Journal: Cancer Cell International

Article Title: Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

doi: 10.1186/s12935-024-03557-1

Figure Lengend Snippet: ADSCs-Exos IFN−γ+TNF−α promoted immune escape of TNBC cells by transporting UCHL1. ADSCs were treated with 10 ng/mL IFN-γ and TNF-α, and transfected with si-NC or si-UCHL1. The exosomes were extracted from the supernatant. TNBC cells were treated with 10 μg/mL of the ADSCs-CM and different exosomes for 24 h, and then co-cultured with THP-1 cells for 48 h or with T cells for 16 h. A UCHL1 protein levels in TNBC cells in the indicated groups. B Viability of TNBC cells in the indicated groups. C Representative images showing Ki-67 expression in TNBC cells in the indicated groups. Scale bar = 100/25 μm. D Representative images showing migration of TNBC cells in the indicated groups. Scale bar = 100 μm. * P < 0.05 vs . MDA-MB-231 or MDA-MB-468. # P < 0.05 vs . Exos + MDA-MB-231 or Exos + MDA-MB-468. & P < 0.05 vs . Exos IFN−γ+TNF−α+con + MDA-MB-231 or Exos IFN−γ+TNF−α+con + MDA-MB-468. E The proportion of CD68 + CD206 + THP-1 cells in the indicated groups. F Arg-1, CD206, TNF-α, and iNOS expression in THP-1 cells. * P < 0.05 vs . THP-1. # P < 0.05 vs . MDA-MB-231 + THP-1 or MDA-MB-468 + THP-1. & P < 0.05 vs . Exos + THP-1. @ P < 0.05 vs . Exos IFN−γ+TNF−α+con + THP-1. G The proportion of CD3 + CD8 + T cells in the indicated groups. H Perforin and GZMB levels in T cells. * P < 0.05 vs . T cells. # P < 0.05 vs . MDA-MB-231 + T cells or MDA-MB-468 + T cells. & P < 0.05 vs . Exos + T cells. @ P < 0.05 vs . Exos IFN−γ+TNF−α+con + T cells

Article Snippet: Granular enzyme B (GZMB) and perforin levels were analyzed using specific GZMB (CSB-E08718h, CUSABIO, China) and perforin (CSB-E09313h, CUSABIO, China) ELISA kits according to the instructions.

Techniques: Transfection, Cell Culture, Expressing, Migration

Journal: Cancer Cell International

Article Title: Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

doi: 10.1186/s12935-024-03557-1

Figure Lengend Snippet: ADSCs-Exos IFN−γ+TNF−α promoted tumor growth in vivo. BALB/c mice were subcutaneously injected with 4T1 cells (2 × 10 6 ) and treated with 100 μg of the different exosomes every 3 days. A Tumor weight and volume in the indicated groups. B In-situ expression of Ki-67 in the tumor tissues of indicated groups. Scale bar = 100/25 μm. C The F4/80 + /CD206 + cells in the tumor tissues of indicated groups. Scale bar = 25 μm. D The proportion of CD3 + CD8 + T cell in the tumor tissues of indicated groups. E Perforin and GZMB levels in the tumor tissues of indicated groups. F and G CD8 + /GZMB + cells and CD8 + /perforin + cells in the tumor tissues of indicated groups. (H) HDAC6, UCHL1, PD-L1, p-STAT3 and STAT3 levels in the tumors of indicated groups. * P < 0.05 vs . 4T1. # P < 0.05 vs . Exos + 4T1. & P < 0.05 vs . Exos IFN−γ+TNF−α+con + 4T1

Article Snippet: Granular enzyme B (GZMB) and perforin levels were analyzed using specific GZMB (CSB-E08718h, CUSABIO, China) and perforin (CSB-E09313h, CUSABIO, China) ELISA kits according to the instructions.

Techniques: In Vivo, Injection, In Situ, Expressing

Journal: Frontiers in Immunology

Article Title: NK cell function down regulated by HMGB2 through ANGPT1/PI3K/AKT pathway and its effect on esophageal squamous carcinoma cells

doi: 10.3389/fimmu.2025.1666199

Figure Lengend Snippet: Down-regulation of HMGB2 enhances the function of NK cells. (a) qRT-PCR analysis of HMGB2 mRNA levels in NK-92 cells transfected with siNC, siHMGB2 #1, siHMGB2 #2, and siHMGB2 #3. (b) Western blot analysis of HMGB2 protein levels in NK-92 cells transfected with siNC or siHMGB2. (c) The secretion levels of perforin-1 and granzyme B were measured by ELISA in NK-92 cells after HMGB2 knockdown. (d) Cytokine release (TNF-α and IFN-γ) from NK-92 cells with HMGB2 knock down were showed in histogram. (e) Cytotoxicity of NK-92 cells with HMGB2 knockdown against various ESCC cell lines (EC109, KYSE70, KYSE150, KYSE450) was assessed at 24h and 48h using a cytotoxicity assay. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test for normally distributed data, or Kruskal–Wallis tests followed by Dunn’s post hoc tests for non-normally distributed data. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent the mean ± SD.

Article Snippet: After centrifugation, the culture supernatant was collected, and the concentrations of granzyme B and perforin 1 were measured using the Human Granzyme B ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human Perforin 1 ELISA Kit (E-EL-H1123c, Elabscience), respectively, according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: NK cell function down regulated by HMGB2 through ANGPT1/PI3K/AKT pathway and its effect on esophageal squamous carcinoma cells

doi: 10.3389/fimmu.2025.1666199

Figure Lengend Snippet: Overexpression of HMGB2 inhibits the function of NK-92 cells. (a) qRT-PCR analysis of HMGB2 mRNA levels in NK-92 cells transfected with pcDNA3.1-Vector or pcDNA3.1-HMGB2. (b) Western blot analysis of HMGB2 protein expression in NK-92 cells transfected with pcDNA3.1-Vector or pcDNA3.1-HMGB2. (c) The secretion levels of perforin-1 and granzyme B were measured by ELISA in NK-92 cells after HMGB2 overexpression. (d) Cytokine release (TNF-α and IFN-γ) from NK-92 cells with HMGB2 overexpression was showed in histogram. (e) Cytotoxicity of NK-92 cells with HMGB2 overexpression against various ESCC cell lines (EC109, KYSE70, KYSE150, KYSE450) at 24h and 48h was assessed using a cytotoxicity assay. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test for normally distributed data, or Kruskal–Wallis tests followed by Dunn’s post hoc tests for non-normally distributed data. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent the mean ± SD.

Article Snippet: After centrifugation, the culture supernatant was collected, and the concentrations of granzyme B and perforin 1 were measured using the Human Granzyme B ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human Perforin 1 ELISA Kit (E-EL-H1123c, Elabscience), respectively, according to the manufacturer’s instructions.

Techniques: Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay

Journal: Frontiers in Immunology

Article Title: NK cell function down regulated by HMGB2 through ANGPT1/PI3K/AKT pathway and its effect on esophageal squamous carcinoma cells

doi: 10.3389/fimmu.2025.1666199

Figure Lengend Snippet: Confirmation of HMGB2 knockout and functional assessment of KO-HMGB2 NK-92 cells. (a) Sanger sequencing chromatograms showing two distinct allelic edits in the KO-HMGB2 NK-92 cells. (b) Western blot analysis of HMGB2 protein levels in wild-type (WT) and KO-HMGB2 NK-92 cells. (c) ELISA analysis of perforin-1 and granzyme B secretion in supernatants collected from KO-HMGB2 and WT NK-92 cells co-cultured with KYSE450 cells for 12 hours. (d) qRT-PCR analysis of activating receptors (NKG2D, NKp44, NKp46, and CD107a) in KO-HMGB2 and WT NK-92 cells. (e) Cytotoxicity of NK-92 cells with HMGB2 knock out against various ESCC cell lines (EC109, KYSE70, KYSE150, KYSE450) at 24h and 48h was assessed using a cytotoxicity assay. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test for normally distributed data, or Kruskal–Wallis tests followed by Dunn’s post hoc tests for non-normally distributed data. * p < 0.05, ** p < 0.01, *** p < 0.001. Error bars represent the mean ± SD.

Article Snippet: After centrifugation, the culture supernatant was collected, and the concentrations of granzyme B and perforin 1 were measured using the Human Granzyme B ELISA Kit (E-EL-H1617c, Elabscience, Beijing, China) and Human Perforin 1 ELISA Kit (E-EL-H1123c, Elabscience), respectively, according to the manufacturer’s instructions.

Techniques: Knock-Out, Functional Assay, Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Cytotoxicity Assay

Journal: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 1. miR-218 expression is downregulated and inversely correlated with the expression of SH3GL1 in MB cancer cell lines. (A) qPCR analysis of the miR-218 expression levels in a panel of human MB cells and in normal cerebellum. (B and C) SH3GL1 expression at the mRNA and protein levels in different human MB cell lines and normal cerebellum. MB, medulloblastoma; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 2. SH3GL1 is a target of miR-218. (A) Predicted miR-218 target sequences in the 3'-UTR of SH3GL1 (SH3GL1-UTR-WT) and mutants containing three mutated nucleotides in the 3'-UTR of SH3GL1 (SH3GL1-UTR-MUT). (B and C) HEK‑293T cells were cotransfected with miR‑218 or anti‑miR‑218 and psicheck2 vector with SH3GL1 3'-UTR, WT or mutated. After 36 h, the luciferase activity was measured. Renilla luciferase activity was normalized to firefly luciferase expression for each sample. Each experiment was performed in triplicate. *P<0.05. (D) Relative miR-218 expression levels in PSFK and UW228 cells stably expressing miR-218 by qPCR. (E and F) qPCR and western blot analysis were performed to detect the expression of SH3GL1 mRNA and protein in PSFK and UW228 cells stably expressing miR-218. UTR, untranslated region; MUT, mutated; WT, wild type; NC, normal cerebellum.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Expressing, Stable Transfection, Western Blot

Journal: Molecular medicine reports

Article Title: miR-218 is downregulated and directly targets SH3GL1 in childhood medulloblastoma.

doi: 10.3892/mmr.2013.1639

Figure Lengend Snippet: Figure 4. SH3GL1 knockdown phenocopies the miR‑218 overexpression in MB cells. (A and B) Expression of SH3GL1, p‑ERK and p‑Jun were detected by western blot analysis in PSFK or UW228 cells following miR-218 overexpression and transfection with siSH3GL1. (C and D) Cell proliferation analysis of transfected PSFK or UW228 cells with siSH3GL1 or NC by MTT assay at 24, 48 and 72 h following transfection. MB, medulloblastoma; NC, normal cerebellum; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase; p‑ERK, phosphorylated‑extracellular signal‑regulated kinases.

Article Snippet: The sequence of the SH3GL1 siRNA was obtained from Origene (SR304356; Rockville, MD, USA).

Techniques: Knockdown, Over Expression, Expressing, Western Blot, Transfection, MTT Assay

Journal: Oncotarget

Article Title: PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer.

doi: 10.18632/oncotarget.25091

Figure Lengend Snippet: Figure 5: The N-terminal EK piece both stabilizes and inhibits EK-PSA-TRP and EK-PSA-GZMB. Enzymatic activity of pre-incubated EK-PSA-TRP + PSA (red dashed), PSA-TRP + PSA (black dashed), EK-PSA-TRP alone (red solid), and PSA-TRP alone (black solid) (A). Enzymatic activity of EK-PSA-TRP +/- a 4 hour EK treatment (B). Enzymatic activity of pre-incubated EK-PSA-GZMB + PSA (red dashed), PSA-GZMB + PSA (black dashed), EK-PSA-GZMB alone (red solid), and PSA-GZMB alone (black solid) (C). Enzymatic activity of EK-PSA-GZMB +/- a 4 hour EK treatment (D).

Article Snippet: Cloning and mutagenesis of GZMB and TRP constructs In order to express hGZMB in reasonable amounts using a mammalian expression system, the EK-C248 gene containing an N-Terminal enterokinase activation site was generated using by modification of the WT GZMB gene (Origene Plasmid SC321693).

Techniques: Activity Assay, Incubation