Journal: Dermatology Practical & Conceptual

Article Title: GRWD1 Drives Melanoma Growth Through NF-κB Signaling Pathway

doi: 10.5826/dpc.1504a5918

Figure Lengend Snippet: GRWD1 knockdown suppresses invasion and oncogenic signaling in melanoma cells. (A) Matrigel invasion assay shows significantly reduced invasion capacity in GRWD1 siRNA-treated melanoma cells (**** P <0.001). (B) Western blot analysis shows reduced Bcl-2 expression and increased cleaved PARP levels in GRWD1 knockdown cells, indicating apoptosis. (C) Western blot analysis of key oncogenic signaling proteins. GRWD1 knockdown leads to decreased NF-κB, Src, and MDM2 expression while increasing p53 levels. (D) Validation of GRWD1 knockdown effects in A2780 ovarian cancer cells. Cell viability is significantly reduced after GRWD1 knockdown (* P <0.05, * P <0.01). Flow cytometry and Western blot data represent mean ± SD from three independent experiments. Statistical comparisons were made using one-way ANOVA (Tukey’s post hoc test).

Article Snippet: Membranes were incubated overnight at 4° C with primary antibodies against GRWD1 (Biolegend, Cat. No: 645702), Bcl-2 (Elabscience, Cat. No: E-AB-22004), cleaved PARP (Cell Signaling, Cat. No: D214, 19F4), P53 (Biolegend, Cat. No: 645702), p21 (BT LAB, Cat. No: BT-AP12602), MDM2 (Biogend, Cat. No: E-AB-60076), NF-κB p65 (St John’s Laboratory, Cat. No: STJ903553-100), Src (Cell Signaling, Cat. No: 2108S), and β-Actin (Santa Cruz, Cat. No: sc-47778) as a loading control.

Techniques: Knockdown, Invasion Assay, Western Blot, Expressing, Biomarker Discovery, Flow Cytometry

Journal: Dermatology Practical & Conceptual

Article Title: GRWD1 Drives Melanoma Growth Through NF-κB Signaling Pathway

doi: 10.5826/dpc.1504a5918

Figure Lengend Snippet: GRWD1 promotes the ability of proliferation, survival and invasion in melanoma cells through the activation of NF-κB pathway. Proposed mechanism underlying GRWD1-mediated melanoma progression. GRWD1 enhances NF-κB signaling, which transcriptionally upregulates Bcl-2 and activates Src. NF-κB also induces MDM2 expression, promoting p53 degradation. The downregulation of p53 relieves transcriptional repression of Bcl-2, further supporting cell survival and proliferation. This GRWD1–NF-κB–MDM2/p53–Bcl-2 axis contributes to melanoma pathogenesis. Arrows represent activation (solid) and inhibition (dashed).

Article Snippet: Membranes were incubated overnight at 4° C with primary antibodies against GRWD1 (Biolegend, Cat. No: 645702), Bcl-2 (Elabscience, Cat. No: E-AB-22004), cleaved PARP (Cell Signaling, Cat. No: D214, 19F4), P53 (Biolegend, Cat. No: 645702), p21 (BT LAB, Cat. No: BT-AP12602), MDM2 (Biogend, Cat. No: E-AB-60076), NF-κB p65 (St John’s Laboratory, Cat. No: STJ903553-100), Src (Cell Signaling, Cat. No: 2108S), and β-Actin (Santa Cruz, Cat. No: sc-47778) as a loading control.

Techniques: Activation Assay, Expressing, Inhibition

Journal: Nature Communications

Article Title: The histone demethylase Kdm6b regulates subtype diversification of mouse spinal motor neurons during development

doi: 10.1038/s41467-022-28636-7

Figure Lengend Snippet: a , b Unsupervised clustering analysis of the single-cell transcriptomes of Slc18a3 + cells from E12.5 control spinal cord ( Kdm6b f/+ ;Olig2-Cre;mTmG ). 11 c ontrol c lusters, CC0 to CC10, were visualized using tSNE. Cell types were assigned according to the expression of specific marker genes. Each dot represents a single cell and was color-coded based on the cluster identity. c , d Violin plots for selected marker genes in CC0-CC10. e – j tSNE plots to show the expression of the indicated genes in each plot. The color intensity indicates the expression levels of the genes. The tSNE plots in f , g , and i reveal the co-expression pattern of the two genes. f CC8 (V2IN) and CC9 (V3IN) co-express Slc18a3 and Slc17a6 (cyan, Double+), while the remaining clusters express only Slc18a3 (red, Slc18a3+). g CC1 (nbMN) and CC2 (MMC) co-express Isl1 and Lhx3 (cyan, Double+), whereas CC3 (HMC), CC4 (LMCm), and CC6 (PGC-Isl1 + ) express Isl1 only (red, Isl1+). CC8 (V2IN) express Lhx3 only (green, Lhx3+). i CC4 (LMCm) and CC5 (LMCl) co-express Foxp1 and Aldh1a2 (cyan, Double+), whereas CC6 (PGC-Isl1 + ) and CC7 (PGC-Isl1 − ) express only Foxp1 (red, Foxp1+). k Immunohistochemical analyses with Isl1 and Nos1 antibodies in E13.5 wild-type spinal cords at thoracic levels. Nos1 + PGC cells were divided into Isl1 + and Isl1 − subpopulations. Scale bar, 50 μm. 3–6 sections in 3 embryos were analyzed and the representative images were shown.

Article Snippet: Primary antibodies were listed as follow: goat anti-Olig2 (R&D Systems AF2418, 1:500), rabbit anti-Olig2 (Millipore AB9610, 1:1000), guinea pig anti-Mnx1 (Homemade, 1:500), rabbit anti-Isl1 (Abcam ab109517, 1:250), guinea pig anti-Isl1 (Homemade, 1:2000), rabbit anti-Lhx3 (Abcam ab14555, 1:1000), guinea pig anti-Lhx3 (Homemade, 1:250), guinea pig anti-Vsx2 (Homemade, 1:1000) , guinea pig anti-Sox14 (Homemade, 1:500) , rabbit anti-Nkx2-2 (Abcam ab191077, 1:50), rabbit anti-Foxp1 (Abcam ab16645, 1:2000), rabbit anti-Nos1 (Immunostar 24287, 1:1000), rabbit anti-Kdm6b (Novus Biologicals NBP1-06640, 1:100), guinea pig anti-Klf5 , rabbit anti-Mecom (Cell Signaling Technology #2593, 1:500), sheep anti-Zfhx3 (Novus Biologicals AF7384, 1:200), and rabbit anti-Zfhx4 (Sigma HPA023837, 1:200).

Techniques: Expressing, Marker, Immunohistochemical staining

Journal: Nature Communications

Article Title: The histone demethylase Kdm6b regulates subtype diversification of mouse spinal motor neurons during development

doi: 10.1038/s41467-022-28636-7

Figure Lengend Snippet: a Immunohistochemical analyses for Lhx3, FoxP1, and Nos1 and in situ hybridization analyses for Aldh1a2 in E12.5 spinal cords. Foxp1 + MNs, Aldh1a2 + LMC, and Nos1 + PGC were markedly reduced in Mnx1p::Lhx3 transgenic mice, in which Lhx3 expression was expanded to all MNs. Scale bars, 50 μm. b Immunohistochemical analysis with Kdm6b antibody. Kdm6b protein was detected at E11.5 and substantially removed by E12.5 in Kdm6b -cKO; Isl1-Cre mice. Scale bars, 50 μm. c–g Immunohistochemical analyses for Lhx3, FoxP1, Mnx1, and Isl1, in situ hybridization analyses for Aldh1a2, Slc18a3, Klf5, and Kcnip4 in E13.5 spinal cords, and the quantification of specific marker-labeled MNs per 12 μm thick section. In Kdm6b -cKO; Isl1-Cre mice, Lhx3 + and Mnx1 + MNs were significantly reduced, but neither Foxp1 + nor Isl1 + MNs changed significantly. Scale bars, 50 μm. The error bars represent the standard deviation of the mean. ** p < 0.01; ns non-significant in the two-tailed Student’s t -test. n = 3–6 mice per genotype and 9 slices per genotype. Source data are provided as a Source Data file. The exact p -values are as follows. Lhx3, brachial, 1.44 × 10 −8 ; thoracic, 8.05 × 10 −10 . Foxp1, brachial, 0.68; thoracic, 0.94. Mnx1, brachial, 1.79×10 −9 ; thoracic, 2.36 × 10 −7 . Isl1, brachial, 0.55; thoracic, 0.76.

Article Snippet: Primary antibodies were listed as follow: goat anti-Olig2 (R&D Systems AF2418, 1:500), rabbit anti-Olig2 (Millipore AB9610, 1:1000), guinea pig anti-Mnx1 (Homemade, 1:500), rabbit anti-Isl1 (Abcam ab109517, 1:250), guinea pig anti-Isl1 (Homemade, 1:2000), rabbit anti-Lhx3 (Abcam ab14555, 1:1000), guinea pig anti-Lhx3 (Homemade, 1:250), guinea pig anti-Vsx2 (Homemade, 1:1000) , guinea pig anti-Sox14 (Homemade, 1:500) , rabbit anti-Nkx2-2 (Abcam ab191077, 1:50), rabbit anti-Foxp1 (Abcam ab16645, 1:2000), rabbit anti-Nos1 (Immunostar 24287, 1:1000), rabbit anti-Kdm6b (Novus Biologicals NBP1-06640, 1:100), guinea pig anti-Klf5 , rabbit anti-Mecom (Cell Signaling Technology #2593, 1:500), sheep anti-Zfhx3 (Novus Biologicals AF7384, 1:200), and rabbit anti-Zfhx4 (Sigma HPA023837, 1:200).

Techniques: Immunohistochemical staining, In Situ Hybridization, Transgenic Assay, Expressing, Marker, Labeling, Standard Deviation, Two Tailed Test