Journal: Cell chemical biology

Article Title: Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter.

doi: 10.1016/j.chembiol.2021.07.016

Figure Lengend Snippet: Figure 3. Carebastine inhibits VCP to enhance NIS function and radioiodide uptake (A) Connectivity Map (L1000 assay) hierarchical cluster analysis comparing the transcriptional signatures of 47 drug treatments with 45 genetic perturbations. Blue, major clusters; green, validated drugs. (B) NanoBiT evaluation of protein:protein interaction between VCP and NIS (left). Normalized NanoBiT assay results at 10 min after addition of Nano-Glo live cell assay solution to HeLa cells treated with VCP inhibitor CB5083 for 24 h (right, n = 4). (C and D) Radioiodide uptake (C) and relative NIS protein levels (D) in TPC-1-NIS cells treated with VCP inhibitors (ebastine [EBT], clotrimazole [CLOT], and astemizole [AST]), either alone or in combination with SAHA. (E) Radioiodide uptake of TPC-1-NIS cells treated with carebastine (CBT) at indicated doses versus EBT. See also Figure S2C.

Article Snippet: For western blotting, primary antibodies were used against LC3B (1:1000; Cell Signalling Technology, cat# 2775), NIS (1:1000; Proteintech, cat# 24324-1-AP), p62/SQSTM1 (1:4000; Proteintech, cat# 18420-1-AP), VCP (1:1000; Proteintech, cat# 10736-1-AP), HA (1: 1000; BioLegend, cat# 901501), and b-actin (1:2000; Sigma-Aldrich, cat# A1978).

Techniques:

Journal: Cell chemical biology

Article Title: Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter.

doi: 10.1016/j.chembiol.2021.07.016

Figure Lengend Snippet: Figure 4. Drug combinations targeting non-canonical pathways augment NIS function (A) Western blot analysis of LC3B-I, LC3B-II, p62, and NIS protein levels in TPC-1-NIS or 8505C-NIS cells treated with chloroquine (CQ). (B and C) Radioiodide uptake in TPC-1-NIS (B) or 8505C-NIS (C) cells treated with CQ at different time points.

Article Snippet: For western blotting, primary antibodies were used against LC3B (1:1000; Cell Signalling Technology, cat# 2775), NIS (1:1000; Proteintech, cat# 24324-1-AP), p62/SQSTM1 (1:4000; Proteintech, cat# 18420-1-AP), VCP (1:1000; Proteintech, cat# 10736-1-AP), HA (1: 1000; BioLegend, cat# 901501), and b-actin (1:2000; Sigma-Aldrich, cat# A1978).

Techniques: Western Blot

Journal: Cell chemical biology

Article Title: Targeting non-canonical pathways as a strategy to modulate the sodium iodide symporter.

doi: 10.1016/j.chembiol.2021.07.016

Figure Lengend Snippet: Figure 6. Putative model of key targetable processes to enhance NIS function and radioiodide therapy NIS maintains a fine balance between protein synthesis, folding, assembly, trafficking, and degradation. (A) The HDAC inhibitor SAHA and MEK inhibitor selumetinib stimulate transcriptional expression of NIS. Surveillance pathways target misfolded NIS protein for ER-associated degradation (ERAD), as well as performing homeostatic regulation of correctly folded NIS protein production (UPR). (B and C) p97/VCP is a critical component of (B) ubiquitin-proteasome and (C) autophagy-lysosome pathways. (D) Disulfiram inhibits proteasomal degradation and autophagy. (E) Vesicular trafficking (e.g., ADP-ribosylation factor 4 [ARF4]) promotes transport of NIS to the PM where it is active. (F) Late endosomes merge with autophagosomes prior to fusing with lysosomes for degradation of protein cargoes. Chloroquine inhibits the fusion of auto- phagosomes and lysosomes, and components of the proteasome. (G) NIS is internalized away from the PM (e.g., PBF) in a clathrin-dependent process, which diminishes radioiodide uptake. Created with BioRender.com.

Article Snippet: For western blotting, primary antibodies were used against LC3B (1:1000; Cell Signalling Technology, cat# 2775), NIS (1:1000; Proteintech, cat# 24324-1-AP), p62/SQSTM1 (1:4000; Proteintech, cat# 18420-1-AP), VCP (1:1000; Proteintech, cat# 10736-1-AP), HA (1: 1000; BioLegend, cat# 901501), and b-actin (1:2000; Sigma-Aldrich, cat# A1978).

Techniques: Expressing, Ubiquitin Proteomics

Journal: Cell Stress

Article Title: Sensitive, non-immunogenic in vivo imaging of cancer metastases and immunotherapy response

doi: 10.15698/cst2023.08.288

Figure Lengend Snippet: ( A and B ) Schematic illustration of the workflow to obtain stable mNIS expression in transduced tumor cell lines without additional expression of potentially immunogenic in vitro selection markers. (C) Flow cytometry of expanded monoclonal sgKRT19 and sgScramble cancer cells prior to transduction (Ctrl), then before (Pre) and after (Post) adeno-Flpo transduction. (D) Western blot analysis for mNIS and KRT19 expression in monoclonal sgKRT19 and sgScramble cells following Ad-Flpo transfection and selection for the absence of GFP fluorescence (left two lanes). Blotting results on protein extracts from both non-mNIS expressing cells (NIS ctrl-) and mNIS expressing cells (NIS ctrl+) are shown in the right two lanes.

Article Snippet: Removal of the positive selection cassette was further confirmed by PCR analysis, while the preservation of mNIS expression was assessed by Western blotting with an anti-NIS antibody (1:200 dilution; #514487, Santa Cruz, Dallas, TX).

Techniques: Expressing, In Vitro, Selection, Flow Cytometry, Transduction, Western Blot, Transfection, Fluorescence

Journal: Cell Stress

Article Title: Sensitive, non-immunogenic in vivo imaging of cancer metastases and immunotherapy response

doi: 10.15698/cst2023.08.288

Figure Lengend Snippet: ( A and B ) A representative SPECT/CT maximum intensity projection (MIP) and a 2D coronal slice image of metastatic mNIS+/GH-pancreatic tumors developing predominantly in the liver, 5 weeks after tumor cell introduction via the portal vein. Note that the thyroid and salivary glands, stomach and bladder (denoted by green arrows in A ) are sites of endogenous NIS expression or probe excretion and do not represent sites of tumor development. (C) Photograph of tumors (yellow arrows) in the liver of the same mouse imaged in A and B, taken 5 days later at necropsy (also see Figures S2 and S3).

Article Snippet: Removal of the positive selection cassette was further confirmed by PCR analysis, while the preservation of mNIS expression was assessed by Western blotting with an anti-NIS antibody (1:200 dilution; #514487, Santa Cruz, Dallas, TX).

Techniques: Single Photon Emission Computed Tomography, Expressing