Journal: Frontiers in oncology

Article Title: CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

doi: 10.3389/fonc.2022.931749

Figure Lengend Snippet: FIGURE 1 Effects of CAPRIN2 on the ferroptosis and survival of ECM-detached NPC cells and the migration and invasion of NPC cells. (A) Viability assay of ECM-detached NPC cell lines treated with erastin (5 mM) and/or ferrostatin-1 (1 mM) for 24 h (B) Viability assay of the indicated stable 5-8F or C666-1 cell lines cultured under ECM detachment conditions for 72 h For (A, B), the experiments were repeated three times, and the data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001. (C, D) Migration and invasion assays of the indicated stable 5-8F (C) and C666-1 (D) cell lines. These assays were conducted in triplicate. Representative images are displayed. The data are presented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies against CAPRIN2 (NBP1-88318, Novus), HMGCR (sc-271595, Santa Cruz) and b-actin (66009-1-Ig, Proteintech, Wuhan, China) were commercially obtained. frontiersin.org

Techniques: Migration, Viability Assay, Cell Culture

Journal: Frontiers in oncology

Article Title: CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

doi: 10.3389/fonc.2022.931749

Figure Lengend Snippet: FIGURE 2 CAPRIN2 promotes the ferroptosis resistance, survival, migration and invasion of NPC cells through HMGCR. (A) Overexpression of HMGCR partially reverses the effects of CAPRIN2 on the ferroptosis of ECM-detached 5-8F (left panel) and C666-1 (right panel) cells. The NPC cell lines were treated with erastin (5 mM) for 24 h (B, C) MDA assay (B) and GSH assay (C) results of erastin-treated NPC stable cell lines as indicated. (D) Ectopic expression of HMGCR partially rescues the effects of CAPRIN2 knockdown on ECM-detached NPC cell survival. For (A–C) and (D), the experiments were conducted in triplicate, and the data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01. (E, F) Stable overexpression of HMGCR partially reverses the effects of CAPRIN2 knockdown on 5-8F (E) and C666-1 (F) cell migration and invasion. Representative images of three independent experiments are shown. The data are expressed as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies against CAPRIN2 (NBP1-88318, Novus), HMGCR (sc-271595, Santa Cruz) and b-actin (66009-1-Ig, Proteintech, Wuhan, China) were commercially obtained. frontiersin.org

Techniques: Migration, Over Expression, Multiple Displacement Amplification, GSH Assay, Stable Transfection, Expressing, Knockdown

Journal: Frontiers in oncology

Article Title: CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

doi: 10.3389/fonc.2022.931749

Figure Lengend Snippet: FIGURE 3 Regulation of lung colonization capacity via the CAPRIN2/HMGCR axis in NPC cells. (A, B) The inhibitory effect of erastin on the lung metastasis of NPC cells was enhanced by knockdown of the CAPRIN2/HMGCR axis. (C, D) CAPRIN2 promotes the lung colonization of NPC cells through HMGCR. For (A, C), representative images of lungs and HE staining are shown. The location of lung metastatic nodules is indicated by the arrow. For (B, D), the number of lung metastases (left panel) and the weight of the lungs (right panel) are given. * p < 0.05, ** p < 0.01.

Article Snippet: Primary antibodies against CAPRIN2 (NBP1-88318, Novus), HMGCR (sc-271595, Santa Cruz) and b-actin (66009-1-Ig, Proteintech, Wuhan, China) were commercially obtained. frontiersin.org

Techniques: Knockdown, Staining

Journal: Frontiers in oncology

Article Title: CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

doi: 10.3389/fonc.2022.931749

Figure Lengend Snippet: FIGURE 4 LINC00941 acts as an upstream molecule to regulate the biological functions of CAPRIN2. (A) LINC00941 downregulation promoted the ferroptosis of ECM-detached NPC cells, which was partially rescued by CAPRIN2 overexpression. The NPC cells were treated with erastin (5 mM) for 24 h (B) Knockdown of LINC00941 decreased the survival of ECM-detached NPC cells, which could be partially reversed by CAPRIN2 overexpression. For (A) and (B), the assays were conducted in triplicate, and the data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01. (C, D) LINC00941 knockdown inhibited the migration and invasion of 5-8F (C) and C666-1 (D) cells, and overexpression of CAPRIN2 partially reversed this effect. Representative images are shown. The data are provided as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies against CAPRIN2 (NBP1-88318, Novus), HMGCR (sc-271595, Santa Cruz) and b-actin (66009-1-Ig, Proteintech, Wuhan, China) were commercially obtained. frontiersin.org

Techniques: Over Expression, Knockdown, Migration

Journal: Frontiers in oncology

Article Title: CAPRIN2 upregulation by LINC00941 promotes nasopharyngeal carcinoma ferroptosis resistance and metastatic colonization through HMGCR.

doi: 10.3389/fonc.2022.931749

Figure Lengend Snippet: FIGURE 5 CAPRIN2 is overactivated in NPC tissues and is associated with a poor prognosis in patients. (A) The expression level of CAPRIN2 in 20 NPC tissues and 5 nasopharyngeal tissues. (B) The correlation between LINC00941/CAPRIN2, CAPRIN2/HMGCR or LINC00941/HMGCR in 20 NPC tissues. For (A) and (B), the expression levels of CAPRIN2, HMGCR and LINC00941 were determined by qRT-PCR. The levels were normalized to those of b-actin and shown as the mean ± SEM. * p < 0.05. (C) Representative immunohistochemical images of normal nasopharyngeal tissues (left panel) and NPC tissues (middle panel). The boxes represent the magnified region. The representative image of negative stained control (right panel, top) shows the negative staining result of NPC tissues incubated with antibody-free serum. The representative image of positive stained control (right panel, bottom) shows the positive staining result of CAPRIN2 in NPC tissues incubated with the primary antibody of CAPRIN2. (D) Kaplan-Meier survival analysis of the association between CAPRIN2 expression and the PFS or OS of NPC patients (log-rank test).

Article Snippet: Primary antibodies against CAPRIN2 (NBP1-88318, Novus), HMGCR (sc-271595, Santa Cruz) and b-actin (66009-1-Ig, Proteintech, Wuhan, China) were commercially obtained. frontiersin.org

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Control, Negative Staining, Incubation

Journal: Hepatology (Baltimore, Md.)

Article Title: Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex.

doi: 10.1002/hep.27243

Figure Lengend Snippet: Figure 2. Wnt/β-catenin activation in HB. A. Schematics of the CTNNB1 and CAPRIN2 proteins showing the positions of individual somatic alterations

Article Snippet: Samples were incubated at room temperature for 1 h with an antibodies against β-catenin (1:100; BD Biosciences), SPOP (1:600; Novus) and Caprin2 (1:00; Novus) followed by incubation with the secondary antibody (Dako); sections were counterstained with hematoxylin.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

doi: 10.1101/2022.07.28.501815

Figure Lengend Snippet: Arginine vasopressin ( Avp ), Caprin family member 2 ( Caprin2) , Cocaine and Amphetamine Regulated Transcript ( Cartpt) , Cellular retinoic acid-binding protein 1 ( Crabp1 ), Galanin ( Gal ), glucagon-like peptide-1 receptor ( Glp1r ), 5-Hydroxytryptamine Receptor 2A ( Htr2a ), oxytocin ( Oxt ), Prodynorphin ( Pdyn ), Pro-Melanin Concentrating Hormone ( Pmch ), ras-related dexamethasone induced 1 ( Rasd1 ), and Transthyretin ( Ttr ) were corrected for Glyceraldehyde-3-phosphate dehydrogenase ( Gapdh ) RNA content. Data are expressed as mean ± SD.

Article Snippet: Following this, sections were incubated overnight at 4°C with primary antibodies against AVP neurophysin II (NP-II; MERCK, MABN845, PS41; 1:200); OXT NP-I (PS38; 1:100; ; CAPRIN2 (Proteintech, 20766-1-AP, 1:500), PDYN (Proenkephalin B (D-6); Santa Cruz Biotechnology, sc-398808, 1:100); CART (Bio-techne, AF163, 1:200); TTR (Bio-techne, NBP2-41101, 1:200), 5-HT2A (Alomone labs, ASR-033, 1:200).

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

doi: 10.1101/2022.07.28.501815

Figure Lengend Snippet: Detection of green fluorescent protein (GFP), arginine vasopressin (AVP), oxytocin (OXT), Caprin family member 2 (CAPRIN2), Prodynorphin (PDYN), Cocaine- and amphetamine-regulated transcript protein (CART), Transthyretin (TTR) and Serotonin 5-HT2A receptor (5-HT2A). Images are representative of n = 4. Scale bar represents 40 µm.

Article Snippet: Following this, sections were incubated overnight at 4°C with primary antibodies against AVP neurophysin II (NP-II; MERCK, MABN845, PS41; 1:200); OXT NP-I (PS38; 1:100; ; CAPRIN2 (Proteintech, 20766-1-AP, 1:500), PDYN (Proenkephalin B (D-6); Santa Cruz Biotechnology, sc-398808, 1:100); CART (Bio-techne, AF163, 1:200); TTR (Bio-techne, NBP2-41101, 1:200), 5-HT2A (Alomone labs, ASR-033, 1:200).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 promotes constitutive LRP6 Ser-1490 phosphorylation. A, HEK293 cells were transfected with Caprin-2-HA. Cells were treated with control conditioned medium (CON CM), Wnt-3a protein, or Dkk1 CM for 30 min, and phosphorylation of endogenous LRP6 was then detected using an anti-pLRP6 (p1490) antibody. The anti-LRP6 antibody and anti-α-tubulin were used as the internal controls. The immunoblots were quantified by densitometry, and the intensity values were normalized with those of LRP6; values are given beneath each band. B, HEK293 cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 72 h. Cells were treated with control CM, Wnt-3a protein, or Dkk1 CM for 30 min, and phosphorylation of LRP6 was then detected and quantified as indicated. C, HEK293 cells were transfected with CDK14-GFP, Cyclin Y-GFP, and Caprin-2-HA, and 24 h later, cells were collected and analyzed by Western blotting. D, HEK293 cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 48 h, and then cells were transfected with CDK14-GFP and Cyclin Y-GFP. 24 h later, cells were collected and analyzed by Western blotting.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Transfection, Western Blot, Infection

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2-mediated LRP6 Ser-1490 phosphorylation is cell cycle-dependent. A, HeLa cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 56 h. Then cells were arrested in G2/M by nocodazole (100 ng/ml) treatment for 16 h. Cells were collected in SDS sample buffer and analyzed by Western blotting. Parallel FACS analysis was performed correspondingly. B, HeLa cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 24 h. Then cells were synchronized at G1/S by double thymidine block. G1/S-arrested cells were then washed to progress through the cell cycle. Cells were collected at the indicated times and analyzed by Western blotting. FACS analysis of the cells is shown beneath the corresponding lanes. CCNB was used as a marker for cell cycle progression. C, HeLa cells with or without infection of Caprin-2 shRNAs were arrested at the indicated phases of the cell cycle by nocodazole treatment and then subjected to Western blotting analysis.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Infection, Western Blot, Blocking Assay, Marker

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 interacts with CDK14/Cyclin Y. A, HeLa cells were fractionated and then subjected to Western blotting analysis. LRP6, α-tubulin, and Sp1 served as the loading controls for the membrane (Mem), cytosolic (Cyto), and nuclear (Nuc) fractions, respectively. B, schematic of Caprin-2 fragments. Numbers indicate amino acids. HR, homologous region. N, amino-terminal fragment of Caprin-2; M, middle fragment of Caprin-2; C, carboxyl-terminal fragment of Caprin-2. C and D, HEK293T cells expressing HA-Ca2-FL, HA-Ca2-N, HA-Ca2-M, or HA-Ca2-C were co-transfected with either CDK14-GFP or Cyclin Y-GFP. Then the cell lysates were immunoprecipitated with anti-GFP antibody, and the immunoprecipitates were probed with the indicated antibodies. E and F, GST-tagged CDK14 or Cyclin Y expressed in E. coli was bound to the glutathione-agarose beads. Then the His-tagged Caprin-2 fragments were added as indicated and incubated for 3 h. After three washes with lysis buffer, the proteins were eluted with the SDS loading buffer and analyzed by Western blotting using anti-GST and anti-His monoclonal antibodies.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Western Blot, Expressing, Transfection, Immunoprecipitation, Incubation, Lysis

Journal: The Journal of Biological Chemistry

Article Title: Activation/Proliferation-associated Protein 2 (Caprin-2) Positively Regulates CDK14/Cyclin Y-mediated Lipoprotein Receptor-related Protein 5 and 6 (LRP5/6) Constitutive Phosphorylation *

doi: 10.1074/jbc.M116.744607

Figure Lengend Snippet: Caprin-2 scaffolds the CDK14-Cyclin Y-LRP6 complex. A, HEK293T cells were transfected with CDK14-GFP and Cyclin Y-FLAG with or without 5HA-Caprin-2 as indicated. Cell lysates were then immunoprecipitated with anti-FLAG antibody, and the immunoprecipitates were probed with the indicated antibodies. B, HEK-293T cells were infected by a lentivirus encoding shRNAs of Caprin-2 for 48 h, and then cells were transfected with the indicated plasmids. Co-IP was performed as described in A. C, endogenous interaction of Caprin-2/CDK14/Cyclin Y increased at G2/M. HeLa cells were synchronized by double thymidine block, and the cells lysates from G1/S- or G2/M-synchronized cells were immunoprecipitated by Cyclin Y-specific antibody, followed by Western blotting analysis with the indicated antibodies. D, endogenous Caprin-2/CDK14/Cyclin Y/LRP6 complexes were analyzed in HEK293 cells. Immunoprecipitation was performed with an anti-LRP6 polyclonal antibody. IgG was used as a control. Before co-immunoprecipitation, cells were treated with shCaprin-2 lentivirus for 56 h and nocodazole for another 16 h. E, model for Caprin-2 modulating constitutive LRP6 phosphorylation.

Article Snippet: Caprin-2 antibody was produced by Abgent.

Techniques: Transfection, Immunoprecipitation, Infection, Co-Immunoprecipitation Assay, Blocking Assay, Western Blot