Journal: Molecular Oncology

Article Title: E2F1‐activated SPIN1 promotes tumor growth via a MDM2‐p21‐E2F1 feedback loop in gastric cancer

doi: 10.1002/1878-0261.12778

Figure Lengend Snippet: E2F1 promotes transcription of SPIN1. (A) Schematic diagram of the putative SPIN1 promoter regions (−2022/0, −1004/0, −508/0, −374/0, −253/0, −150/0, and −75/0). These promoter fragments were PCR‐amplified from the genomic DNA of BGC823 cells, which were then inserted into the HindIII‐NheI sites upstream of the firefly luciferase in the pGL3‐Basic vector. (B) Transcriptional activity analysis of the putative SPIN1 promoter fragments in 293T cells by dual‐luciferase reporter assays. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t ‐test with Welch's correction,* P < 0.05). (C, D) Dual‐luciferase reporter assays show that E2F1 and SP1 increase promoter activities of pGL3‐374/0. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t ‐test, ** P < 0.01). (E, F) RT‐qPCR assay indicates that E2F1 promotes the expression level of SPIN1 in MKN45 cells, whereas SP1 does not. Three independent experiments were performed, and data are presented as mean ± SD (unpaired t ‐test, ** P < 0.01). (G) Western blot assay indicates that E2F1 promotes the expression level of SPIN1, as well as its target genes MDM2, P‐RB, while the expression level of p21 is decreased. Three independent experiments were performed. (H) ChIP‐PCR assay shows that PCR products were amplified from the DNA fragment which was immunoprecipitated by the anti‐E2F1 antibody using primers that covered the four E2F1‐binding sites. Three independent experiments were performed. (I, J) Approximate 150‐fold (MKN45) and 20‐fold (BGC823) enrichment of the promoter amplifications of SPIN1 in the four binding sites in GC cells was observed. Three independent experiments were performed, and data are presented as mean ± SD.

Article Snippet: Then, cDNA was synthesized from 1 μg of the total RNA with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan).

Techniques: Amplification, Luciferase, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Immunoprecipitation, Binding Assay

Journal: Molecular Oncology

Article Title: E2F1‐activated SPIN1 promotes tumor growth via a MDM2‐p21‐E2F1 feedback loop in gastric cancer

doi: 10.1002/1878-0261.12778

Figure Lengend Snippet: SPIN1 promotes tumorigenesis and proliferation via activation of MDM2‐p21‐E2F1 pathway through binding with H3K4me3. (A–D) RT‐qPCR revealed that the mRNA levels of MDM2, CDK4, CyclinD1, and E2F1 were suppressed by SPIN1 siRNA, while little effect on the mRNA expression was found when SPIN1 was overexpressed. Three independent experiments were performed, and data are presented as mean ± SD. (E, F) Western blot analysis verified that the protein levels of MDM2, CDK4, CyclinD1, P‐RB, and E2F1 were increased, and p21 was decreased after SPIN1 overexpressed in MKN45 and BGC823 cells, while SPIN1 knockdown exhibited the opposite result. Three independent experiments were performed. (G) CHIP‐PCR assay showed that H3K4me3 directly interacted with the H3K4me3 binding sites within MDM2 promoter in MKN45 cells. M1, M2, M3, M5, M6, M7, M8, M10, M11, and M14 represent primers that covered the H3K4me3 binding sites respectively. (H) CHIP‐qPCR analysis indicated higher fold enrichment of promoter amplifications of H3K4me3 in the anti‐H3K4me3 group than that of the anti‐IgG group in MKN‐45 cells. Three independent experiments were performed, and data are presented as mean ± SD. (I) Co‐IP assay validated the interaction between endogenous SPIN1 and H3K4me3 in MKN45 cells using anti‐SPIN1 antibody. Three independent experiments were performed. (J) MDM2 was negative in nontumorous tissues ( n = 33), and expression of MDM2 in GC with LNM ( n = 33) was significantly higher than that without LNM ( n = 36) (magnification ×400). (K) Kaplan–Meier analysis showed that high MDM2 expression was correlated with poorer OS and DFS. (L) The results of Spearman's correlation analysis showed a positive relationship between MDM2 and SPIN1.

Article Snippet: Then, cDNA was synthesized from 1 μg of the total RNA with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan).

Techniques: Activation Assay, Binding Assay, Quantitative RT-PCR, Expressing, Western Blot, Knockdown, ChIP-qPCR, Co-Immunoprecipitation Assay

Journal: Molecular Oncology

Article Title: E2F1‐activated SPIN1 promotes tumor growth via a MDM2‐p21‐E2F1 feedback loop in gastric cancer

doi: 10.1002/1878-0261.12778

Figure Lengend Snippet: SPIN1 promotes tumorigenesis and proliferation via activation of MDM2‐p21‐E2F1 pathway in vivo . (A) After 3 times injection of LV‐NC ( n = 5, 0.1 mL per mouse) and LV‐ShRNA‐SPIN1 ( n = 5, 0.1 mL per mouse), on day 30, photographs of representative tumors are shown. (B) HE staining results showed that the tumors of LV‐Sh‐SPIN1 group ( n = 5) were well‐encapsulated while LV‐NC group ( n = 5) displayed local invasion into the muscle tissues (magnification ×100). Immunohistochemistry for Ki‐67 revealed that the GC cells in the LV‐shRNA‐SPIN1 group ( n = 5) had a lower positivity rate than those in the LV‐NC group ( n = 5) (magnification ×100). (C) Tumor size was measured using vernier caliper, and tumor volume was determined as described in Materials and methods section for 30 days. Each data point is the mean value of primary tumors ( n = 5). Data are presented as mean ± SD. (D) RNA from fresh tumor tissues was extracted. qPCR showed that MDM2, CDK4, CyclinD1, and E2F1 mRNA expression were suppressed in the LV‐ShRNA‐SPIN1 group, but there was no significant difference in the p21 mRNA expression between two groups. Three independent experiments were performed, and data are presented as mean ± SD. (E) Western blotting confirmed the result that MDM2, CDK4, CyclinD1, P‐RB, and E2F1 protein expression were decreased in LV‐ShRNA‐SPIN1 group, while p21 was increased. Three independent experiments were performed. (F) Working model of the upstream regulatory and function mechanisms of SPIN1 in GC progression.

Article Snippet: Then, cDNA was synthesized from 1 μg of the total RNA with a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan).

Techniques: Activation Assay, In Vivo, Injection, shRNA, Staining, Immunohistochemistry, Expressing, Western Blot

Journal: JID Innovations

Article Title: Isolation of Lymphocytes from Human Skin and Murine Tissues: A Rapid and Epitope-Preserving Approach

doi: 10.1016/j.xjidi.2022.100155

Figure Lengend Snippet: Detection of innate and adaptive lymphocytes in the skin during digestion at different time points. ( a ) Representative flow cytometry plots showing the gating strategy for B cells, T cells, NK cells, and ILCs. ( b ) Summary graphs depicting the absolute numbers of live cells for CD45 + CD14 ‒ lymphocytes, T cells, B cells, NK cells, and ILCs after the digestion of skin biopsies at different incubation periods (15, 30, 45, 60, and 90 minutes) with 1.7 mg/ml of collagenase and 0.2 mg/ml of DNase (median; n = 6). Statistical significance was tested using Kruska‒Wallis test, followed by Dunn’s multiple comparisons test. ∗∗ P < 0.01. ( c ) Relative frequency of different immune cell subsets in peripheral blood and skin biopsies at different incubation periods of digestion (mean + SEM; n = 6). Statistical significance was tested using Wilcoxon matched-pairs signed rank test. Statistical significance between blood and skin ∗ P < 0.05. ( d ) UMAP illustrating the differences between skin and blood and the respective immune cell populations for all cells (upper row) and for the respective incubation periods used for skin (lower row; mean, n = 6). ( e ) Representative flow cytometry plot showing the identification of counting beads in the FSC-A and SSC-A. Numbers of live CD45 + CD14 ‒ lymphocytes, T cells, B cells, NK cells, and ILCs were extrapolated using the absolute number of events and counting beads in the skin during digestion (mean, n = 2). Statistical significance was tested using Mann‒Whitney test; ns. ( f ) Numbers of live CD45 + CD14 ‒ lymphocytes, T cells, B cells, NK cells, and ILCs identified by flow cytometry in the skin during the digestion of skin using either a commercially available whole-skin dissociation kit Commercial kit (black-lined bar filled with red, n = 2) or the rapid isolation protocol with collagenase IV and DNase Manual cell isolation (unfilled red-lined bar, mean, n = 2). Statistical significance was tested using Mann‒Whitney test. ( g ) Representative histograms showing the expression of CD69, CD8, CD45RA, CCR7, CXCR3, CCR6, CCR4, and CLA in the blood (unfilled black line) compared with that of skin-derived T cells processed with a commercially available whole-skin dissociation kit Commercial kit (black line filled with red) and the rapid isolation protocol with collagenase and DNase Manual cell isolation (unfilled red line). Histograms depict one representative dataset of two independent experiments. Differences did not reach significance. FSC-A, forward scatter area; ILC, innate lymphoid cell; ns, not significant; SSC-A, side scatter area; UMAP, unsupervised uniform manifold approximation and projection analysis.

Article Snippet: The optimized rapid digestion protocol was compared with a commercially available whole skin dissociation kit, following the manufacturers’ instructions with minor modifications (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Flow Cytometry, Incubation, Isolation, Cell Isolation, Expressing, Derivative Assay