoding cdk1, cdk2, cdk3, cdk4, p27Kip, p21Cip, PISSLRE, p16INK and the housekeeping gene products L32 and GAPDH, were synthesized from the hCC-1 Human Cell Cycle Multiprobe Template Set. Total RNA was extracted from CML or NL CD34+ cells and hybridized to the labeled probes. Free probe and remaining single-stranded mRNA were digested with RNase, and the “RNase-protected” probes were resolved on denaturing polyacrylamide gels and transferred by electroblotting to a positively charged nylon membrane. The nonisotopic probe was visualized with Ambion's BrightStar BioDetect Kit. A representative example of three separate experiments is shown. Quantitative differences in protein levels were evaluated by scanning images by using a GS-700 Imaging Densitometer and quantitated by using molecular analyst software. Compared with normal CD34+ cells, levels of p27Kip and GADPH in CML cells were 1.02-fold and 1.05-fold higher (densitometry values added to the figure: values are OD × mm).

Article Snippet: High-specific-activity biotin-labeled, anti-sense RNA probes were synthesized from the hCC-1 Human Cell Cycle Multiprobe Template Set (PharMingen) by using the MAXIscript In Vitro Transcription Kit (Ambion, Austin, TX).

Techniques: Activity Assay, Labeling, Synthesized, Imaging, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C is involved in the ECM remodeling and induces the HSCs activation. a GO analysis based on intersection proteins binding to Sema3C and NRP1. b Representative images of collagen gel contraction assays with MHCC-97L cells in either MHCC-97L supernatants or OE-Sema3C-MHCC-97L supernatants. Graph quantifying the change in the percentages of gel contraction by MHCC-97L cells in different conditions. Scale bar, 5 mm. c Representative images showing H&E, Masson’s trichrome and picrosirius red staining, and IHC staining for α-SMA and collagen I expression in serial sections of orthotopic liver xenograft tumors of BALB/C nude mice injected with MHCC-97L-Vector or MHCC-97L-OE-Sema3C cells. Gross tissue image scale bar, 1 cm, microscope image scale bar, 200 μm. d Percent of collagen fibers and α-SMA per field were counted in at least three random fields per animal, (n = 5). e Representative immunofluorescence images for α-SMA and collagen I in HSCs treated with dosage rhSema3C or supernatants of different Sema3C expression levels. Scale bar, 50 μm. LX-2 cells were treated with a gradient dose of rhSema3C or supernatants from Sema3C-overexpressed Hep3B and MHCC-97L cells. The α-SMA expression levels were detected by western blotting analysis ( f ). The TGF-β1 secretion was examined by ELISA assay ( g ). The chemoresistance ability was reflected by an MTT assay ( h ). The ability of migration and invasion was performed by Transwell assays, scale bar, 200 μm ( i ). 5 × 10 5 Hep3B cells or OE-Sema3C Hep3B cells were injected subcutaneously into nude mice alone or mixed with LX-2 cells in a 1:1 ratio. The mice were sacrificed, and the xenograft tumors were excised 32 days after inoculation ( j ). The bar chart showed the tumor weight in each treatment group ( k ). The tumor volumes were monitored for 32 days ( l ). n = 5 per group. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Activation Assay, Binding Assay, Staining, Immunohistochemistry, Expressing, Injection, Plasmid Preparation, Microscopy, Immunofluorescence, Western Blot, Enzyme-linked Immunosorbent Assay, MTT Assay, Migration

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: IL-6 and cholesterol biosynthesis are responsible for Sema3C-mediated HSCs activation. a The volcano map showed the differentially expressed genes of LX-2 cells treated with rhSema3C or PBS in vitro. HSCs were treated with rhSema3C or supernatants of MHCC-97L cells with different Sema3C expression levels. mRNA levels of IL6 and IL8 were detected using qRT-PCR ( b ). ELISA was used to examine the IL6 secretion level ( c ). d GSEA identified an enrichment of genes involved in the NF-κB pathway in rhSema3C-treated LX-2 cells. e Western blotting showed phosphorylated and total p65 protein expression levels in LX-2 cells treated with rhSema3C. LX-2 cells were pre-treated with Bay 11-7082 and subsequently stimulated with rhSema3C, the levels of phosphorylated p65, total p65, and α-SMA expression were determined by western blotting ( f ), and IL6 secretion levels were examined by ELISA assay ( g ). h LX-2 cells were treated with OE-Sema3C-Hep3B cell-derived supernatants, Co-IP assay was used to detect the interaction between ITGB1 and NRP1. LX-2 cells were transfected with siNRP1 and subsequently stimulated with rhSema3C, IL6 secretion levels were detected by ELISA ( i ). The phosphorylated p65, total p65, α-SMA, and HMGCR expression levels were determined by using western blotting ( j ). k LX-2 cells were treated with a dosage of rhSema3C or supernatants of Sema3C-overexpressed HCC cells, and ITGB1 protein expression was determined by western blotting. LX-2 cells were transfected with siITGB1 and subsequently stimulated with rhSema3C, and the IL6 secretion levels were detected by ELISA assay ( l ), the phosphorylated p65, total p65, and α-SMA expression levels were determined by using Western blotting ( m ). LX-2 cells were treated with siNRP1, siITGB1 or rhSema3C as indicated, the secretion of TGF-β1 in each group was detected by ELISA ( n ), the chemotherapy resistance of LX-2 cells was evaluated by MTT assay ( o ), and migration and invasion were performed by Transwell assay, scale bar, 200 μm ( p ). rhSema3C recombinant human Sema3C; Data are presented as means ± SD. ns not significantly; *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Activation Assay, In Vitro, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Co-Immunoprecipitation Assay, Transfection, MTT Assay, Migration, Transwell Assay, Recombinant

Journal: Signal Transduction and Targeted Therapy

Article Title: Semaphorin 3C (Sema3C) reshapes stromal microenvironment to promote hepatocellular carcinoma progression

doi: 10.1038/s41392-024-01887-0

Figure Lengend Snippet: Sema3C expression is closely correlated with CAFs in HCC and is mediated by CAFs-derived TGF-β1. a Correlation between Sema3C expression and CAFs-associated genes ( ACTA2, VIM, FAP, PDGFRB , and S100A4 ) and collagen-associated genes ( COL1A1, COL1A2, FLNA, ELN , and LOX ) in HCC using the HCCDB datasets. The dots in the figure indicate p < 0.05. The color intensity and size of the circle are proportional to the correlation coefficients. b Representative images of IF staining for low or high Sema3C expression (red) with different degrees of stromal infiltration (α-SMA-green) in HCC tissues or DEN+CCl 4 -induced mouse liver tissues. Cell nuclei were stained with DAPI (blue). c The correlation analysis revealed a positive correlation between high Sema3C expression and the stromal percentage in patients with HCC (total n = 27). d TCGA-LIHC analysis of the correlation between patients with T stage or stage and FAP high /Sema3C low or FAP high /Sema3C high in HCC. e Western blotting showed that treatment with CAFs-conditioned media (CAFs-CM) significantly increased Sema3C expression compared with the control in HCC cells. f AP1 (including c-Jun/c-Fos) transcriptional binding sites in the Sema3C gene promoter. g , h MHCC-97L and Huh7 cells were transfected with si-c-Fos or si-c-Jun as indicated and the levels of Sema3C, c-Fos, and c-Jun were validated by western blotting. i ChIP-qPCR analysis showed that TGF-β1 stimulation facilitated the binding of phosphorylated c-Jun (p-c-Jun) and phosphorylated c-Fos (p-c-Fos) to the Sema3C gene promoter. j The levels of Sema3C, phosphorylated c-Jun (p-c-Jun), total c-Jun, p-c-Fos, and total c-Fos in MHCC-97L and Huh7 cells treated with different concentrations of TGF-β1 were determined by using western blotting. k Western blotting indicated that TGF-β1-induced Sema3C expression and AP1 signal activation could be inhibited by galunisertib (TGF-β1 receptor type I Inhibitor). l , m Western blotting confirmed that galunisertib and the TGF-β1 neutralizing antibody (anti-TGF-β1) inhibited the Sema3C upregulation stimulated by treatment with CAFs-CM. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: To verify the effect of TGF-β1 on Sema3C expression in HCC, 1 μg/mL anti-TGF-β1 antibody or an IgG control (Proteintech, China) was added to the CM of CAFs and incubated at room temperature for 1 h. The treated CM was then used to co-culture with the corresponding HCC cells.

Techniques: Expressing, Derivative Assay, Staining, Western Blot, Control, Binding Assay, Transfection, ChIP-qPCR, Activation Assay