Journal: The Journal of Neuroscience

Article Title: Cyclin-Dependent Kinase 5 Is Required for Control of Neuroblast Migration in the Postnatal Subventricular Zone

doi: 10.1523/JNEUROSCI.1014-07.2007

Figure Lengend Snippet: Emx1-mediated Cdk5 conditional KO mice have a defect in neuroblast migration, demonstrated by BrdU labeling. A, C, E, G, One hour after BrdU pulse labeling. There was no significant difference in the density of BrdU+ cells between Cdk5 ECKO and control brains in either the SVZ (A, C; control, 88,319 ± 16,806 cells/mm3; Cdk5 ECKO, 70,817 ± 6089 cells/mm3; n = 3; p = 0.3829) or the OB (E, G; control, 4219 ± 1513 cells/mm3; Cdk5 ECKO, 5582 ± 1008 cells/mm3; n = 3; p = 0.4949). B, D, F, H, Five days after BrdU pulse labeling. B, D, The number of labeled cells retained in the SVZ was greater in the Cdk5 ECKO brains (D; 7380 ± 849 cells, n = 3) than in the controls (B; 4665 ± 401 cells, n = 3). F, H, The number of labeled cells in the OB was lower in the Cdk5 ECKO brains (H; 66,472 ± 6525 cells, n = 3) than in the controls (F; 119,071 ± 7569 cells, n = 3). A′–D′, Higher-magnification views of the portion of SVZ marked by rectangles in A–D. Scale bars: A–D, E–H, 200 μm; A′–D′, 50 μm.

Article Snippet: The following primary antibodies were used: rabbit anti-β-galactosidase antibody, 1:2000 (Biogenesis, Poole, UK); mouse anti-GFAP antibody, 1:200 (Sigma, St. Louis, MO); mouse anti-Mash1 (mammalian achaete-scute homolog 1) antibody, 1:100 (PharMingen, San Diego, CA); goat anti-Dcx antibody, 1:200 (Santa Cruz Biotechnology, Santa Cruz, CA); rat anti-bromodeoxyuridine (BrdU) antibody, 1:100 (AbCam, Cambridge, MA); rabbit anti-Ki67 antibody, 1:800 (Novocastra, Newcastle upon Tyne, UK); and rabbit anti-green fluorescent protein (GFP) antibody, 1:100 (MBL, Aichi, Japan).

Techniques: Migration, Labeling

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 2. Down-regulation of G9a represses cell proliferation of GC. A) Western blotting and real-time qPCR assays were performed to detect the G9a expression in 3 G9a-knockdown GC cell lines. Tubulin was used as a loading control. B) G9a knockdown repressed the growth and proliferation of HGC-27, MKN-45, and SGC-7901 cells. Cell viability was detected using CCK8 assays. C) GC cells were treated with BIX01294 and analyzed for cell viability using CCK8 assays. D) Ki67 immunofluorescence assays were performed after G9a knockdown or inhibition. Cells were treated with 7 mM BIX01294 for 48 h. Representative images show immunofluorescence. Scale bars, 20 mm. All data are shown as means 6 SD; n = 3. *P , 0.05, **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Expressing, Knockdown, Control, Inhibition

Journal: The FASEB Journal

Article Title: G9a promotes cell proliferation and suppresses autophagy in gastric cancer by directly activating mTOR

doi: 10.1096/fj.201900233rr

Figure Lengend Snippet: Figure 3. Down-regulation of G9a represses colony formation in vitro and tumor formation of GC cells in vivo. A, B) The effects of G9a on the colony formation in 3 G9a-knockdown GC cell lines. Scale bars, 20 mm. The colony numbers in plate were quantified. C, D) The tumor growth curve and tumor weight of G9a knockdown GC cells injected into NOD-SCID mice. E) Immunohistochemical (IHC) staining of G9a expression (left) and Ki67 expression (right). Scale bars, 20 mm. F) The quantification of G9a-positive cells (left) and Ki67-positive cells (right). All data are shown as means 6 SD; n = 3. **P , 0.01, ***P , 0.001.

Article Snippet: G9a (ab40542), Ki67 (ab92742), Unc-51–like autophagyactivating kinase 1 (ULK1; ab128859), Beclin-1 (ab207612), p62/ SQSTM1 (ab207305), mTOR (ab32028), H3K9me1 (ab9045), H3K9me2 (ab1220), and p-H3S10 (5176) antibodies were purchased from Abcam (Cambridge, MA, USA). p(Ser2448)-mTOR (D9C2), CyclinA2 (BF683), CyclinB1 (D5C10), p(Ser757)-ULK1 (D7O6U), p70S6K (D5U1O), CDK1 (POH1), and LC3B (D11) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vitro, In Vivo, Knockdown, Injection, Immunohistochemical staining, Immunohistochemistry, Expressing

Journal: Cell Reports Medicine

Article Title: Neoadjuvant Fc-enhanced anti-CTLA-4 targets Tregs to augment androgen deprivation in high-risk prostate cancer: A randomized phase I trial

doi: 10.1016/j.xcrm.2026.102638

Figure Lengend Snippet: Phenotypic modulation of T cells and enhanced priming by anti-CTLA4-NF (A) PaCMAP plot of NeoRED-P patient tumor-infiltrating CD8 T cells by CyTOF. Clusters were derived from FlowSOM. (B) Pseudocolor density plots of CD8 T cells in PaCMAP space stratified by treatment group. (C) Expression of CD39 and 4-1BB by geometric MI on CD8 T cells as represented by color mapping on PaCMAP plot. (D) Violin plot representing frequency of manually gated CD39 + 4-1BB + CD8 T cells as a percentage of all CD8 T cells stratified by treatment group. Untreated, n = 7; ADT, n = 8; ADT + anti-CTLA4-NF, n = 8. Single patient with MSI hi status called out in plot. For this and all violin plots to follow, solid lines denote group medians, while dashed lines denote quartiles. (E) PaCMAP plot of NeoRED-P patient tumor-infiltrating CD4 + FoxP3 - Tconv cells by CyTOF. Clusters were derived from FlowSOM. (F) Pseudocolor density plots of CD4 Tconv cells in PaCMAP space stratified by treatment group. (G) Expression of CD39 and 4-1BB by geometric MI on CD4 Tconv as represented by color mapping on PaCMAP plot. (H) Violin plot representing frequency of manually gated CD39 + 4-1BB + CD4 Tconv cells as a percentage of all CD8 T cells stratified by treatment group. Untreated, n = 7; ADT, n = 8; ADT + anti-CTLA4-NF, n = 8. Single patient with MSI hi status called out in plot. (I) PaCMAP plot of MycCaP-infiltrating CD8 T cells by 45-parameter flow cytometry in response to ADT, ADT + anti-CTLA4 (ND), or ADT + anti-CTLA4 (D). Clusters were derived from FlowSOM. (J) Pseudocolor density plots of CD8 T cells in PaCMAP space stratified by treatment group. (K) Expression of CD39 and 4-1BB by geometric MFI on CD8 T cells as represented by color mapping on PaCMAP plot. (L) Violin plot representing frequency of manually gated CD39 + 4-1BB + CD8 T cells as a percentage of all CD8 T cells stratified by treatment group. (M) Biaxial plots representing expression of CD44 and Ki67 on CD8 T cells in tumor-draining lymph nodes of mice shown in (I–L). (N) Biaxial plots representing expression of CD44 and Ki67 on CD4 + FoxP3 − Tconv cells in tumor-draining lymph nodes of mice shown in (L–O). (O) Violin plots representing frequencies of CD44 + Ki67 + CD8 and CD4 Tconv cells as a percentage of parent populations stratified by treatment group. Two-tailed Welch’s t test was used to assess statistical significance. All murine data shown are n = 7 per group and representative of two independent experiments each for survival and immune profiling studies.

Article Snippet: Anti-Human Ki67 (B56) 172Yb , Standard BioTools , Cat# 3172024B.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Tissue-resident macrophage survival depends on mitochondrial function regulated by SerpinB2 in chronic inflammation

doi: 10.1038/s41467-026-69196-4

Figure Lengend Snippet: A Flowcytometric analysis of the macrophage subsets in visceral adipose tissue of CX 3 CR1 +/GFP mice which express GFP under the CX 3 CR1promoter. B tdTomato + (CX 3 CR1 high ) macrophages were quantified at day 7 after a single i.p injection of tamoxifen in CX 3 CR 1 CreER/+ ROSA tdTomato/+ mice that express YFP under the CX 3 CR1 promoter and tdTomato in CX 3 CR1-expressing cells upon tamoxifen injection using intravital microscopy (n = 6/group). C Parabiosis between C57BL/6 and CX 3 CR1 GFP/+ mice was performed. Flow cytometry was conducted to enumerate chimerism in the macrophage subsets in the C57BL/6 mice six months after parabiosis. (n = 5 for CX 3 CR1 − and 6 for CX 3 CR1 + ). D Heatmap displaying the genes with at least a two-fold difference between the VAT macrophage subsets and with FDR < 0.01 (n = 3/group). E – G Heatmaps displaying the expression of the genes using bulk RNA sequencing comparing CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − macrophages (n = 3/group) and CD206 − and CD206 + macrophage subsets of VAT (n = 3/group). H q-PCR quantification of the genes associated with glycemia and diabetes in CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − macrophages sorted from VAT of lean mice (n = 6–12/group). I Bar graph representing the frequency of CX 3 CR1 + CCR2 + and CX 3 CR1 − CCR2 − VAT macrophages enriched in the insulin sensitivity and resistance genes shown in ( G , H ) (n = 3-6 /group). J PCA plot showing the relations among the genes responsible for insulin sensitivity, survival, resident macrophage (ATM) markers, inflammation, insulin resistance, and monocyte-derived macrophages (MDM) markers in the VAT macrophage subsets using bulk RNA sequencing. K , L Frequencies of CCR2 + and CCR2 − macrophage subsets in human VAT as measured by flow cytometry ( K ) (n = 5/group) and confocal microscopy ( L ) (n = 15 for lean and 13 for obese). M – O Quantification of the VAT macrophage subsets in HFD-fed mice by flow cytometry (n = 4/group) ( M ) and serial intravital microscopy (Scale bar = 10 µm) ( N , O ) was performed in lean and obese CX 3 CR1 CreER/+ ROSA tdTomato/+ mice (n = 3 for CD, 4 for HFD 2 months, and 5 for HFD 4 months). P – R Apoptosis in VAT resident and monocyte-derived macrophages in lean and obese mice was examined using annexin V by flow cytometry ( P ) (n = 4/group), and caspase 3 staining by flow cytometry ( Q ) (n = 5 for CD and 4 for HFD/group) and confocal microscopy ( R ) (n = 14/group). S Quantification of the VAT macrophage subsets in lean and obese CX 3 CR1 creER/+ ROSA tdTomato mice before and after removal of HFD (n = 7/group). T Evaluation of Ki-67 + VAT resident macrophages after HFD withdrawal (n = 10/group). Mean ± s.e.m. * P < 0.05, ** P < 0.01, *** P < 0.001. The Mann–Whitney test (two-tailed) was used to determine the significance between two groups. One-way ANOVA with Bonferoni’s post hoc correction test was performed to determine differences among data obtained from more than two groups (Fig. 1O and S).

Article Snippet: Tissues were incubated for 48 hours with the primary antibodies against proteins like F4/80 (Invitrogen, #MAI-91124), cleaved caspase 3 (Abcam, #ab13847), CD11b (Abcam, #ab133357), CX 3 CR1 (Abcam, #ab8021), CCR2 (Bio-Rad, #AAM72), Ki67 , , , CD68 (ThermoFisher Scientifics, # 14-0688-82), and SerpinB2 (Invitrogen, #PA5-27857) followed by washing with PBS and incubation for 24 hours at 4 °C with Alexa fluor 488, 594 and 647-conjugated secondary antibodies.

Techniques: Injection, Expressing, Intravital Microscopy, Flow Cytometry, RNA Sequencing, Derivative Assay, Confocal Microscopy, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 1 TKI therapy alters the tumor immune microenvironment. (A) Representative mIF images of pretreatment tumor and resected samples analyzed for immune-related biomarkers. (B) Densities (cells/mm2) of CD8+, CD8+GB+, CD8+PD-1+, CD163+, CD68+, and CD163+CD68+ by mIF quantification in paired pretreatment tumor samples and resected tumors. (C) Cell viability CCK-8 assay for cells treated with TKIs (osimertinib: 10 nM, lorlatinib: 10 nM), activated T cells (1:1 ratio to cancer cells), or the combination. (D) T cell-mediated cancer cell-killing assay. PC-9 and H3122 cells co-cultured with activated T cells for 48 hours with or without TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were subjected to crystal violet staining. Ratio of cancer cells to T cells: 1:1. (E) Ki67 incorporation assay on PC-9 and H3122 cells treated as indicated. Activated T cells (1:1 ratio to cancer cells) or TKIs (osimertinib: 10 nM, lorlatinib: 10 nM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into mice via the tail vein on day 7, and osimertinib was administered as indicated. (G) Macroscopic appearance of tumors after drug application for 4 weeks. (H) The tumor weight (g) for each mouse is shown. *p<0.05, **p<0.01. ns, no significance. (I) Immunofluorescence staining with an antibody against CD8 to detect T cells and antibodies against CD68 and CD206 to detect macrophages in TKI-resistant non-small cell lung cancer tissues (11 cases of EGFR-TKI resistance, 5 cases of ALK-TKI resistance). Scale bar: 50 µm. ALK, anaplastic lymphoma kinase; DAPI, 4′,6-diamidino-2-phenylindole; EGFR, epidermal growth factor receptor; ALKi,anaplastic lymphoma kinase inhibitor; EGFRi,epidermal growth factor receptor inhibitor; hu-PBMC,human-Peripheral blood mononuclear cell; mIF, multiplex immunofluorescence; PD-1, programmed cell death protein 1; TKI, tyrosine kinase inhibitor.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: CCK-8 Assay, Cell Culture, Staining, Injection, Immunofluorescence, Multiplex Assay

Journal: Journal for immunotherapy of cancer

Article Title: Understanding the dynamics of TKI-induced changes in the tumor immune microenvironment for improved therapeutic effect.

doi: 10.1136/jitc-2024-009165

Figure Lengend Snippet: Figure 6 Aspirin enhances the antitumor immunity response. (A) PC-9 cells were injected into mice (n=3 mice per group) on day 0, hu-PBMCs were injected into the tail veins of mice on day 7, and osimertinib/osimertinib plus aspirin was administered as indicated. (B) Macroscopic appearance of tumors after drug application for 4 weeks. The tumor weight (g) of each mouse is shown. *p<0.05, ***p<0.001. (C) Cell viability CCK-8 assay for cells treated with aspirin (200 µM), activated T cells (1:2 ratio to cancer cells), or the combination for 48 hours. Data are shown as the mean±SEM. *p<0.01. (D) T cell-mediated cancer cell-killing assay. PC-9OR and HCC827OR cells co-cultured in the indicated groups for 48 hours were subjected to crystal violet staining. Ratio of cancer cells to T cells: 2:1. (E) Ki67 incorporation assay on PC-9OR and HCC827OR cells treated as indicated. Activated T cells (1:2 ratio to cancer cells) or aspirin (200 µM) were added to the culture medium for 48 hours. Cells were then counterstained with DAPI. (F) Flow cytometry analysis of the exhaustion- and activation-related molecule Foxp3 in activated T cells co-cultured with the indicated cells (ratio of cancer cells to T cells: 1:1) for 48 hours with or without aspirin (200 µM). (G) PD-L1 levels in total protein extracts from indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (H) LAMC2 levels in total protein extracts from the indicated cells treated with aspirin for 48 hours, analyzed by western blotting. (I) Immunofluorescence staining with an antibody against LAMC2 in PC-9 xenografts treated with osimertinib or osimertinib plus aspirin. (J) Schematic diagram of a T-cell chemotaxis assay directly regulated by the treatment of PC-9OR cells with aspirin (200 µM). Representative images of infiltrating T cells stained with CFSE dye. (K) Representative images of the immunofluorescence staining of CD68 (red), CD206 (green), and DAPI (blue) in mouse tumor tissue sections. (L) Immunofluorescence staining with antibodies against CD8 (red) and LAMC2 (green) in non-small cell lung cancer tissues treated with osimertinib or osimertinib plus aspirin. Scale bar: 50 µm.hu-PBMC,human-Peripheral blood mononuclear cell; S.C,Subcutaneous; CFSE, carboxyfluorescein diacetate succinimidyl ester; DAPI, 4′,6-diamidino-2-phenylindole; Foxp3, forkhead box P3; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; LAMC2, laminin subunit γ−2; PBS, phosphate-buffered saline; PD-L1, programmed death ligand 1.

Article Snippet: Then, the cells were fixed and incubated overnight with Ki67 (#M00254- 8, Boster, Wuhan, China).

Techniques: Injection, CCK-8 Assay, Cell Culture, Staining, Flow Cytometry, Activation Assay, Western Blot, Immunofluorescence, Chemotaxis Assay, Saline