Journal: International Journal of Molecular Medicine

Article Title: Exogenous spermine attenuates diabetic kidney injury in rats by inhibiting AMPK/mTOR signaling pathway

doi: 10.3892/ijmm.2021.4860

Figure Lengend Snippet: Spermine attenuates podocyte injury and promotes autophagy in T1D rats. (A) Representative immunoblotting of nephrin, CD2-AP and podocin protein levels in kidney sections. (B) Representative immunohistochemical staining for podocin in kidney sections (scale bar, 100 µ m) and its quantification. (C) Representative immunoblotting for Atg5, P62, Beclin1 and LC3II/LCI in kidney sections. (D) Representative immunohistochemical staining for LC3 in kidney sections (scale bar, 100 µ m) and its quantification. Data are expressed as the mean ± standard error of the mean (n=6). * P<0.05 vs. the control group; # P<0.05 vs. the T1D group. T1D, type 1 diabetic; CD-2AP, CD2-associated protein; Atg5, autophagy protein 5; LC3, microtube-associated proteins 1A/1B light chain 3; Sp, spermine-treated group.

Article Snippet: After blocking with TBS with 0.1% Tween-20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C, the membrane was incubated overnight at 4°C with antibodies against ODC (1:1,000; cat. no. ab97395; Abcam), SSAT (1:1,000; cat. no. ab105220; Abcam), nephrin (1:400; cat. no. BA1669; Boster Biological Technology), podocin (1:400; cat. no. BA3416; Boster Biological Technology), CD2-associated protein (CD-2AP; 1:1,000; cat. no. bs-0512R; BIOSS), microtube-associated proteins 1A/1B light chain 3 (LC3; 1:1,000; cat. no. 3868T; Cell Signaling Technology, Inc.), Beclin 1 (1:1,000; cat. no. 3495T; Cell Signaling Technology, Inc.), P62 (1:1,000; cat. no. 8025T; Cell Signaling Technology, Inc.), autophagy protein 5 (Atg5; 1:1,000; cat. no. 12994T; Cell Signaling Technology, Inc.), cleaved caspase-3 (1:1,000; cat. no. 9661T; Cell Signaling Technology, Inc.), total (t)-mTOR (1:1,000; cat. no. A00003-2; Boster Biological Technology), phosphorylated (p)-mTOR (1:600; cat. no. BM4840; Boster Biological Technology), t-AMPK (1:1,000; cat. no. A00994-6; Boster Biological Technology), and p-AMPK (1:1,000; cat. no. P00994; Boster Biological Technology).

Techniques: Western Blot, Immunohistochemical staining, Staining

Journal: International Journal of Molecular Medicine

Article Title: Exogenous spermine attenuates diabetic kidney injury in rats by inhibiting AMPK/mTOR signaling pathway

doi: 10.3892/ijmm.2021.4860

Figure Lengend Snippet: Spermine reduces HG-induced apoptosis via activating autophagy in podocytes. (A) Cell Counting Kit-8 assays were performed using podocytes incubated under HG conditions in the presence of spermine or rapamycin. (B) Hoechst 33342 staining (scale bar, 500 µ m). (C) Representative immunoblotting analyses of the proteins levels of nephrin, CD-2AP and podocin protein levels. (D) Representative images of immunofluorescence staining for nephrin under different culture conditions (scale bar, 200 µ m). (E) Representative immunoblotting analyses for P62, Beclin1, LC3II/LC3I and cleaved caspase-3. Data are expressed as the mean ± standard error of the mean (n=6-8). * P<0.05 vs. the NG group; # P<0.05 vs. the HG group. HG, high glucose; CD-2AP, CD2-associated protein; LC3, microtube-associated proteins 1A/1B light chain 3; Sp, spermine-treated group; NG, normal glucose.

Article Snippet: After blocking with TBS with 0.1% Tween-20 (TBST) containing 5% non-fat dry milk for 1 h at 4°C, the membrane was incubated overnight at 4°C with antibodies against ODC (1:1,000; cat. no. ab97395; Abcam), SSAT (1:1,000; cat. no. ab105220; Abcam), nephrin (1:400; cat. no. BA1669; Boster Biological Technology), podocin (1:400; cat. no. BA3416; Boster Biological Technology), CD2-associated protein (CD-2AP; 1:1,000; cat. no. bs-0512R; BIOSS), microtube-associated proteins 1A/1B light chain 3 (LC3; 1:1,000; cat. no. 3868T; Cell Signaling Technology, Inc.), Beclin 1 (1:1,000; cat. no. 3495T; Cell Signaling Technology, Inc.), P62 (1:1,000; cat. no. 8025T; Cell Signaling Technology, Inc.), autophagy protein 5 (Atg5; 1:1,000; cat. no. 12994T; Cell Signaling Technology, Inc.), cleaved caspase-3 (1:1,000; cat. no. 9661T; Cell Signaling Technology, Inc.), total (t)-mTOR (1:1,000; cat. no. A00003-2; Boster Biological Technology), phosphorylated (p)-mTOR (1:600; cat. no. BM4840; Boster Biological Technology), t-AMPK (1:1,000; cat. no. A00994-6; Boster Biological Technology), and p-AMPK (1:1,000; cat. no. P00994; Boster Biological Technology).

Techniques: Cell Counting, Incubation, Staining, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: Glia multitask to compensate for neighboring glial cell dysfunction

doi: 10.1101/2024.09.06.611719

Figure Lengend Snippet: ( A-B ) Control VNCs with CG (green), astrocytes (magenta), and Crz neurons (anti-Crz, gray) show normal Crz+ neuronal cell bodies and projections at L3 (A), but by 6 hours after pupal formation (h APF) the cell bodies are mostly cleared by CG and the projections are cleared by astrocytes (B). ( C-D ) When CG expressing RNAi against Spz3 exhibit disrupted CG morphology, more Crz+ neuron cell bodies remain, while the projections are still cleared. Scale bars = 10 µm. White arrowheads denote Crz+ neuronal projections while the yellow arrowheads denote the Crz+ neuronal cell bodies. ( E ) Quantification of the number of Crz+ neuronal cell bodies at L3 and 6h APF and ( F ) Crz neurite debris remaining at 6h APF. ( G-H ) Control brains with CG (green), EG (magenta), neuronal nuclei (blue), and or88a-CD2 (anti-ratCD2, gray) show or88a+ neuronal projections in the antenna lobe in uninjured animals (G), but these projections are cleared by EG following injury partially at 3 days post injury (DPI) and fully by 5 DPI (H). ( I-J ) When CG morphology is disrupted with Spz3 RNAi, or88a+ neurons are still cleared by EG. Scale bars = 10 µm. ( K-L ) Quantification of the or88a+ volume normalized to the volume of the antennal lobe in uninjured control and Spz3 knockdown animals (K) and at 3 and 5 DPI (L). ( M ) Control brains with CG (green) exposed to 70 kDa Dextran (magenta) successfully block Dextran from the L3 CNS. ( N ) When CG express Spz3 RNAi , Dextran is still excluded from the L3 CNS, suggesting an intact BBB and functioning SPG. ( O ) Quantification of the area of Dextran within the L3 CNS. N = numbers of animals, denoted in each corresponding bar. (n.s.) Not significant. (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. (E, F, K) Unpaired t-test, (L,O) One-Way ANOVA with multiple comparisons.

Article Snippet: The following primary antibodies were used: chicken anti-GFP (1:1000; Antibodies Inc), rat anti- mCherry (1:1000, Invitrogen); rabbit anti-DsRed (1:1000; Clontech) rat anti-Elav (1:100; Developmental Studies Hybridoma Bank [DSHB], 7E8A10), mouse anti-Elav (1:100, DSHB, 9F8A9), Anti-β-Galactosidase (1:1000; Promega, Z3781), mouse anti-Repo (1:5; DSHB, 8D12), mouse anti-Prospero (1:100; DSHB, MR1A), rabbit anti-DCP1 (1:100; Cell Signaling, 9578S), rabbit anti-corazonin (1:500, Freeman Lab), mouse anti-nc82 (1:20; DSHB, nc82), mouse anti- ratCD2 (1:200; BioRad, MCA154GA), rabbit anti-p35 (1:1000, Fisher Scientific, NB10056153T).

Techniques: Control, Expressing, Knockdown, Blocking Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Impaired Lymphocytes Development and Xenotransplantation of Gastrointestinal Tumor Cells in Prkdc -Null SCID Zebrafish Model 1

doi: 10.1016/j.neo.2016.06.007

Figure Lengend Snippet: IF and RT-PCR analyses for activated lymphocytes. (A) IF detected by CD2AP marker. The CD2AP-reactive lymphocytes are present in the peripheral blood from both SCID and control zebrafish, suggesting that the cells are either activated T lymphocytes or NK cells. DAPI staining was used to detect the blood cells. (B) Double IF detected by CD2AP (Cy2) and NKP46 (Cy3) markers. The NKP46 is a specific marker for NK cells, whereas the CD2AP marker is reactive to both T lymphocytes and NK cells. (C) RT-PCR analysis. Glyceraldehyde 3-phosphate dehydrogenase gene detection was used as control. Nonspecific cytotoxic cell receptor protein-1 and NK specific protein (NK cell triggering receptor) gene detections indicate NK cell. TCR V(D)JC (Vb12-Cb1) and immunoglobulin (IgVH1-Cm) gene transcript detections reveal V(D)J recombination and heavy-chain rearrangement. (D) Counted numbers of cells depending on CD2AP or NKP46 positivity. Green arrowhead, positive for CD2AP; red arrowhead, positive for NKP46; white arrowhead, positive for both CD2AP and NKP46; blue arrowhead, lymphocytes negative for both CD2AP and NKP46; yellow arrowhead; red blood cells.

Article Snippet: Immunofluorescence (IF) experiments were performed by using the cell smears with the primary antibodies rabbit anti–CD2-associated protein (CD2AP) (sc-9137, Santa Cruz Biotechnology Inc.) and mouse anti-NKP46 (sc-53,599, Santa Cruz Biotechnology Inc.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Marker, Control, Staining

Journal: Academic radiology

Article Title: Natural Killer Cell-Associated Radiogenomics Model for Hepatocellular Carcinoma: Integrating CD2 and Enhanced CT-Derived Radiomics Signatures.

doi: 10.1016/j.acra.2024.10.043

Figure Lengend Snippet: Figure 3. Identification and validation of CD2 an NK cell-related prognostic biomarker for HCC patients. (a) Selection of the λ parameter in LASSO-Cox regression. Vertical lines indicate partial likelihood deviance for each λ value, with the minimum deviance at an optimal λ of 0.05. (b) Process of deriving the four candidate genes with non-zero coefficients at the optimal λ value. The vertical axis shows the regression coefficients, and the horizontal axis shows the log(λ) values. (c) Forest plot of multivariable Cox regression analysis, confirming CD2 as an independent prognostic factor among the four selected genes. Kaplan–Meier analysis of the TCGA-LIHC cohort. High CD2 expression is significantly associated with longer (d) OS and (e) PFS. (f) Kaplan–Meier analysis of the GSE76427 cohort, showing that high CD2 expression is significantly associated with longer OS. Kaplan–Meier analysis of the GSE14520 cohort. High CD2 expression is significantly associated with longer (g) OS and (h) RFS. (i) Kaplan–Meier analysis of the TCIA-TCGA-LIHC cohort, showing that high CD2 expression is significantly associated with longer OS. (j) Ridgeline plot illustrating GSEA results, highlighting significant positive correlations between CD2 expression and KEGG processes. (k) GSEA indicated that high CD2 expression was enriched in NK cell-mediated cytotoxicity signaling. HCC, Hepatocellular carcinoma; KEGC, Kyoto Encyclopedia of Genes and Genomes; LASSO, Least absolute shrinkage and selection operator; NK, Natural killer; RFS, Recurrence-free survival; TCGA-LHIC, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma.

Article Snippet: Sections were then incubated overnight at 4 °C with a primary rabbit monoclonal antibody against CD2 (ABclonal, China, A23199, 1:200 dilution).

Techniques: Biomarker Discovery, Selection, Expressing

Journal: Academic radiology

Article Title: Natural Killer Cell-Associated Radiogenomics Model for Hepatocellular Carcinoma: Integrating CD2 and Enhanced CT-Derived Radiomics Signatures.

doi: 10.1016/j.acra.2024.10.043

Figure Lengend Snippet: Figure 4. Radiomics model construction and validation. (a) Representative enhanced CT arterial phase image of a patient with HCC, with the white arrow indicating the location of the tumor. (b) ROI outlining, with the green area showing the ROI, and the 3D image of the ROI displayed in the upper-right corner. (c) Selection of the λ parameter for the LASSO-Cox model. The vertical lines indicate partial likelihood deviance for each λ value, with the optimal λ at 0.12. (d) Use of the optimal value of λ to select three features. (e, g, i) Kaplan–Meier analysis of OS based on radioscore stratification in the TCIA-TCGA-LIHC, TCIA-TACE-seg, and Tongji cohorts. (f, h, j) Distribution of radioscores, survival time, survival status, and heatmaps of selected radiomics features for each cohort. (k) Representative immunohistochemistry images for CD2 expression in samples from the Tongji cohort. (l) Differences in radioscores among HCC patients with different TNM or BCLC stages across the three cohorts. HCC, Hepatocellular carcinoma; LASSO, Least absolute shrinkage and selection operator; OS, Overall survival; TCIA-TCGA-LIHC, The Cancer Imaging Archive-The Cancer Genome Atlas-Liver Hepatocellular Carcinoma; TACE, Transarterial chemoembolization .

Article Snippet: Sections were then incubated overnight at 4 °C with a primary rabbit monoclonal antibody against CD2 (ABclonal, China, A23199, 1:200 dilution).

Techniques: Biomarker Discovery, Selection, Immunohistochemistry, Expressing, Imaging

Journal: Academic radiology

Article Title: Natural Killer Cell-Associated Radiogenomics Model for Hepatocellular Carcinoma: Integrating CD2 and Enhanced CT-Derived Radiomics Signatures.

doi: 10.1016/j.acra.2024.10.043

Figure Lengend Snippet: Figure 5. Overview of the comparison of the four HCC radiogenomics subtypes defined by CD2 expression and radioscore. (a) Sankey diagram of samples distribution in four radiogenomics subtypes based on CD2 expression and radioscore. (b) Kaplan–Meier analysis of OS in the four radiogenomics subgroups of patients with HCC patients. p values for comparisons between each pair of groups are shown in the upper left corner. (c) KEGG enrichment analysis of the overlapping genes from the differential gene expression analysis of the high CD2 plus low radioscore subgroup and the other three subgroups. (d) Heatmap of GSVA results for NK cell-related pathways and biological processes for each radiogenomics subgroup. (e) Mutation waterfall plot of the top 20 genes with the highest mutation frequency in the patients of the four subgroups. GSVA, Gene Set Variation Analysis; KEGC, Kyoto Encyclopedia of Genes and Genomes; NK, Natural killer.

Article Snippet: Sections were then incubated overnight at 4 °C with a primary rabbit monoclonal antibody against CD2 (ABclonal, China, A23199, 1:200 dilution).

Techniques: Comparison, Expressing, Gene Expression, Mutagenesis

Journal: Frontiers in Genetics

Article Title: Identification of Novel Prognostic Biomarkers Relevant to Immune Infiltration in Lung Adenocarcinoma

doi: 10.3389/fgene.2022.863796

Figure Lengend Snippet: The correlations between PSTPIP1/PILRA and immune biomarkers in LUAD. Scatter plots showing the correlation between (A) PSTPIP1 and PD-L1, (B) PSTPIP1 and IFN-γ, (C) PILRA and PD-L1, and (D) PILRA and IFN-γ in the LUAD samples ( n = 515). Violin plots showing the expression of (E) PSTPIP1 and (F) PILRA in the LUAD samples with different degrees of differentiation (including 167 with poor differentiation, 209 with moderate differentiation, and 60 with well differentiation). ns indicates p ≥ 0.05, * p < 0.05.

Article Snippet: Next, the slices were incubated with the primary antibodies anti-PSTPIP1 (11951-1-AP, rabbit, polyclonal, dilution 1:50, Proteintech, Wuhan, China), anti-PILRA (orb38981, rabbit, polyclonal, dilution 1:200, Biorbyt, Cambridge, United Kingdom), and PBS (blank control) overnight at 4°C, washed three times with PBS, and incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody (ab205718, Abcam, Cambridge, United Kingdom) for 30 min.

Techniques: Expressing

Journal: Frontiers in Genetics

Article Title: Identification of Novel Prognostic Biomarkers Relevant to Immune Infiltration in Lung Adenocarcinoma

doi: 10.3389/fgene.2022.863796

Figure Lengend Snippet: The influence of PSTPIP1 and PILRA on immune infiltration and prognosis in LUAD. Violin plots showing (A) the immune score in samples with low or high expression of PSTPIP1 and PILRA. GSVA-derived clustering heat maps of differentially infiltrated immune cell populations between the high and low expression groups of (B) PSTPIP1 and (C) PILRA. Only lymphocytes with log(fold change) > 0.2 are shown. The influence of (D) PSTPIP1 and (E) PILRA on the overall survival time of LUAD patients. The yellow line indicates samples with highly expressed genes and the blue line indicates samples with lowly expressed genes. Violin plots showing (F) the expression of PSTPIP1 and PILRA in the LUAD patients with different responses to nivolumab (including five responders and five nonresponders) and (G) the expression of PSTPIP1 in SKCM mice with different responses to anti–PD-L1 treatment (including 27 responders and 23 nonresponders). ns indicates p ≥ 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Next, the slices were incubated with the primary antibodies anti-PSTPIP1 (11951-1-AP, rabbit, polyclonal, dilution 1:50, Proteintech, Wuhan, China), anti-PILRA (orb38981, rabbit, polyclonal, dilution 1:200, Biorbyt, Cambridge, United Kingdom), and PBS (blank control) overnight at 4°C, washed three times with PBS, and incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody (ab205718, Abcam, Cambridge, United Kingdom) for 30 min.

Techniques: Expressing, Derivative Assay

Journal: Frontiers in Genetics

Article Title: Identification of Novel Prognostic Biomarkers Relevant to Immune Infiltration in Lung Adenocarcinoma

doi: 10.3389/fgene.2022.863796

Figure Lengend Snippet: Histopathological examples of PSTPIP1/PILRA expression and tumor-infiltrating lymphocytes (TILs) in LUAD. (A) PSTPIP1 and PILRA expression is demonstrated by brown staining, and the bar plot shows the average optical density of PSTPIP1 and PILRA in the PD-L1–positive or PD-L1–negative LUAD tissue samples. (B) The TILs are displayed as purple spots in hematoxylin and eosin staining, and bar plots show the stromal TILs in tissue samples with high and low PSTPIP1/PILRA expressions. * p < 0.05.

Article Snippet: Next, the slices were incubated with the primary antibodies anti-PSTPIP1 (11951-1-AP, rabbit, polyclonal, dilution 1:50, Proteintech, Wuhan, China), anti-PILRA (orb38981, rabbit, polyclonal, dilution 1:200, Biorbyt, Cambridge, United Kingdom), and PBS (blank control) overnight at 4°C, washed three times with PBS, and incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody (ab205718, Abcam, Cambridge, United Kingdom) for 30 min.

Techniques: Expressing, Staining