Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Western blotting to detect the expression levels of LMNA and LMNB1 in cells. (A) The protein signal bands for LMNA, LMNB1, and GAPDH. (B, C) The relative expression levels of LMNB1 and LMNA (normalized to GAPDH expression) in different cell lines. The mean ± SEM of two independent experiments are shown (P = 0.0005 and P < 0.0001 for LMNB1 and LMNA, respectively; ANOVA). The results show that LMNB1-overexpressing EP156T (LMN-EP156T) cells express significantly higher amounts of LMNB1 than the parental and Mock EP156T cells. All three prostate cancer cell lines express high levels of LMNB1. All cells express LMNA, with PC-3 and DU145 expressing higher amounts of LMNA than the other cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Western Blot, Expressing

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Immunohistochemical (IHC) staining of LMNB1 expression. Staining results were shown for (A) parental EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells (positive control). Both LMN-EP156T and PC-3 cells expressed strong nuclear staining of LMNB1, which was higher than either parental EP156T or Mock EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Staining, Positive Control

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: MTS assay to measure the rate of cell growth. A total of 6.5 × 103 cells in 0.1 ml of complete medium were seeded onto each well of 96 well-plates in triplicate for each cell line. Five repeated plates were prepared for measurements at 5 time points. At each time point, one plate was used for this assay. MTS reagent (20 μl) was added to each well, and the plates were incubated at 37°C for 1 h. The absorbance at 490 nm is shown as the mean ± SEM of three independent experiments. LMNB1-overexpressing EP156T (LMN-EP156T) cells show significantly faster growth compared to the other two cell lines (P < 0.01; ANOVA).

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: MTS Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Matrigel-coated transwell assay (37°C for 48 h, 40 × magnification). A total of 5 × 104 cells of each cell line in 0.1 ml medium + 0.1% FBS were seeded onto an insert well (pore size, 8 µm) for a 24-well plate pre-coated with Matrigel. The insert wells were placed on receiver wells with 0.65 ml of medium + 10% FBS per well. After incubation at 37°C for 48 h, the cells on the apical side of the insert wells were removed with swabs. Only cells that had passed the membrane could be visualized after staining in 20% methanol with 0.25% crystal violet for 10 min. The results are shown for (A) EP156T, (B) Mock EP156T, and (C) LMN-EP156T cells. (D) The number of cells that penetrated the wells are shown as the mean ± SEM of three independent experiments. Statistical significance was determined by ANOVA (P < 0.0001). Significantly more LMNB1-overexpressing EP156T (LMN-EP156T) cells passed through the 8-µm pores than parental and Mock EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Transwell Assay, Pore Size, Incubation, Membrane, Staining

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Colony-formation assay. Results are shown for (A) EP156T, (B) Mock EP156T, (C) LMNB1-overexpressing EP156T (LMN-EP156T), and (D) PC-3 cells. A total of 5 × 103 cells of each cell line were mixed in 0.3% soft agar with complete growth medium, and plated onto a culture dish (diameter, 3.5 cm). Numerous colonies can be seen in the plates inoculated with PC-3 after 2 weeks of incubation (magnification, 100 ×) at 37°C. However, no colonies have formed in the plates inoculated with the other cell lines. White arrows indicate PC-3 cell colonies.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Colony Assay, Incubation

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Xenograft mouse model showing evident subcutaneous tumors in the right chest 7 weeks after the injection of PC-3 cells. (A-D) Four of the five mice injected with PC-3 cells formed subcutaneous tumors (red arrows). The tumor sizes range between 13 mm and 19 mm in diameter. No tumors were seen in mice injected with EP156T, Mock EP156T, or LMNB1-overexpressing EP156T cells (photos not shown).

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Injection

Journal: American Journal of Cancer Research

Article Title: LMNB1, a potential marker for early prostate cancer progression

doi:

Figure Lengend Snippet: Hematoxylin and eosin-stained tissue sections from xenografted mice. (A) An enlarged and metastasized lymph node from the PC-3-injected mouse. (B-E) Lung sections from mice injected with PC-3, EP156T, Mock EP156T, and LMNB1-overexpressing EP156T cells, respectively. Each row shows the same section with increasing magnification. Red arrows indicate PC-3 metastatic cell nests with clear nucleoli in either the lymph node (A) or lung (B). There are no subcutaneous growths or metastatic lesions in mice injected with the parental, Mock, or LMNB1-overexpressing EP156T cells.

Article Snippet: EP156T prostate epithelial cells and transfection with lentiviral vectors harboring LMNB1 A human TERT -immortalized EP156T prostatic epithelial cell line was purchased from the American Type Culture Collection (CRL3289TM, ATCC, Manassas, VA, USA) and grown in MCDB-153 medium supplemented with bovine pituitary extract (25 mg/500 ml medium), hEGF (5 ng/ml medium), 1% fetal bovine serum (FBS), and 0.5 μg/ml puromycin.

Techniques: Staining, Injection

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3 and eotaxins are expressed in choroidal neovascularization. a,b, Immunofluorescence shows that CCR3 (green) receptor expression colocalizes with CD31 + (red) expressing blood vessels in surgically excised human age-related macular degeneration (AMD) choroidal neovascular (CNV) tissue. Nuclei stained blue by DAPI. b, Specificity of CCR3 staining is confirmed by absence of staining with isotype control IgG ( a ). Individual red and green fluorescence channels are shown in . c,d, CCR3 is not immunolocalized in CD31 + (red) blood vessels (white arrowheads) in the choroid of patients with atrophic AMD who do not have CNV ( c ) or in aged patients without AMD ( d ). Autofluorescence of retinal pigmented epithelium (white arrow) and Bruch's membrane (asterisks) overlying choroid is seen ( c,d ). e,f, CCR3 is not expressed in surgically excised avascular retinal fibrosis tissue ( e ) or in blood vessel of choroidal melanoma ( f ). g-j, Immunohistochemistry (golden brown reaction product) shows expression of CCL11 ( g ), CCL24 ( h ), and CCL26 ( i ) in surgically excised AMD CNV tissue, primarily in the stroma (red arrowheads) but also in the blood vessels (yellow arrows). Specificity of staining is confirmed by absence of staining with isotype control IgG ( j ). Scale bars, 10 μm.

Article Snippet: Immunofluorescent staining was performed with antibodies against human CCR3 (rat monoclonal, R&D Systems) or human CD31 (mouse monoclonal, Dako) and identified with Alexa 488 (Molecular Probes) or Cy3 secondary antibodies (Jackson ImmunoResearch).

Techniques: Immunofluorescence, Expressing, Staining, Control, Fluorescence, Membrane, Immunohistochemistry

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3 activation promotes angiogenesis. a, Tube formation of primary human choroidal endothelial cells (CECs) in Matrigel in vitro was reduced by neutralizing anti-human CCR3 antibodies (Ab) compared to isotype IgG. n = 6, * P < 0.05 compared to isotype IgG. b, Fraction of CD31 + VEGFR2 + gated mouse CECs in vivo in proliferative state (S phase) was increased 5 days after laser injury in wild-type mouse eyes compared to control (uninjured eyes), and was reduced by intraocular administration of neutralizing anti-mouse CCR3 Ab compared to isotype IgG. n = 6–10, * P < 0.05 compared to IgG treatment. c, Stimulation with eotaxins for 24 h induced human CEC proliferation. n = 4, * P < 0.05 compared to bovine serum albumin (BSA) treatment. d,e, Stimulation with eotaxins, but not PBS, induced actin polymerization in human CECs. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone-stimulated cells ( d ). Rhodamine-phalloidin staining (red) shows F-actin fibre formation in eotaxin-stimulated cells ( e ). Nuclei stained blue by DAPI. Data representative of 3–4 independent experiments are shown. c,e, CCL11 (10 ng/ml), CCL24 (100 ng/ml), CCL26 (2 μg/ml). f, Stimulation with eotaxins for 16 hours induces dose-dependent migration of human CECs across 8 μm pore size Transwells. n = 5–10, * P < 0.05 compared to BSA treatment. ( a–c, f ) Significance by Mann-Whitney U test. Error bars depict s.e.m.

Article Snippet: Immunofluorescent staining was performed with antibodies against human CCR3 (rat monoclonal, R&D Systems) or human CD31 (mouse monoclonal, Dako) and identified with Alexa 488 (Molecular Probes) or Cy3 secondary antibodies (Jackson ImmunoResearch).

Techniques: Activation Assay, In Vitro, In Vivo, Control, Fluorescence, Staining, Migration, Pore Size, MANN-WHITNEY

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CNV reduced by CCR3 or eotaxin ablation or blockade independent of leukocyte modulation. a,b, Laser-induced CNV in wild-type mice was reduced by neutralizing anti-mouse CCR3 Ab compared to isotype IgG ( a ) and by the CCR3 receptor antagonist (RA) SB328437 ((S)-Methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate) compared to vehicle (PBS/DMSO) ( b ) in a dose-dependent fashion. n = 8–12, * P < 0.05 compared to no antibody or receptor antagonist. c, Representative examples of CNV in drug-treated mice. d, Laser-induced CNV was reduced in Ccr3 −/− mice compared to wild-type mice. n = 9, * P < 0.05 compared to wild-type mice. e, Eotaxin-1 (Ccl-11) and eotaxin-2 (Ccl-24) protein levels, measured by ELISA, were increased following laser injury in wild-type mice. n = 6, * P < 0.05, # P < 0.01 compared to 0 h baseline. f, Ccl-11 and Ccl-24 immunofluorescence (green) was localized in the retinal pigmented epithelial cell layer (arrows) adjacent to CD31+ (red) choroidal endothelial cells (arrowheads) on day 1 after laser injury in wild-type mice. Nuclei stained blue by DAPI. No specific immunofluorescence was detected with isotype control IgGs. Images representative of 3 independent experiments are shown. g, Laser-induced CNV was reduced in Ccl11 −/− and in Ccl24 −/− mice compared to wild-type mice. n = 8–10, * P < 0.05 compared to wild-type mice. CNV is further reduced in Ccl11 −/− × Ccl24 −/− mice compared to single null mice. # P < 0.05 compared to single null mice. h, Laser-induced CNV in wild-type mice was reduced by neutralizing antibodies against mouse CCL11 or CCL24 compared to isotype IgG. n = 7–10, * P < 0.05 compared to no injection (control) or IgG. i, Representative examples of CNV in eotaxin-neutralized mice. j, Neutralizing anti-CCR3 antibodies (Ab) reduced laser-induced CNV in mice deficient in eosinophils (Δdbl GATA) or mast cells ( Kit w-v ). n = 6–9, * P < 0.05 compared to IgG. Scale bars, ( c,i ), 100 μm; f , 20 μm. Error bars depict s.e.m.

Article Snippet: Immunofluorescent staining was performed with antibodies against human CCR3 (rat monoclonal, R&D Systems) or human CD31 (mouse monoclonal, Dako) and identified with Alexa 488 (Molecular Probes) or Cy3 secondary antibodies (Jackson ImmunoResearch).

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Injection

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3-targeting quantum dots detect subretinal choroidal neovascularization (CNV). a, Images of the fundus taken after intravenous injection of sodium fluorescein in wild-type and Ccl2 −/− × Ccr2 −/− mice showed normal retinal vascular filling but no areas of hyperfluorescence indicative of CNV. b, After intravenous injection of QDot-CCR3 Fab in the same Ccl2 −/− × Ccr2 −/− mouse shown in ( a ), focal branching choroidal hyperfluorescence was visualized (arrow) at 1 h in the same area that was not hyperfluorescent during fluorescein angiography (arrowhead in a ). The intensity of this hyperfluorescence (shown in red pseudocolour in the inset) increased, attaining a peak at 4 h, and then declined in intensity but still persisted at 12 h. Corresponding images of QDot-Isotype Fab angiography showed no hyperfluorescence. c–e, The region corresponding to the area of hyperfluorescence seen on QDot-CCR3 Fab angiography in ( b ) contained multiple CD31 + blood vessels in the choroid (Ch) that were proliferating (Ki67 + ; arrows) and had not invaded the retina (Ret). Individual red (CD31 + , c ), and green (Ki67 + , d ), and merged ( e ) fluorescence channel images are shown. Arrows point to proliferating endothelial cells. Inset shows Ki67 + CD31 + cells in higher magnification. f, QDot-CCR3 Fab hyperfluorescent areas were localized to areas of subretinal CNV with CCR3 + endothelial cells. g, The QDot label was visualized within CD31 + vasculature of subretinal CNV lesions. Images representative of 6 independent experiments. Scale bars, ( c–e ), 10 μm.

Article Snippet: Immunofluorescent staining was performed with antibodies against human CCR3 (rat monoclonal, R&D Systems) or human CD31 (mouse monoclonal, Dako) and identified with Alexa 488 (Molecular Probes) or Cy3 secondary antibodies (Jackson ImmunoResearch).

Techniques: Injection, Fluorescence

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: (A) miR-148a levels were evaluated in 141 medulloblastoma tissues classified in the four molecular subgroups, normal developing (from less than 1year old infants) cerebellar tissues and adult (from individuals >21 yr of age) cerebellar tissues. (B, C, D) Relative Quantity of miR-148a in the indicated medulloblastoma cell line and its polyclonal populations transduced with pTRIPZ control lentiviral vector (Vector) or pTRIPZ-miR-148a construct expressing miR-148a in doxycycline inducible manner (P1, P2) and in the cells transiently transfected with control siGLO or miR-148a mimic. + DOX: doxycycline induced.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Transduction, Control, Plasmid Preparation, Construct, Expressing, Transfection

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: (A) Y axis denotes percentage growth inhibition obtained on doxycycline induction of the vector control and P1, P2 polyclonal populations of the indicated cell line as judged by the MTT assay. (B) Y axis indicates percent reduction in the number of colonies formed on doxycycline induction of the vector control and P1, P2 population cells of Daoy cells and percent reduction in the number of colonies formed on irradiation of doxycycline induced indicated population, in a clonogenic assay. (C, D) Y axis denotes the number of colonies formed in a soft agar assay by the vector control and P1, P2 population of the indicated cell line. The significance of the difference in the growth inhibition or soft agar colony formation observed on doxycycline induction of the polyclonal populations expressing pTRIPZ-miR-148a as compared to that observed in the polyclonal population transfected with control pTRIPZ vector was calculated by the student's t test. ** indicates p < 0.001 while *** indicates p < 0.0001 as determined using Student's t test.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Inhibition, Plasmid Preparation, Control, MTT Assay, Irradiation, Clonogenic Assay, Soft Agar Assay, Expressing, Transfection

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: Invasion potential was determined by studying migration through matrigel coated 8-μm pore size transwell inserts. The study was done using the stable polyclonal populations (P1, P2) of Daoy and D283 cells expressing miR-148a on doxycycline induction. (A) Representative images of the Calcein, AM labeled cells on the lower side of the transwell chamber membrane 36 h after seeding the indicated polyclonal populations of Daoy cells onto the transwell insert, with (d, e & f) or without (a, b & c) induction of miR-148a expression and after restoration of NRP1 expression (g, h) in doxycycline induced cells. (B-C) Y axis denotes the total fluorescence intensity of the invaded cells normalized to the total intensity of the initial cell number seeded of the indicated cell populations. (D) Y axis indicates the percent reduction in the fluorescence of the invaded cells of the indicated stable polyclonal populations on doxycycline induction (+DOX) of Daoy P1 and P2 population cells with (+NRP1) or without restoration of NRP1 expression. ** indicates p < 0.001; *** indicates p < 0.0001.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Migration, Pore Size, Expressing, Labeling, Membrane, Fluorescence

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: (A-B) Y axis denotes the area of subcutaneous tumors formed over a period of 4-6 weeks in nude mice, post injection of doxycycline induced Daoy or D425 P2 polyclonal population (pTRIPZ-miR-148a) and vector (pTRIPZ) control population and after restoration of NRP1 expression in doxycycline induced P2 population of Daoy cells (n = 6 each). (D) shows photographs of these subcutaneous tumors. (F) shows bioluminescence images of nude mice orthotopically injected with D283 stable polyclonal population P1 expressing miR-148a upon doxycycline (DOX) induction and, firefly luciferse under CAG promoter. The images were captured on 1 st and 4 th week after the injection. (C) Y axis shows relative fold increase in the average radiance on 4 th week as compared to that on 1 st week after injection of D283 cells P1 population with (+DOX) or without doxycycline induction (Control) in 7 mice each. (G) shows photographs of hematoxylin-eosin stained paraffin sections of the orthotopic xenografts of control and doxycycline induced D283 cells P1 population. The area marked with rectangle in a & b shows invading margins of the tumor cells in the control and doxycycline induced miR-148a expressing cells; c & d show magnified images (400 X) of the invading margins indicated by rectangles in a & b.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Injection, Plasmid Preparation, Control, Expressing, Staining

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: Y-axis denotes Luciferase activity of 293 FT cells transfected with the construct having 3′-UTR region of the indicated gene cloned downstream luciferase cDNA in a pcDNA 3.0 vector and EGFP expressing vector with or without pcDNA 3.0 construct expressing miR-148a. The luciferase activity is expressed relative to the GFP fluorescence. *** indicates p < 0.0001. (B) Schematic diagram shows miR-148a target site in NRP1 3′-UTR and position of the mutations (indicated by asterisk) introduced in the target site by site directed mutagenesis. (C, D, E, F) Western blot analysis shows reduction in the expression levels of NRP1, ROCK1 and DNMT1 protein in stable polyclonal populations of Daoy (C, E) and D283 (D, F) cells on doxycycline induction of miR-148a expression. γ-tubulin was used as a loading control. +: Doxycycline induction; * indicates exogenous expression of NRP1 in doxycyline induced cells.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Luciferase, Activity Assay, Transfection, Construct, Clone Assay, Plasmid Preparation, Expressing, Fluorescence, Mutagenesis, Western Blot, Control

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: NRP1 expression was studied in FFPE tissue sections belonging to the four molecular subgroups (WNT, SHH, Group 3, Group 4). The staining was scored as ‘negative’ for complete absence of staining, ‘Low’ for weak intensity and focal positive (~10-20% cells positive) areas, ‘Moderate’ for moderate intensity and more than or equal to 50% positive area while, ‘High’ for high intense staining in more than 80% of area. Representative images of NRP1 staining in the medulloblastoma tissues scored as negative (e, i), low (a, b, d, f, h), moderate (c, g, j, l) and high intense staining (k) respectively.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Expressing, Staining

Journal: Oncoscience

Article Title: MiR-148a, a microRNA upregulated in the WNT subgroup tumors, inhibits invasion and tumorigenic potential of medulloblastoma cells by targeting Neuropilin 1

doi:

Figure Lengend Snippet: (A) Percentage distribution of medulloblastoma tissues having NRP1 expressed scored as negative, low, moderate or high is shown across the four subgroups of medulloblastomas. (B) Kaplan Meier survival analysis of 62 medulloblastoma patients segregated into two groups based on NRP1 expression. “NRP1 low” group includes medulloblastoma tissues with no or low NRP1 expression while “NRP1 high” group includes tumor tissues with moderate or high NRP1 expression.

Article Snippet: Human medulloblastoma cell lines Daoy and D283 were obtained from ATCC, Manassas, VA, USA.

Techniques: Expressing

Journal: Cell Death & Disease

Article Title: Chemotherapy enhances HMGA1 secretion through the mutant p53-CK2 axis in pancreatic ductal adenocarcinoma cells

doi: 10.1038/s41419-025-08082-1

Figure Lengend Snippet: A Differentially secreted proteins detected by MS analysis in the CM of PANC-1 cells transiently transfected with si TP53 R273H or siScramble. The 25 proteins significantly downsecreted are indicated by the light lavender-shaded rectangle underlaid on the plot. Vertical dashed lines indicate log2 fold change = ±0.5. Horizontal dashed line indicates the cut-off p-value p = 0.05. TPM1, CSTN1, HMGA1 and CP2R1 proteins are marked on the plot. B Differentially secreted proteins detected by MS analysis in the CM of AsPC-1 cells transiently transfected with TP53 R273H or MOCK plasmid. The 83 proteins significantly hypersecreted are indicated by the light teal-shaded rectangle underlaid on the plot. Among them, TPM1, CSTN1, HMGA1 and CP2R1 proteins are specifically highlighted. Vertical dashed lines indicate log2 fold change = ±0.5. Horizontal dashed line indicates the cut-off p-value p = 0.05. C Venn diagram indicating the overlaps of the differentially mut or wt p53-dependent secreted proteins detected by MS analysis. D Histogram showing 4 proteins (Tropomyosin 1 (TPM1), Calsyntenin 1 (CSTN1), High Mobility Group A1 (HMGA1), Cytochrome P450 Family 2 Subfamily R Member 1 (CP2R1)) hypersecreted by AsPC-1 (p53-null) cells overexpressing TP53 R273H and downsecreted by PANC-1 cells after KD of the same hot-spot mutp53 isoform. E UMAP visualization of all identified cell types present in the pancreatic microenvironment subset by disease state: Adjacent Normal ( n = 3), Healthy ( n = 6) and Tumor ( n = 16). Data source: Pancreatic Tissue Single Cell Atlas. F UMAP visualizations showing HMGA1 expression across the major cell populations subset by disease state (Adjacent Normal, Healthy, Tumor). Data source: Pancreatic Tissue Single Cell Atlas. G HMGA1 gene expression level in tumor derived epithelial cells compared to adjacent normal or healthy epithelial cells (number of cells: 892 Adj.Normal; 14,380 Healthy; 9484 Tumor). Data source: Pancreatic Tissue Single Cell Atlas. H HMGA1 gene expression level in tumor compared to normal tissue. Data source: GEPIA database. * p < 0.01. I HMGA1 expression correlates with the mutational status of TP53 ( n = 66 no-mutation, n = 62 missense mutations). Data source: cBioportal database, TCGA PanCancer Atlas . (Wilcoxon test). **** p < 0.0001. J HMGA1 expression level in PDAC patient with TP53 mut ( n = 24 no-mutation, n = 43 missense mutations Data source: cBioportal database, QCMG Nature 2016 . (Wilcoxon test). **** p < 0.0001. K Kaplan-Meier (KM) plot of survival probability (log-rank test) for PDAC patients only as obtained from KM-plotter database using the default parameters. L Kaplan-Meier survival plot after PDAC patients’ stratification for tumor stage (S2, S3, S4) in KM plotter database showing HMGA1 expression is a prognostic factor in advanced pancreatic cancer. M Volcano plot of differential gene expression (DGE) analysis for metastatic vs primary tumors using the microarray dataset GSE71729 showing HMGA1 is significantly highly expressed in metastatic patients (Log2FC = 2.383837; −log10(Adj. p-value) = 10.47756). Red highlights indicate significant regulated genes; black highlights indicate non-significant genes. Vertical dashed lines indicate log2 fold change = ±1. Horizontal dashed line indicates p = 0.01. N Immunoblot validation of HMGA1 KO in human PANC-1 cell line. KO denotes HMGA1 KO cells, while the minus sign (-) represents the parental cells. Vinculin was used as a loading control. O Representative images, with corresponding magnifications, of PANC-1 (top) and HMGA1-KO PANC-1 (bottom) cells invading through Matrigel-coated transwell inserts (8 μm pore size) after 24 h of incubation at 37 °C. Right panel: bar plot quantification of the percentage of invasive cells. Data are presented as mean ± SD (n = 4). (Unpaired t-test). **p < 0.01. P Tumor volume (mm 3 ) from subcutaneous injection of either PANC-1 or HMGA1 KO PANC-1 cells in immunodeficient mice. Data plotted are mean tumor volumes + SEM ( n = 6 for each cohort). (Two-way ANOVA). ****p < 0.0001. Q Individual tumor volumes + SEM at the endpoint (Unpaired t-test). *p < 0.05.

Article Snippet: The PDAC human cell lines AsPC-1 (p53-null) and PANC-1 ( TP53 R273H ) were purchased from the American Type Culture Collection (ATCC).

Techniques: Transfection, Plasmid Preparation, Expressing, Gene Expression, Derivative Assay, Mutagenesis, Microarray, Western Blot, Biomarker Discovery, Control, Pore Size, Incubation, Injection

Journal: Cell Death & Disease

Article Title: Chemotherapy enhances HMGA1 secretion through the mutant p53-CK2 axis in pancreatic ductal adenocarcinoma cells

doi: 10.1038/s41419-025-08082-1

Figure Lengend Snippet: A Immunoblot of secreted HMGA1 protein validating the MS-data. Amido black staining (a.b.) was used as loading control for WB. B Immunoblot of HMGA1 protein in different human PDAC cell lines. C Immunoblot of secreted HMGA1 protein in different human PDAC cell lines. D Immunoblot validation of TP53 KO in human PANC-1 cell line. KO denotes TP53 KO cells, while the minus sign (−) represents the parental cells. Vinculin was used as a loading control. E Immunoblot of secreted HMGA1 protein in PaCa3, PANC-1 and TP53 KO PANC-1 cells. F Cell growth percentage measured by cristal violet assay in HMGA1 KO PANC-1 cells cultured for 48 h with PANC-1 CM or HMGA1 KO PANC-1 CM. (Unpaired t-test). *** p < 0.001. G Cell growth percentage measured by cristal violet assay in p53-null AsPC-1 cells cultured for 48 h with R273H CM or MOCK CM after the addition of anti-HMGA1 antibody (0.226 µg/µl, 1:100 in growth medium) or the IgG Isotype Control (Cell signaling, 5742). (Two-way ANOVA). *p < 0.05, **p < 0.01. H Immunoblot analysis of HMGA1 protein in PANC-1 CM alongside different µg of rHMGA1 protein. On the right, the slope obtained from linear regression analysis of the average adjusted total band intensity values + SD (arbitrary units, a.u.) of immunoreactivity corresponding to rHMGA1. The adjusted total band intensity values were derived from densitometric analysis of the immunoreactive bands for HMGA1 secreted by PANC-1 cells, as well as rHMGA1 used as a standard with increasing sample loads. Data were analyzed using Image Lab Software (Bio-Rad, version 6.1.0 build 7). The blue square on the slope represents the value corresponding to the secreted HMGA1. I Cell growth percentage measured by cristal violet assay in HMGA1 KO PANC-1 cells cultured for 72 h after treatment with different doses of rHMGA1 protein. (One-way ANOVA). ****p < 0.0001.

Article Snippet: The PDAC human cell lines AsPC-1 (p53-null) and PANC-1 ( TP53 R273H ) were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Staining, Control, Biomarker Discovery, Cell Culture, Derivative Assay, Software

Journal: Cell Death & Disease

Article Title: Chemotherapy enhances HMGA1 secretion through the mutant p53-CK2 axis in pancreatic ductal adenocarcinoma cells

doi: 10.1038/s41419-025-08082-1

Figure Lengend Snippet: A Immunoblot of p-p53, p53 and HMGA1 proteins in PANC-1 cells after treatment with different anti-cancer drugs at sublethal doses (1 µM gemcitabine (GEM), 5 µM 5-fluorouracil (5-FU), 1 µM oxaliplatin (OXA) and 5 µM irinotecan (IRI)). B Immunoblot of HMGA1 protein in PANC-1 cells secretome after treatment with different anti-cancer drugs at sublethal doses. C Bar charts depict A.I. (a.u.) of HMGA1 secreted by PANC-1 cells with or without 1 µM GEM treatment versus a.b. analyzed using Image Lab Software (Bio-Rad, version 6.1.0 build 7). Data plotted are mean of seven independent experiments ± SD. (Unpaired t-test). *p < 0.05. D qPCR showing TP53 expression upon sublethal dose GEM treatment of PANC-1 cells for 6, 10, 24 and 48 h. ***p < 0.001, ****p < 0.0001. E qPCR showing HMGA1 expression upon sublethal dose of GEM treatment of PANC-1 cells for 6, 10, 24, and 48 h. F Immunoblot of p-p53, p53, and HMGA1 proteins in PANC-1 cells upon sublethal dose GEM treatment for 6, 10, 24 and 48 h. G Bar charts of PaCa3, Hs776t, PANC-1 and SUIT-2 cells viability (PI-/Ann V-) after 24 h treatment with 1 µM GEM. H Immunoblot of p53 and HMGA1 proteins in two cell lines carrying TP53 w t (PaCa3 and Hs776t) and two cell lines harboring TP53 R273H (PANC-1 and SUIT-2) after being treated or not with 1 µM of GEM. I Immunoblot of HMGA1 in human PDAC cells secretome after 1 µM GEM treatment.

Article Snippet: The PDAC human cell lines AsPC-1 (p53-null) and PANC-1 ( TP53 R273H ) were purchased from the American Type Culture Collection (ATCC).

Techniques: Western Blot, Software, Expressing

Journal: Theranostics

Article Title: Emodin reduces Breast Cancer Lung Metastasis by suppressing Macrophage-induced Breast Cancer Cell Epithelial-mesenchymal transition and Cancer Stem Cell formation

doi: 10.7150/thno.45395

Figure Lengend Snippet: Emodin inhibits TGF-β1 and macrophage-induced EMT in breast cancer cells. A. A heatmap plot of emodin-modulated EMT gene expression in breast cancer cells following TGF-β1 stimulation (48 h); data for plotting were obtained by qPCR; B. Emodin inhibits EMT gene expression in 4T1 cells cocultured with TAM-like macrophages; * P <0.05; ** P <0.01; C. Flow cytometry analysis of CD206 expression in TCCM-induced macrophages, with or without emodin treatment; D-E. ELISA measurement of TGF-β1 production from macrophages ( D ) or breast cancer cells ( E ) after the indicated stimulation (PMCM: peritoneal macrophage conditioned medium). * P <0.05; ** P <0.01; *** P <0.001; F. Wound-healing assay of the 4T1 cell migration following PMCM stimulation with or without emodin treatment (16 h). Migration index = (Width before migration - Width after migration)/ Width before migration; all data were normalized to the first group; * P <0.05; ** P <0.01; representative images are shown in A. G. Matrigel invasion assay of 4T1 cells following PMCM treatment with or without emodin. Matrigel concentration: 300 µg/ml; Transwell pore size: 8 µm; Invasion time: 24 h; Invasion index was calculated by the normalization of cell number per field in each group to the DMSO control group. * P <0.05; representative images of the invade cells staining with DAPI are shown in B . H. Gene expression of the invadopodia formation markers in breast cancer cells. EO771 cells were treated with vehicle or emodin, or conditioned medium from macrophages treated with EO771 conditioned medium with or without emodin (16 h). * P <0.05; ** P <0.01.

Article Snippet: The breast cancer cell lines EO771, 4T1, MCF7, and MDA-MB-231 were obtained from the American Type Culture Collection.

Techniques: Gene Expression, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Wound Healing Assay, Migration, Invasion Assay, Concentration Assay, Pore Size, Control, Staining

Journal: Theranostics

Article Title: Emodin reduces Breast Cancer Lung Metastasis by suppressing Macrophage-induced Breast Cancer Cell Epithelial-mesenchymal transition and Cancer Stem Cell formation

doi: 10.7150/thno.45395

Figure Lengend Snippet: Emodin suppresses the stemness and progenitor properties of tumor-initiating cells. A. Flow cytometry analysis of the effect of emodin on cancer stem cell population in 4T1 cells with or without TGF-β1 stimulation (24 h). The cancer stem cell percentages are shown. * P <0.05; *** P <0.001; B. Flow cytometry analysis of the effect of emodin on progenitor cell populations in PyMT cells after TGF-β1 stimulation for 48 h; the quantifications of the progenitor cell percentages and the mean fluorescence intensity of CD61 in PyMT cells are shown. The CD61 + subpopulation was gated from the CD24 + CD49f + cell population; * P <0.05; ** P <0.01; *** P <0.001; C. Flow cytometry analysis of the effect of emodin on progenitor cell populations in 4T1 cells after TGF-β1 stimulation for 48 h; the quantifications of the progenitor cell percentages and the mean fluorescence intensity of CD61 in 4T1 cells are shown. The CD61 + subpopulation was gated from CD24 + CD49f + cell population; *** P <0.001; D-E. Emodin inhibited the expression of stemness-related genes in MDA-MB-231 cells ( D ) and 4T1 cells ( E ); data were obtained from qPCR analysis. * P <0.05; ** P <0.01; *** P <0.001; F. Emodin inhibits tumor mammosphere formation of EO771 cells. The 1 st mammospheres were cultured in ultra-low attachment plates for 7 days, and the 2 nd mammospheres were cultured for another 7 days; quantification of the number of formed 1 st mammospheres and 2 nd mammospheres in EO771 culture with or without emodin treatment is shown; the numbers in X-axis indicate the numbers of cells that were added in each well of the plate. G. Limiting dilution assays show the inhibitory effect of emodin on breast cancer stem cells in EO771 cells. The data are presented as the number of mice with detectable tumors (0-4) versus total number of mice injected with cancer cells (4 or 5). The estimated stem cell frequencies were calculated for statistical comparison (right).

Article Snippet: The breast cancer cell lines EO771, 4T1, MCF7, and MDA-MB-231 were obtained from the American Type Culture Collection.

Techniques: Flow Cytometry, Fluorescence, Expressing, Cell Culture, Injection, Comparison

Journal: Theranostics

Article Title: Emodin reduces Breast Cancer Lung Metastasis by suppressing Macrophage-induced Breast Cancer Cell Epithelial-mesenchymal transition and Cancer Stem Cell formation

doi: 10.7150/thno.45395

Figure Lengend Snippet: Emodin suppresses both canonical and noncanonical pathways of TGF-β1 signaling in breast cancer cells. A. 4T1 and EO771 cells were stimulated by TGF-β1 with or without emodin for 1 h. Western blot was used to detect the phosphorylation and total protein of smad2/3; and the band densities were quantified using three samples in each group; B. Total protein of smad4 was detected by western blot and quantified using three samples in each group; C-D. The phosphorylation and total protein levels of STAT3 and AKT were detected in 4T1 cells after 1 h treatment as indicated ( C ), and quantifications of the band densities are shown ( D ); E-H. The transcription factors for EMT were detected in 4T1 and MDA-MB-231 cells using western blot ( E,G ), and the quantifications of the relative band densities are shown ( F,H ). * P <0.05; ** P <0.01.

Article Snippet: The breast cancer cell lines EO771, 4T1, MCF7, and MDA-MB-231 were obtained from the American Type Culture Collection.

Techniques: Western Blot, Phospho-proteomics

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3 and eotaxins are expressed in choroidal neovascularization. a,b, Immunofluorescence shows that CCR3 (green) receptor expression colocalizes with CD31 + (red) expressing blood vessels in surgically excised human age-related macular degeneration (AMD) choroidal neovascular (CNV) tissue. Nuclei stained blue by DAPI. b, Specificity of CCR3 staining is confirmed by absence of staining with isotype control IgG ( a ). Individual red and green fluorescence channels are shown in . c,d, CCR3 is not immunolocalized in CD31 + (red) blood vessels (white arrowheads) in the choroid of patients with atrophic AMD who do not have CNV ( c ) or in aged patients without AMD ( d ). Autofluorescence of retinal pigmented epithelium (white arrow) and Bruch's membrane (asterisks) overlying choroid is seen ( c,d ). e,f, CCR3 is not expressed in surgically excised avascular retinal fibrosis tissue ( e ) or in blood vessel of choroidal melanoma ( f ). g-j, Immunohistochemistry (golden brown reaction product) shows expression of CCL11 ( g ), CCL24 ( h ), and CCL26 ( i ) in surgically excised AMD CNV tissue, primarily in the stroma (red arrowheads) but also in the blood vessels (yellow arrows). Specificity of staining is confirmed by absence of staining with isotype control IgG ( j ). Scale bars, 10 μm.

Article Snippet: 96-well plates were coated with Growth Factor Reduced Matrigel (BD Biosciences) mixed with rat neutralizing antibody against human CCR3 (20 μg/ml, R&D Systems) or control rat IgG2a (Invitrogen) and allowed to solidify in the incubator at 37 °C for 45 min. Human choroidal endothelial cells (CECs) - were plated on top of the Matrigel at 2.25 × 10 4 /cm 2 in EBM-2 basal media (Cambrex) containing 1% FBS with CCR3 antibody or rat IgG2a at the above concentrations and allowed to grow overnight.

Techniques: Immunofluorescence, Expressing, Staining, Control, Fluorescence, Membrane, Immunohistochemistry

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3 activation promotes angiogenesis. a, Tube formation of primary human choroidal endothelial cells (CECs) in Matrigel in vitro was reduced by neutralizing anti-human CCR3 antibodies (Ab) compared to isotype IgG. n = 6, * P < 0.05 compared to isotype IgG. b, Fraction of CD31 + VEGFR2 + gated mouse CECs in vivo in proliferative state (S phase) was increased 5 days after laser injury in wild-type mouse eyes compared to control (uninjured eyes), and was reduced by intraocular administration of neutralizing anti-mouse CCR3 Ab compared to isotype IgG. n = 6–10, * P < 0.05 compared to IgG treatment. c, Stimulation with eotaxins for 24 h induced human CEC proliferation. n = 4, * P < 0.05 compared to bovine serum albumin (BSA) treatment. d,e, Stimulation with eotaxins, but not PBS, induced actin polymerization in human CECs. Relative F-actin content is expressed as the ratio of the mean channel fluorescence between eotaxin- and media alone-stimulated cells ( d ). Rhodamine-phalloidin staining (red) shows F-actin fibre formation in eotaxin-stimulated cells ( e ). Nuclei stained blue by DAPI. Data representative of 3–4 independent experiments are shown. c,e, CCL11 (10 ng/ml), CCL24 (100 ng/ml), CCL26 (2 μg/ml). f, Stimulation with eotaxins for 16 hours induces dose-dependent migration of human CECs across 8 μm pore size Transwells. n = 5–10, * P < 0.05 compared to BSA treatment. ( a–c, f ) Significance by Mann-Whitney U test. Error bars depict s.e.m.

Article Snippet: 96-well plates were coated with Growth Factor Reduced Matrigel (BD Biosciences) mixed with rat neutralizing antibody against human CCR3 (20 μg/ml, R&D Systems) or control rat IgG2a (Invitrogen) and allowed to solidify in the incubator at 37 °C for 45 min. Human choroidal endothelial cells (CECs) - were plated on top of the Matrigel at 2.25 × 10 4 /cm 2 in EBM-2 basal media (Cambrex) containing 1% FBS with CCR3 antibody or rat IgG2a at the above concentrations and allowed to grow overnight.

Techniques: Activation Assay, In Vitro, In Vivo, Control, Fluorescence, Staining, Migration, Pore Size, MANN-WHITNEY

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CNV reduced by CCR3 or eotaxin ablation or blockade independent of leukocyte modulation. a,b, Laser-induced CNV in wild-type mice was reduced by neutralizing anti-mouse CCR3 Ab compared to isotype IgG ( a ) and by the CCR3 receptor antagonist (RA) SB328437 ((S)-Methyl-2-naphthoylamino-3-(4-nitrophenyl)propionate) compared to vehicle (PBS/DMSO) ( b ) in a dose-dependent fashion. n = 8–12, * P < 0.05 compared to no antibody or receptor antagonist. c, Representative examples of CNV in drug-treated mice. d, Laser-induced CNV was reduced in Ccr3 −/− mice compared to wild-type mice. n = 9, * P < 0.05 compared to wild-type mice. e, Eotaxin-1 (Ccl-11) and eotaxin-2 (Ccl-24) protein levels, measured by ELISA, were increased following laser injury in wild-type mice. n = 6, * P < 0.05, # P < 0.01 compared to 0 h baseline. f, Ccl-11 and Ccl-24 immunofluorescence (green) was localized in the retinal pigmented epithelial cell layer (arrows) adjacent to CD31+ (red) choroidal endothelial cells (arrowheads) on day 1 after laser injury in wild-type mice. Nuclei stained blue by DAPI. No specific immunofluorescence was detected with isotype control IgGs. Images representative of 3 independent experiments are shown. g, Laser-induced CNV was reduced in Ccl11 −/− and in Ccl24 −/− mice compared to wild-type mice. n = 8–10, * P < 0.05 compared to wild-type mice. CNV is further reduced in Ccl11 −/− × Ccl24 −/− mice compared to single null mice. # P < 0.05 compared to single null mice. h, Laser-induced CNV in wild-type mice was reduced by neutralizing antibodies against mouse CCL11 or CCL24 compared to isotype IgG. n = 7–10, * P < 0.05 compared to no injection (control) or IgG. i, Representative examples of CNV in eotaxin-neutralized mice. j, Neutralizing anti-CCR3 antibodies (Ab) reduced laser-induced CNV in mice deficient in eosinophils (Δdbl GATA) or mast cells ( Kit w-v ). n = 6–9, * P < 0.05 compared to IgG. Scale bars, ( c,i ), 100 μm; f , 20 μm. Error bars depict s.e.m.

Article Snippet: 96-well plates were coated with Growth Factor Reduced Matrigel (BD Biosciences) mixed with rat neutralizing antibody against human CCR3 (20 μg/ml, R&D Systems) or control rat IgG2a (Invitrogen) and allowed to solidify in the incubator at 37 °C for 45 min. Human choroidal endothelial cells (CECs) - were plated on top of the Matrigel at 2.25 × 10 4 /cm 2 in EBM-2 basal media (Cambrex) containing 1% FBS with CCR3 antibody or rat IgG2a at the above concentrations and allowed to grow overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Injection

Journal: Nature

Article Title: CCR3 is a therapeutic and diagnostic target for neovascular age-related macular degeneration

doi: 10.1038/nature08151

Figure Lengend Snippet: CCR3-targeting quantum dots detect subretinal choroidal neovascularization (CNV). a, Images of the fundus taken after intravenous injection of sodium fluorescein in wild-type and Ccl2 −/− × Ccr2 −/− mice showed normal retinal vascular filling but no areas of hyperfluorescence indicative of CNV. b, After intravenous injection of QDot-CCR3 Fab in the same Ccl2 −/− × Ccr2 −/− mouse shown in ( a ), focal branching choroidal hyperfluorescence was visualized (arrow) at 1 h in the same area that was not hyperfluorescent during fluorescein angiography (arrowhead in a ). The intensity of this hyperfluorescence (shown in red pseudocolour in the inset) increased, attaining a peak at 4 h, and then declined in intensity but still persisted at 12 h. Corresponding images of QDot-Isotype Fab angiography showed no hyperfluorescence. c–e, The region corresponding to the area of hyperfluorescence seen on QDot-CCR3 Fab angiography in ( b ) contained multiple CD31 + blood vessels in the choroid (Ch) that were proliferating (Ki67 + ; arrows) and had not invaded the retina (Ret). Individual red (CD31 + , c ), and green (Ki67 + , d ), and merged ( e ) fluorescence channel images are shown. Arrows point to proliferating endothelial cells. Inset shows Ki67 + CD31 + cells in higher magnification. f, QDot-CCR3 Fab hyperfluorescent areas were localized to areas of subretinal CNV with CCR3 + endothelial cells. g, The QDot label was visualized within CD31 + vasculature of subretinal CNV lesions. Images representative of 6 independent experiments. Scale bars, ( c–e ), 10 μm.

Article Snippet: 96-well plates were coated with Growth Factor Reduced Matrigel (BD Biosciences) mixed with rat neutralizing antibody against human CCR3 (20 μg/ml, R&D Systems) or control rat IgG2a (Invitrogen) and allowed to solidify in the incubator at 37 °C for 45 min. Human choroidal endothelial cells (CECs) - were plated on top of the Matrigel at 2.25 × 10 4 /cm 2 in EBM-2 basal media (Cambrex) containing 1% FBS with CCR3 antibody or rat IgG2a at the above concentrations and allowed to grow overnight.

Techniques: Injection, Fluorescence

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Involvement of BAG5 interactome in regulation of actin cytoskeleton, focal adhesion and RNA metabolism. (A) Coomassie blue staining of SDS-PAGE gel analyzed BAG5 immunocomplex from a panel of immortalized lung epithelial and NSCLC cell lines. (B) SDS-PAGE gel from lung epithelial cell line BEAS-2B and NSCLC cell line A549 were identified by mass spectrometry. Ten members of Hsp70/HSC70 family were identified to interact with BAG5 in A549 cell. (C) Venn diagram showed the different interacting proteins between BEAS-2B and A549 cell. (D, E) GO (D) and KEGG pathways (E) enrichment analysis of 130 unique BAG5-interacting proteins in A549 cell. (F, G) Cytoscape ClueGO visualizing the enriched pathways of TOP 100 (F) and TOP 5 (G) hub proteins unique in A549 cell. (H) Co-IP was performed to confirm the potential interaction of BAG5 with the top 5 of BAG5-interacting proteins influencing protein translation. (I) BAG5-interacting proteins were analyzed by western blot analysis.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Staining, SDS Page, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: BAG5 was highly expressed in tumor epithelial cells and correlated with NSCLC metastasis. (A, B) Expression levels of BAG5 in tumor versus normal tissues were analyzed using a combined TCGA and GTEx dataset. Comparisons include lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), as well as subgroups with lymph node (N) or distant metastasis (M). *** p < 0.001; The statistical difference of two groups was compared through the Wilcox test, significance difference of three groups was tested with Kruskal-Wallis test. (C) Kaplan-Meier survival curve was constructed for NSCLC patients from TCGA based on BAG5 expression levels (BAG5-High vs. BAG5-Low). Significance ( p ) was evaluated by Log-rank test. HR: hazard ratio. (D) Forest plot of hazard ratios (HR) for the association of BAG5 expression with overall survival (OS) across 27 NSCLC datasets from the OSluca online database. Detailed statistics, including HR, 95% CI, and p-values for each dataset, are provided in Supplemental Table OSluca. (E–G) The public single-cell RNA-seq dataset ( GSE119911 ) was reanalyzed. BAG5 expression levels across major cell types in NSCLC versus normal lung tissues are shown. In panel H, average expression levels of BAG5 are presented as a heatmap across annotated cell types. (H) Western blotting analysis was performed to examine BAG5 expression in a panel of normal and NSCLC cell lines, with GAPDH used as the loading control. Representative immunoblots are shown (upper), and densitometric quantification of relative BAG5 protein levels normalized to GAPDH is presented (lower). Statistical significance was evaluated using one-way ANOVA, ****p < 0.0001. (I) BAG5 protein level was investigated using Western blot in paired fresh NSCLC tumor (T) and paratumor (P) normal tissues, and representative images were provided. β-actin was used as a loading control. LUAD (J) Scatter plots showing relative expression of BAG5 in paired NSCLC tumor (T) and paratumor (P) normal tissues. ** p < 0.01.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing, Construct, RNA Sequencing, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: BAG5 knockout inhibited proliferation and invasion of NSCLC in vitro and in vivo . (A, B) Control and BAG5 knockout NSCLC cells were injected subcutaneously on the right flanks of nude mice (n = 5-6 mice per group). Tumors of each group were removed and photographed after sacrifice of animals. Tumor weight and volumes (mean ± SD) were analyzed. * p < 0.05. (C) Xenografts were sectioned and stained with BAG5 and Ki67. Scale bars, 25μm. (D, E) To evaluate spontaneous metastasis, BAG5 knockout and control A549 cells were i.v. injected to nude mice (n = 8 mice per group). Representative images ( D left), quantified values ( D right) and H&E staining (E) of metastatic nodules in lung. ** p < 0.01. Scale bars, 2mm and 250μm. (F) H&E and immunohistochemical staining images of NSCLC cancer tissues, PDX and derived PDO. Scale bars, 50 µm. (G) To evaluate heterogeneous expression of BGA5 in tumor, the constructed PDOs were separated with a filter with 100μm pore size and BAG5 expression was investigated using histochemical staining. (H) The PDO (PDO#1) with higher basal BAG5 expression were infected with gRNA guided BAG5 using CRISPR/Cas9 system for knockout. Western blot analysis confirming effective BAG5 knockout in PDO#1. β-actin was used as a loading control. (I) Control and BAG5 knockout PDOs were seeded and maintained for 1 week. Representative photographs (left) of colony formation and their quantitative analysis (right) were presented. Data represent the mean ± SD of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test; **p < 0.001. (J) Invasion of cells to the lower compartment and chemotaxis of the upper compartment organoids was evaluated by Matrigel-uncoated Transwell. Representative photographs (left) and their numbers (right). These data show mean ± SD of three independent experiments. * p < 0.05.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Knock-Out, In Vitro, In Vivo, Control, Injection, Staining, Immunohistochemical staining, Derivative Assay, Expressing, Construct, Pore Size, Infection, CRISPR, Western Blot, Two Tailed Test, Chemotaxis Assay

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Enrichment of hallmark genes involved in EMT and metabolic reprogramming in BAG5 + NSCLC tumor cells. (A) The t-SNE plot of BAG5 expression landscape in NSCLC tumor tissues. Heterogeneity in the expression of BAG5 was shown in tumor epithelial cells. (B) The relative proportion of BAG5 + cells from different cell types in NSCLC tissues. (C, D) Single sample GSVA (ssGSVA) analysis (BAG5 + vs. BAG5 - tumor epithelial cells) showing most enriched KEGG pathways (C) and Hallmark annotation (D) .

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Regulation of metabolic reprogramming by BAG5 in NSCLC. (A) Heat map of differentially expressed proteins including metabolic reprogramming, EMT and mitodynamics-related ones in CON and BAG5-KD A549 cells by quantitative proteomics. (B) Downregulation of GLUT3 by BAG5 knockout was confirmed by western blot analysis in NSCLC cells. (C) Glucose uptake by NSCLC cells was analyzed by 2-NBDG incorporation experiments. (D, E) OCR (D) and ECAR (E) were measured using seahorse instrument in control and BAG5 knockout A549 and PC9 cells. (F, G) Glucose consumption (F) and lactate production (G) was analyzed by spectrophotometric methods. Data represent mean ± SD; * p < 0.05.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Quantitative Proteomics, Knock-Out, Western Blot, Control

Journal: Frontiers in Immunology

Article Title: Mechanistic insights into promotion of non-small cell lung cancer by BAG5 using integrative multi-omics approaches

doi: 10.3389/fimmu.2025.1648139

Figure Lengend Snippet: Regulation of mitochondrial dynamics by BAG5 in NSCLC. (A) co-expression of BAG5 and DRP1 transcripts was explored using the single cell transcriptome. (B) Regulation of NFN2 and DRP1 by BAG5 in NSCLC cells was investigated via western blot analysis. (C) Representative images of the mitochondrial morphological change by mitotracker staining in A549 cells with control or BAG5 knockout (left). The proportion of cells (n = 100 cells for each sample) with fragmented, intermediate and elongated mitochondria was quantifified (right).Scale bars, 1 μm or 2 μm. (D) MFI of mitotracker staining was measured using flow cytometry in control or BAG5 knockout NCSCLs. (E) Representative TEM images of A549 cells confirmed the mitochondrial morphological changes by BAG5 downregulation. (F) Cells with control or BAG5 knockout were incubated with 10 μM DCFH-DA for 30 min. The MFI of intracellular ROS level was measured with flow cytometry. Data represent mean ± SD; *p < 0.05; n.s. means no significance (Student’s t test). (G) A proposed model for function and mechanism of BAG5 in NSCLC. Based on the multi-omics data, BAG5, as an oncogene, was shown to affect the malignant phenotype of NSCLC cancer cells, and participate in multiple tumor pathways, including regulation of mitochondrial morphology, metabolic reprogramming, RNA metabolism, cytoskeleton, and EMT, which consequently promoting tumor growth and metastasis.

Article Snippet: NSCLC cells (A549, HCC817, PC9, H1299, H1975, SK-MES-1), the normal human lung epithelial cell line BEAS-2B and 16HBE were obtained from ATCC.

Techniques: Expressing, Western Blot, Staining, Control, Knock-Out, Flow Cytometry, Incubation, Biomarker Discovery

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: Cell viability following the exposure of U87MG ( A ) and MIAMI cells ( B ) to various concentrations of Fc-diOH, blank LNCs or Fc-diOH-LNCs (0.01–100 μM). Notes: Data are expressed as the mean ± SEM (n=3). The results obtained for U87MG cells or MIAMI cells cultured with culture medium alone were considered to correspond to 100% survival. Abbreviations: LNCs, lipid nanocapsules; Fc-diOH, ferrociphenol; MIAMI, marrow-isolated adult multilineage inducible; SEM, standard error of the mean.

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: Cell Culture, Isolation

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: In vitro toxicity of Fc-diOH-LNCs and Fc-diOH-LNC-loaded MIAMI cells to U87MG cells. Notes: ( A ) Schematic diagram of the principle of coculture experiments to assess the cytotoxicity to U87MG cells of Fc-diOH-LNCs or Fc-diOH-LNC-loaded MIAMI cells. The pore size of the insert (0.4 μm) allowed the passage of LNCs. ( B ) Viability of U87MG cells following exposure to Fc-diOH-LNCs (0.03–3 μg) or MIAMI cells loaded with blank LNCs or Fc-diOH-LNCs. Two doses of MIAMI cells were tested: 5×10 3 and 40×10 3 cells, corresponding to Fc-diOH doses of about 0.1 μg and 0.8 μg, respectively. The results obtained for U87MG cells cultured with culture medium alone were considered to correspond to 100% survival. Data are expressed as the mean of three wells ± SEM (n=2) (* P <0.05, versus U87MG cells cultured with culture medium alone). ( C ) Viability of MIAMI cells loaded with blank LNCs or Fc-diOH-LNCs 4 days after their addition to the upper compartment of the Transwell system. Abbreviations: LNCs, lipid nanocapsules; Fc-diOH, ferrociphenol; MIAMI, marrow-isolated adult multilineage inducible; SEM, standard error of the mean.

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: In Vitro, Pore Size, Cell Culture, Isolation

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: Analysis of Fc-diOH-LNC-loaded MIAMI cell migration toward U87MG cells. Notes: ( A ) Schematic diagram of the principle of the migration assay. The pore size of the insert (8 μm) allowed the passage of MIAMI cells. ( B ) Percentage of unloaded or Fc-diOH-LNC-loaded MIAMI cells migrating, after 4 hours of incubation with the control medium and U87MG CM. The number of MIAMI cells added to the upper compartment was taken as 100%. Data are expressed as the mean of three wells ± SEM (n=2). ( C ) Schematic representation of the experimental model. ( D ) Fluorescence microscopy images of tissue sections after the intratumoral injection of MIAMI cells in U87MG-bearing mice. MIAMI cells were detected by labeling the Y-chromosome with a red fluorescent probe. Nuclei were stained with 4′,6-diamidino-2-phenylindole (scale bar =100 μm). Abbreviations: Fc-diOH-LNCs, ferrociphenol lipid nanocapsules; MIAMI, marrow-isolated adult multilineage inducible; SEM, standard error of the mean; CM, conditioned medium.

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: Migration, Pore Size, Incubation, Control, Fluorescence, Microscopy, Injection, Labeling, Staining, Isolation

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: Efficacy of Fc-diOH-LNCs and Fc-diOH-LNC-loaded MIAMI cells in the orthotopic U87MG glioma model

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: Control

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: In vivo toxicity of Fc-diOH-LNCs and Fc-diOH-LNC-loaded MIAMI cells to U87MG cells. Notes: ( A ) Representation of the treatment protocol applied to U87MG-bearing mice. The T2-weighted image shows the presence of the U87MG tumor on day 6, the day of the treatment. ( B ) Representative T2-weighted images of control and treated mice on day 20. ( C ) Tumor volume distribution in each group, calculated by MRI on day 20. ( D ) Kaplan–Meier survival curves for U87MG-bearing mice receiving HBSS, Fc-diOH-LNCs or Fc-diOH-LNC-loaded MIAMI cells, 6 days after the injection of U87MG cells. *Significantly different from the control group ( P <0.05) (each group contained eight mice). Abbreviations: Fc-diOH-LNCs, ferrociphenol lipid nanocapsules; MIAMI, marrow-isolated adult multilineage inducible; MRI, magnetic resonance imaging; HBSS, Hank’s balanced salt solution.

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: In Vivo, Control, Injection, Isolation, Magnetic Resonance Imaging

Journal: International Journal of Nanomedicine

Article Title: Targeting and treatment of glioblastomas with human mesenchymal stem cells carrying ferrociphenol lipid nanocapsules

doi: 10.2147/IJN.S69175

Figure Lengend Snippet: Effect of Fc-diOH-LNCs and Fc-diOH-LNC-loaded MIAMI cells on the number of Ki67 + proliferative cells and the number of CD31 + vessels present in the U87MG tumor. Notes: ( A ) Schematic representation of the experimental model. ( B ) Immunofluorescence staining for Ki67 and CD31 in the tumor 7 days after the intratumoral injection of HBSS, Fc-diOH-LNCs or Fc-diOH-LNC-loaded MIAMI cells (scale bar =100 μm). ( C ) and ( D ) Quantitative results for Ki67 and CD31 immunofluorescence. Results are expressed as the mean ± SEM number of Ki67 + cells in the tumor/mm 2 ( C ) and the mean ± SEM number of CD31 + vessels/mm 2 ( D ). *Significantly different from the control group ( P <0.05). Abbreviations: Fc-diOH-LNCs, ferrociphenol lipid nanocapsules; MIAMI, marrow-isolated adult multilineage inducible; SEM, standard error of the mean; HBSS, Hank’s balanced salt solution.

Article Snippet: The human U87MG GB cell line was obtained from the (American Type Culture Collection, Manassas, VA, USA).

Techniques: Immunofluorescence, Staining, Injection, Control, Isolation