Journal: Frontiers in pharmacology

Article Title: Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

doi: 10.3389/fphar.2023.1331687

Figure Lengend Snippet: FIGURE 2 ABCG1 is upregulated in the MTX resistance cell line Reh-MTXR. (A) Illustration of the GSEA of upregulated ABC transporters pathway in Reh-MTXR cells. FDR <0.25 were defined as the significantly enriched gene sets. (B) RNA-seq analysis of differentially expressed genes upregulated and downregulated more than two folds in Reh cells and Reh-MTXR cells. Red dots represent upregulated genes, and blue dots represent downregulated genes. (C) mRNA levels of FPGS, GGH, DHFR, RFC1, ABCG1, ABCG2, ABCC1, ABCC2, ABCC3, ABCC4, ABCC5, and ABCB1 genes in Reh and Reh- MTXR cells were analyzed by RT-qPCR. Expression levels were normalized to a reference mRNA of Actin. Expression of genes in Reh was set as 1. Data are presented as mean ± SD of three independent tests, ****p < 0.0001, two-tailed Student’s t-test. (D) Cellular ABCG1 protein levels were detected by Western blot. Expression of Actin was measured as a loading control.

Article Snippet: The primary antibodies included β-actin (66009-1-Ig, Proteintech, Wuhan, China, 1:8000 dilution), ABCG1 (13578-1-AP, Proteintech, Wuhan, China, 1:5000 dilution), FPGS (ab184564, Abcam, Cambridge, United Kingdom, 1:1000 dilution), GGH (13264-1-AP, Proteintech, Wuhan, China, 1:2000 dilution).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Control

Journal: Frontiers in pharmacology

Article Title: Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells.

doi: 10.3389/fphar.2023.1331687

Figure Lengend Snippet: FIGURE 4 ABCG1 promotes MTX efflux in Reh-MTXR cells. (A, B) Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells were exposed to 2 μM MTX, and after 24 h of accumulation of MTX, the levels of MTX polyglutamated metabolites were determined by LC-MS. (A) Diagram used for identifying MTX polyglutamated metabolites by LC-MS in various Reh cells. (B) Relative levels of MTX, MTX-Glu2, MTX-Glu3, and MTX-Glu4 in various Reh cells, each group was normalized to the Reh group, which was set as 100%. The experiment was performed in triplicates, and data are presented as mean ± SD. ***p < 0.001, ****p < 0.0001, two-tailed Student’s t-test. (C) Western blot analyses of FPGS and GGH in Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells. We measured the expression of Actin as a loading control. (D) After 20 min of incubation in the medium containing 10 μM MTX, we washed the Reh, Reh-MTXR, and Reh-MTXR ABCG1 knockdown cells and incubated them in the drug-free medium. Efflux was measured by determining the level of MTX remaining in the cells after 0, 5, 15, and 30 min of incubation in the drug-free medium. The data are represented as the relative amount of MTX remaining in cells and are shown as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed Student’s t-test, Reh-MTXR group compared with Reh group; MTXR-shABCG1 group compared with Reh-MTXR group.

Article Snippet: The primary antibodies included β-actin (66009-1-Ig, Proteintech, Wuhan, China, 1:8000 dilution), ABCG1 (13578-1-AP, Proteintech, Wuhan, China, 1:5000 dilution), FPGS (ab184564, Abcam, Cambridge, United Kingdom, 1:1000 dilution), GGH (13264-1-AP, Proteintech, Wuhan, China, 1:2000 dilution).

Techniques: Knockdown, Liquid Chromatography with Mass Spectroscopy, Two Tailed Test, Western Blot, Expressing, Control, Incubation

Journal: Cell reports

Article Title: Pituitary adenomas evade apoptosis via noxa deregulation in Cushing’s disease

doi: 10.1016/j.celrep.2022.111223

Figure Lengend Snippet: (A) A sublabial transsphenoidal approach was used for resection of microadenomas (white arrowheads) in patients with Cushing’s disease (CD). Coronal, post-gadolinium contrast enhanced magnetic resonance image from patient P1. Adenoma and adjacent normal pituitary gland were separately annotated during surgery and dissociated into a single-cell suspension, followed by GEM embedding, sequencing, and computational analysis. Scale bar: 2 cm. (B) Summary of demographic data and analysis for patients included in the study. (C) UMAP embedding of cells from the 6 patient samples with CD, GH, NFPA, and PRL adenomas used in scRNA-seq analysis. Cells cluster according to dominant secretory phenotype. (D) UMAP embedding showing the cell-type identities cells from the 6 patient samples. (E) UMAP embedding showing CD sample identity for cells from patients P1, P2, and P3. (F) UMAP plot showing CD patient cells clustering by cell-type identity. Cell types: leukocytes (Les), endothelial cells (ECs), pericytes (Pes), folliculostellate cells (FSs), corticotrophs (Cs), gonadotroph (Gs), somatotrophs (Ss), lactotrophs (Ls), ambiguous/somato-lactotrophs (SLs), and POU1F1 -positive, hormone-negative (P + H − ) cells. GH, growth hormone; NFPA, non-functioning pituitary adenoma; PRL, prolactinoma.

Article Snippet: GH1 , Novus Biologicals , Cat #NBP2–53262.

Techniques: Suspension, Sequencing

Journal: Cell reports

Article Title: Pituitary adenomas evade apoptosis via noxa deregulation in Cushing’s disease

doi: 10.1016/j.celrep.2022.111223

Figure Lengend Snippet: (A) The UMAP plot from split by patient (top, P1; middle, P2; bottom, P3), colored by cell type. (B) Dot plot showing the expression (marker color) and percentage of cells expressing (marker size) for the top 6 cell-type upregulated genes with highest min.logFC.detected from a filtered group of robustly upregulated cell-type marker genes . Genes used as a priori known classification marker genes are indicated by bold font. (C) Multiplexed immunohistochemistry of tissue from P3, showing localization of key hormone markers in margin and core regions of the sample. White bar: 100 μm. PC, pituitary cells; HPC, hormone-producing cells; Mar, tumor margin; POMC, pro-opiomelanocortin; GH, growth hormone; PRL, prolactin; LH, luteinizing hormone; FSH, follicle-stimulating hormone.

Article Snippet: GH1 , Novus Biologicals , Cat #NBP2–53262.

Techniques: Expressing, Marker, Immunohistochemistry

Journal: Cell reports

Article Title: Pituitary adenomas evade apoptosis via noxa deregulation in Cushing’s disease

doi: 10.1016/j.celrep.2022.111223

Figure Lengend Snippet: (A) UMAP plot demonstrating PMAIP1 abundance in CD adenomas (C) but not in PRL, G, or NFPA adenomas. PMAIP1 was also detected at lower levels in Les and ECs. (B) UMAP plot showing MYC upregulation in CD corticotrophs but not other hormone-producing cells (left panel). MYC expression was also detected in Pes, ECs, and Les. Middle panel: UMAP plot showing overlap of MYC and PMAIP1 expression is mostly limited to CD corticotrophs (yellow dots). Right panel: UMAP plot showing UCHL1 abundance in most hormone-producing cell types in CD. (C) Bulk RNA-seq of CD and non-CD samples verified overexpression of pro-apoptotic genes including PMAIP1 in CD tissues. (D) DNA methylation levels (beta values) at CpG sites associated with the PMAIP1 promoter methylation demonstrating hypomethylation in CD (n = 3) compared with normal (autopsy-derived, n = 20) pituitary glands. *p < 0.05. (E) Multiplex immunohistochemistry (mIHC) of 5 μm thick sections from a CD adenoma. Insets from the core-margin boundary represented by white dashed lines. White bar: 100 μm. Core-margin boundary identified by overlaying expression of POMC, TBX19, and DAPI. Compared with the margin, core adenoma cells show robust overexpression of POMC, TBX19, and MYC; however, noxa expression is decreased within the adenoma core. (F) Representative image from noxa IHC in independent adenoma/normal pairs (n = 10). Pairwise analysis of noxa deconvoluted IHC images (absorbance = mean pixel intensity count per pixel) showing suppressed noxa signal in CD adenomas compared with normal (margin) tissues (p = 0.0013; 95% confidence interval [CI] −0.027 to −0.007). Scale bar: 100 μM. (G) Expected epithelial growth factor (EGF) signaling upregulation and ERK1/2 phosphorylation were found in human CD adenoma primary cell lines (P6_CD and P26_CD). A, adenoma (core); N, normal (margin) pituitary gland. Noxa was undetectable or decreased in core adenomas. (H) A survey of human primary CD adenoma cell lines revealed variable noxa expression compared with sCD adenoma, GH adenoma, and NFPA. CD, corticotroph adenoma causing Cushing’s disease; sCD, hormonally silent corticotroph adenoma; GH, growth-hormone-secreting adenoma; NFPA, non-functioning pituitary adenoma.

Article Snippet: GH1 , Novus Biologicals , Cat #NBP2–53262.

Techniques: Expressing, RNA Sequencing, Over Expression, DNA Methylation Assay, Methylation, Derivative Assay, Multiplex Assay, Immunohistochemistry, Phospho-proteomics

Journal: Cell reports

Article Title: Pituitary adenomas evade apoptosis via noxa deregulation in Cushing’s disease

doi: 10.1016/j.celrep.2022.111223

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: GH1 , Novus Biologicals , Cat #NBP2–53262.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing

Journal: FEBS Open Bio

Article Title: Growth hormone receptor promotes osteosarcoma cell growth and metastases

doi: 10.1002/2211-5463.12761

Figure Lengend Snippet: GHR promotes OS cell colony formation and OS tumor growth through the PI3K/AKT pathway. (A) The expression of GHR was detected by western blotting. (B) Control and GHR knockdown 143B and U2OS cells were analyzed by colony formation. (C) Statistical analysis of the colony formation in (B). Data represent mean ± SEM ( n = 3); two‐tailed Student’s t ‐test was used for statistical analysis, ** P < 0.01, *** P < 0.001 in silenced GHR cells (D, E) Knockdown of GHR by two independent siRNAs inhibited the expression of p‐PI3K/AKT in 143B (D) and U2OS (E) cells.

Article Snippet: The used antibodies were as follows: actin (Sigma‐Aldrich, St. Louis, MO, USA), GHR (Proteintech, Shanghai, China), p‐PI3K (Cell Signaling, Shanghai, China), p‐AKT (Cell Signaling), PI3K (Cell Signaling), AKT (Cell Signaling), Goat anti‐Rabbit (Thermo, Danvers, MA, USA) and Goat anti‐Mouse (Thermo).

Techniques: Expressing, Western Blot, Control, Knockdown, Two Tailed Test

Journal: FEBS Open Bio

Article Title: Growth hormone receptor promotes osteosarcoma cell growth and metastases

doi: 10.1002/2211-5463.12761

Figure Lengend Snippet: GHR modulates the proliferation of OS cells in vivo . (A–C) Control and GHR knockdown U2OS cells were injected into nude mice subcutaneously (2 × 10 6 cells per mouse); tumor volume and weight were measured at the indicated day. Data represent mean ± SEM ( n = 5); two‐tailed Student’s t ‐test was used for statistical analysis, *** P < 0.001. (D) Western blotting analysis of p‐PI3K/AKT for GHR knockdown (sh GHR) or control (sh NC) tumors.

Article Snippet: The used antibodies were as follows: actin (Sigma‐Aldrich, St. Louis, MO, USA), GHR (Proteintech, Shanghai, China), p‐PI3K (Cell Signaling, Shanghai, China), p‐AKT (Cell Signaling), PI3K (Cell Signaling), AKT (Cell Signaling), Goat anti‐Rabbit (Thermo, Danvers, MA, USA) and Goat anti‐Mouse (Thermo).

Techniques: In Vivo, Control, Knockdown, Injection, Two Tailed Test, Western Blot

Journal: Human Vaccines & Immunotherapeutics

Article Title: Cross-reactive humoral immunity of clade 2 Oka and MAV/06 strain-based varicella vaccines against different clades of varicella–zoster virus

doi: 10.1080/21645515.2023.2210961

Figure Lengend Snippet: Image of FAMA test with anti-VZV glycoprotein monoclonal antibodies using six different VZV strains as FAMA antigens (400× magnification). (a) gH, (b) gB, (c) gI, (d) gE.

Article Snippet: Sera were twofold serially diluted with Dulbecco’s phosphate-buffered saline (DPBS, Lonza, Breda, Netherlands), and mouse monoclonal anti-VZV glycoprotein H (gH), gB, gI, and gE antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were diluted 1:50 in DPBS.

Techniques: Bioprocessing