Journal: Cell death & disease

Article Title: Heparin-binding EGF-like growth factor via miR-126 controls tumor formation/growth and the proteolytic niche in murine models of colorectal and colitis-associated cancers.

doi: 10.1038/s41419-024-07126-2

Figure Lengend Snippet: Fig. 3 HB-EGF and EGFR control miR-221 expression that targets AP2a expression. A Fold change in AP2a expression in colon tissues from AOM DSS mice compared to untreated control mice at day 84 (n = 3/group) by qPCR. B Representative immunoblot for AP2a and the loading control b-actin in AP2a OE and Mock cells. C Fold change in AP2a, Egfl7, and miR-126 expression in AP2a OE compared to Mock CMT93 cells as determined by qPCR (n = 3/group). D and E Fold change in miR-221, AP2a, and miR-126 expression in miR-221 OE (D) and miR-221 (E) silenced CMT93 compared to Mock cells (n = 3/group). F Fold change in miR-221 expression in colon tissues of AOM/DSS mice compared to untreated control mice at day 84 (n = 3/group) by qPCR. G Fold change in miR-221 expression in si-HB-EGF, si-EGFR, or si-HB-EGF/si-EGFR CMT93 cells treated with or without rec. HB-EGF compared to the gene expression in si-ctrl CMT93 by qPCR. H Cell proliferation of untreated si-Ctrl, si-miR- 221, and miR-221 OE cells as determined by cell counting (n = 6/group). Mean ± SEM, unpaired Student’s t test. *p < 0.05 and **p < 0.01.

Article Snippet: Cell lysates (2–50 μg proteins) were generated as described previously [6, 17] Membranes (Millipore, Immobilon) were probed with one of the following primary antibodies (1 μg/ml) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, sc1414), ADAM9 precursor (Santa Cruz Biotech, sc-377233), MMP7 (Santa Cruz Biotech, sc-515703), huADAM117 (Santa Cruz Biotech, sc-6416), p-ERK (Cell Signaling, #4370), CCL2 (Santa Cruz Biotech, sc-52701); AP2a (Cell Signaling, #3215); mouse b-actin (Cell Signaling, #4967).

Techniques: Control, Expressing, Western Blot, Gene Expression, Cell Counting

Journal: Cell death & disease

Article Title: Heparin-binding EGF-like growth factor via miR-126 controls tumor formation/growth and the proteolytic niche in murine models of colorectal and colitis-associated cancers.

doi: 10.1038/s41419-024-07126-2

Figure Lengend Snippet: Fig. 4 Silencing of MMP7, ADAM9, and ADAM17 or MMPi treatment enhances miR-126 expression, while miR-126 restoration impairs their expression. A Fold change in ADAM17, miR-221, AP2a, or miR-126 expression in si-ADAM17, si-ADAM9, and si-MMP7 cells compared to expression in si-ctrl CMT93 cells (n = 3/group). B Representative immunoblots for MMP7, ADAM9, ADAM28 and b-actin in si-ctrl, si-MMP7, or si- ADAM9 and si-ADAM28 CMT93 cells. C Fold change in ADAM28, miR-221, AP2a, or miR-126 expression in si-ADAM28 CMT 93 cells (n = 3/group). D Fold change in miR-221, AP2a, and miR-126 expression in wild-type CMT93 cells treated with indicated concentrations of batimastat compared to expression in untreated ctr cells (n = 3/group). E Representative immunoblots for HB-EGF and b-actin in cell lysates from CRC cells treated without or with batimastat. F Fold change in HB-EGF, miR-221, miR-126, ADAM9, and CCL2 expression in Mock, miR-126 OE, and miR-126 KD cells treated with/without batimastat (n = 3/group). B-actin was run on a separate gel from the other proteins for all immunoblots. Mean ± SEM unpaired Student's t test. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Cell lysates (2–50 μg proteins) were generated as described previously [6, 17] Membranes (Millipore, Immobilon) were probed with one of the following primary antibodies (1 μg/ml) overnight at 4 °C: C-terminal cytoplasmic domain of HB-EGF of mouse origin (Santa Cruz Biotech, sc1414), ADAM9 precursor (Santa Cruz Biotech, sc-377233), MMP7 (Santa Cruz Biotech, sc-515703), huADAM117 (Santa Cruz Biotech, sc-6416), p-ERK (Cell Signaling, #4370), CCL2 (Santa Cruz Biotech, sc-52701); AP2a (Cell Signaling, #3215); mouse b-actin (Cell Signaling, #4967).

Techniques: Expressing, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Hyperglycemia-independent neonatal streptozotocin-induced retinopathy (NSIR) in rats

doi: 10.3389/fphar.2024.1395887

Figure Lengend Snippet: Intravitreal STZ injection on P1 inhibits the proliferation of neonatal rat retinal progenitors and delays their cell cycle exit and differentiation. (A) Horizontal P4 retinal sections of indicated locations and groups were stained for nuclear (DAPI, blue) and cell proliferation (Ki67, green). (B) Quantification of Ki67+ cells per mm 2 area of P4 and P8 retinas. (C) Horizontal P8 retinal sections of indicated locations and groups were stained for nuclear (DAPI, blue) and cell proliferation (Ki67, green). (D) Flow cytometry analysis of P4 retinal cells. (E) Whole-mount or horizontal sections of P8 retinas of indicated groups were stained for nuclear (DAPI, blue) and cell type markers, including Ganglion cells (Brn3, green, whole-mount), Cone (ARR3, green), Horizontal cells (OC2, green), Amacrine cells (Ap2a, green), Rod (Rho, green), Bipolar cells (Chx10, green) and Müller glia (Sox9, green). (F) Quantifying all seven retinal cell types (relative to the control group, %). Error bars represent SD of measurements from three animals or three retinas of three animals (n = 3), and asterisks indicate significant differences between control and STZ-treated groups (* p < 0.05, ** p < 0.01, one-way ANOVA followed by Bonferroni’s correction). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; Ivit, Intravitreal injection; PI, Propidium iodide. Scale bar: 50 μm.

Article Snippet: Slides were incubated with blocking solution (1% donkey serum and 0.1% Triton X-100 in PBS) for 1 h, and then with primary antibodies including Ap2a (Santa Cruz, SC-8975); Chx10 (Bremner lab, University of Toronto); Cleaved caspase-3 (Cell Signaling 9661); Cone arrestin or ARR3 (Millipore, AB15282); γ-H2ax (Millipore, 05–636); Onecut2 or OC2 (R&D system AF6294); Ki67 (BD science Pharmingen 550609); PH3 (Upstate 05–598); Rhodopsin (Santa Cruz SC-57433) and Sox9 (Millipore AB5535), overnight at 4°C.

Techniques: Injection, Staining, Flow Cytometry, Control

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet: (A) Schematic showing a doxycycline (dox)-inducible FLAG-MYCN (TRE- MYCN ) lentivirus vector is transduced into human iPSCs, differentiated toward tNCCs, treated with dox, and implanted orthotopically into immunocompromised NSG mice. (B) Western blot showing FLAG-tagged MYCN expression can by modulated with addition of 0.1 μg/mL dox for 24 h. (C and D) Empty vector and TRE-MYCN iPSCs were differentiated toward tNCCs and analyzed for expression of B3GAT1 /HNK1, NGFR /p75, SOX9 , and TFAP2A /AP2A via (C) RT-qPCR (n = 3, error bars represent standard error of mean) and (D) immunofluorescence. Scale bar: 90 μm. (E) Kaplan-Meier survival curve of mice injected with empty vector or TRE-MYCN tNCCs and fed with dox chow (n = 10). p < 0.05 (log-rank test). (F) Immunohistochemical staining for H&E and PHOX2B in TRE- MYCN tumors. Scale bar: 190 μm. See also and .

Article Snippet: Primary antibodies for immunofluorescence were obtained commercially for HNK1 (Sigma Aldrich, 1:500), p75 (Advance Targeting Systems, 1:250), SOX9 (Cell Signaling Technology, 1; 100), AP2A (Thermo Fisher, 1:100), OCT4 (Santa Cruz, 1:500), NANOG (Thermo Fisher, 1:250), SOX2 (Thermo Fisher, 1:500), YAP (Santa Cruz, 1:100), β-catenin (Cell Signaling Technology, 1:300), non-phosphorylated β-catenin (Cell Signaling Technology, 1:300).

Techniques: Plasmid Preparation, Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Injection, Immunohistochemical staining, Staining

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet: (A) Schematic of iPSCs transduced with inducible MYCN expression and/or ALK F1174L and implanted in renal capsules of NSG mice. (B) Western blot validating expression of ALK in ALK F1174L iPSCs. (C and D) Empty vector and ALK iPSCs were differentiated toward tNCCs and analyzed for expression of B3GAT1 /HNK1, NGFR /p75, SOX9 , and TFAP2A /AP2A via (C) RT-qPCR (n = 3, error bars represent standard error of mean) and (D) immunofluorescence. Scale bar: 90 μm. (E) Kaplan-Meier survival curve of mice injected withempty vector (black), ALK tNCCs (green), MYCN tNCCs (blue), or MYCN/ALK tNCCs (red) (n = 10 per group). p < 0.05 between MYCN and MYCN/ALK groups by log-rank test. (F) Western blot analysis validating expression of ALK and MYCN in two separate MYCN and ALK/MYCN tumors. See also and .

Article Snippet: Primary antibodies for immunofluorescence were obtained commercially for HNK1 (Sigma Aldrich, 1:500), p75 (Advance Targeting Systems, 1:250), SOX9 (Cell Signaling Technology, 1; 100), AP2A (Thermo Fisher, 1:100), OCT4 (Santa Cruz, 1:500), NANOG (Thermo Fisher, 1:250), SOX2 (Thermo Fisher, 1:500), YAP (Santa Cruz, 1:100), β-catenin (Cell Signaling Technology, 1:300), non-phosphorylated β-catenin (Cell Signaling Technology, 1:300).

Techniques: Transduction, Expressing, Capsules, Western Blot, Plasmid Preparation, Quantitative RT-PCR, Immunofluorescence, Injection

Journal: Cell reports

Article Title: ALK upregulates POSTN and WNT signaling to drive neuroblastoma

doi: 10.1016/j.celrep.2024.113927

Figure Lengend Snippet:

Article Snippet: Primary antibodies for immunofluorescence were obtained commercially for HNK1 (Sigma Aldrich, 1:500), p75 (Advance Targeting Systems, 1:250), SOX9 (Cell Signaling Technology, 1; 100), AP2A (Thermo Fisher, 1:100), OCT4 (Santa Cruz, 1:500), NANOG (Thermo Fisher, 1:250), SOX2 (Thermo Fisher, 1:500), YAP (Santa Cruz, 1:100), β-catenin (Cell Signaling Technology, 1:300), non-phosphorylated β-catenin (Cell Signaling Technology, 1:300).

Techniques: Recombinant, Membrane, Saline, Western Blot, Blocking Assay, Staining, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Flow Cytometry, Plasmid Preparation, Software