Journal: Open Life Sciences

Article Title: SPOP regulates the expression profiles and alternative splicing events in human hepatocytes

doi: 10.1515/biol-2022-0755

Figure Lengend Snippet: Overexpression of SPOP could facilitate the proliferation of human normal hepatocytes (WRL68). (a) Transfection efficiency of SPOP in WRL68 cells was measured by qRT-PCR. (b) Proliferative abilities of WRL68 cells in the SPOP group and the vec group were detected by CCK8 assay. (c) Colony formation assay detected the proliferations of WRL68 cells in the SPOP group and the vec group. * p < 0.05, ** p < 0.01.

Article Snippet: Human normal hepatocytes WRL68 (ATCC, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (cat. no. PM150210, Procell, Wuhan, China) with 10% GibcoTM fetal bovine serum (cat. no. 10099141C, Invitrogen, AUS) and 1% penicillin–streptomycin (10,000 U/mL) (cat. no. 15140122, Invitrogen, USA), at 37°C in a 5% CO 2 humidified atmosphere.

Techniques: Over Expression, Transfection, Quantitative RT-PCR, CCK-8 Assay, Colony Assay

Journal: Open Life Sciences

Article Title: SPOP regulates the expression profiles and alternative splicing events in human hepatocytes

doi: 10.1515/biol-2022-0755

Figure Lengend Snippet: Overexpression of SPOP could change the gene expression profiles of WRL68. (a) Boxplot of sample gene expression distribution. (b) Correlation between SPOP overexpression (SPOP rep1/2/3) and control (vec rep1/2/3) samples. (c) DEGs between the SPOP group and the vec group. (d) Correlation of log2 fold-change between RNA-Seq and RT-qPCR for significantly DEGs.

Article Snippet: Human normal hepatocytes WRL68 (ATCC, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (cat. no. PM150210, Procell, Wuhan, China) with 10% GibcoTM fetal bovine serum (cat. no. 10099141C, Invitrogen, AUS) and 1% penicillin–streptomycin (10,000 U/mL) (cat. no. 15140122, Invitrogen, USA), at 37°C in a 5% CO 2 humidified atmosphere.

Techniques: Over Expression, Gene Expression, Control, RNA Sequencing, Quantitative RT-PCR

Journal: Open Life Sciences

Article Title: SPOP regulates the expression profiles and alternative splicing events in human hepatocytes

doi: 10.1515/biol-2022-0755

Figure Lengend Snippet: Potential biological functions of SPOP-related genes in WRL68 cells. (a) Top 30 most enriched GO terms of the upregulated DEGs. (b) Top 30 most enriched GO terms of the downregulated DEGs. (c) Top 20 most enriched KEGG pathways of all DEGs. (d) Top 20 most enriched DisGeNET lists of all DEGs.

Article Snippet: Human normal hepatocytes WRL68 (ATCC, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (cat. no. PM150210, Procell, Wuhan, China) with 10% GibcoTM fetal bovine serum (cat. no. 10099141C, Invitrogen, AUS) and 1% penicillin–streptomycin (10,000 U/mL) (cat. no. 15140122, Invitrogen, USA), at 37°C in a 5% CO 2 humidified atmosphere.

Techniques:

Journal: Open Life Sciences

Article Title: SPOP regulates the expression profiles and alternative splicing events in human hepatocytes

doi: 10.1515/biol-2022-0755

Figure Lengend Snippet: Enrichment biological function of SPOP RASEs in WRL68 cells. (a) Number of significant RASEs. (b) GO functional enrichment of different RASEs. (c) KEGG pathway enrichment analysis results: A3SS events, 11 A5SS events, 20 MXE events, 11 RI events, and 100 SE events.

Article Snippet: Human normal hepatocytes WRL68 (ATCC, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (cat. no. PM150210, Procell, Wuhan, China) with 10% GibcoTM fetal bovine serum (cat. no. 10099141C, Invitrogen, AUS) and 1% penicillin–streptomycin (10,000 U/mL) (cat. no. 15140122, Invitrogen, USA), at 37°C in a 5% CO 2 humidified atmosphere.

Techniques: Functional Assay

Journal: Open Life Sciences

Article Title: SPOP regulates the expression profiles and alternative splicing events in human hepatocytes

doi: 10.1515/biol-2022-0755

Figure Lengend Snippet: HCC pathway modified by SPOP overexpression in WRL68 (the figure was drawn according to the information from KEGG PATHWAY database. The molecules in red were upregulated with SPOP overexpression and the molecules in green were downregulated with SPOP overexpression).

Article Snippet: Human normal hepatocytes WRL68 (ATCC, Manassas, USA) were cultured in Dulbecco’s Modified Eagle Medium (cat. no. PM150210, Procell, Wuhan, China) with 10% GibcoTM fetal bovine serum (cat. no. 10099141C, Invitrogen, AUS) and 1% penicillin–streptomycin (10,000 U/mL) (cat. no. 15140122, Invitrogen, USA), at 37°C in a 5% CO 2 humidified atmosphere.

Techniques: Modification, Over Expression

Journal: The Journal of Cell Biology

Article Title: A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

doi:

Figure Lengend Snippet: Rat DLP1 is expressed in all tissues examined. ( A ) Northern blot analysis showing ubiquitous expression of rat DLP1. Total RNA (15 μg/lane) from various rat tissues was hybridized with a DLP1-specific probe. An unknown lower band was detected in testis. The same blot was also hybridized with a GAPDH probe for internal control. ( B and C ) Western blot analyses showing that DLP1 is present ubiquitously in rat tissues ( B ) and cultured mouse and human cells ( C ). Total protein from various rat tissues and tissue cultured cells was run on SDS–polyacrylamide gels, and DLP1 was detected by an affinity-purified anti-DLP1 antibody (DLP-MID). Each lane was loaded with 20 μg total protein, except the brain lane (10 μg). Note that DLP1 in brain, mouse hepatocytes, and human fibroblasts runs differently in SDS-PAGE from DLP1 in other tissues. B , brain; H , heart; Lg , lung; K , kidney; Sp , spleen; T , testis; Lv , liver; P , pancreas; MH , mouse hepatocyte cell line; HF , cultured human fibroblasts.

Article Snippet: Mouse hepatocytes (normal mouse liver cell line BNL CL.2; American Type Culture Collection [ATCC], Rockville, MD) and primary human foreskin fibroblasts were grown in MEM α medium with l-glutamine, ribonucleosides, and deoxyribonucleosides ( GIBCO BRL , Gaithersburg, MD) containing 10% fetal bovine serum.

Techniques: Northern Blot, Expressing, Control, Western Blot, Cell Culture, Affinity Purification, SDS Page

Journal: The Journal of Cell Biology

Article Title: A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

doi:

Figure Lengend Snippet: DLP1 is associated with numerous punctate vesicular structures in cultured mammalian cells. Immunofluorescence microscopy of two distinct cultured cell lines, human fibroblasts ( A ) and a rat hepatocyte cell line, clone 9 ( B ), stained with affinity-purified antibodies to DLP1 (DLP-N). DLP1 is associated with numerous vesicular structures that are distributed throughout the cytoplasm and appear aligned along cytoskeletal filaments that extend outward from perinuclear foci ( arrows ). Higher magnification images of boxed regions in A and B are shown in A′ and B′ , respectively, and strongly suggest a vesicular association. N , nucleus. Bar, 10 μm.

Article Snippet: Mouse hepatocytes (normal mouse liver cell line BNL CL.2; American Type Culture Collection [ATCC], Rockville, MD) and primary human foreskin fibroblasts were grown in MEM α medium with l-glutamine, ribonucleosides, and deoxyribonucleosides ( GIBCO BRL , Gaithersburg, MD) containing 10% fetal bovine serum.

Techniques: Cell Culture, Immunofluorescence, Microscopy, Staining, Affinity Purification

Journal: The Journal of Cell Biology

Article Title: A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

doi:

Figure Lengend Snippet: DLP1-associated organelles do not colocalize with multiple endocytic organelle markers but are fractionated into light membrane and ER fractions. ( A ) Cultured mouse hepatocytes (top two panels of A ) or human cholangiocytes (bottom two panels of A ) were double stained with antibodies to DLP1 (DLP1-N) and FITC-conjugated dextran ( Dx ) to label endocytic compartments and lysosomes, and FITC-conjugated transferrin ( Tf ) to label the recycling endosomal compartment. Little, if any, colocalization between DLP1 and the labeled endocytic compartments is observed. N , nucleus. ( B ) Immunoblots of rat liver subcellular fractions with DLP1 and various endocytic marker antibodies. Rat liver membranes were fractionated by differential centrifugation and sucrose gradient centrifugation. Each fraction was separated by SDS-PAGE and immunoblotted with anti-DLP1 antibody (DLP-MID) and endocytic marker antibodies including: MC63 for conventional dynamin, anti–α-adaptin, anti-Rab5, and anti–β-galactosidase. While DLP1 is abundant in cytosol and a light vesicle fraction ( LM * ), conventional dynamin is enriched in the plasma membrane ( PM ) with little seen in the LM fraction. α-adaptin and Rab5 are enriched in the plasma membrane fraction, and β-galactosidase is in multiple fractions. ( C ) Same fractions (as prepared in B ) were immunoblotted with anti-DLP1 antibody (DLP-MID) and marker antibodies for secretory vesicles including anti–γ-adaptin for Golgi-associated clathrin-coated vesicles, anti-Sec23p for COPII-coated vesicles, anti-Rab8 for post-Golgi secretory vesicles, and anti-μ3 for vesicles associated with the newly identified nonendocytic adaptor complex AP-3. There is substantial coenrichment of DLP1 with the secretory vesicle marker proteins in the LM fraction. Cyt , cytosol; PM , plasma membranes; LM , light microsomes; G , Golgi fraction. ( D ) Rat liver total microsomes were fractionated by sucrose step gradient centrifugation and fractions were immunoblotted with anti-DLP1 (DLP-MID). DLP1 was enriched in the ER and 1.22 M sucrose fraction, in addition to GL. LS , low speed spin supernatant; LP , low speed spin pellet, TM , total microsomes; GL , light Golgi fraction; GH , heavy Golgi fraction; 1.15 , 1.15 M sucrose fraction; 1.22 , 1.22 M sucrose fraction; ER , endoplasmic reticulum fraction. ( E ) Extended fractionation for the liver microsomes (as prepared in B and C ). Fractions were immunoblotted with anti-DLP1 and anti-Rab1, showing DLP1 enrichment in ER. HM , heavy microsomes; SM , smooth microsomes; RM , rough microsomes. 40 μg protein from each fraction was loaded onto the gels in B–E . Bars, 20 μm.

Article Snippet: Mouse hepatocytes (normal mouse liver cell line BNL CL.2; American Type Culture Collection [ATCC], Rockville, MD) and primary human foreskin fibroblasts were grown in MEM α medium with l-glutamine, ribonucleosides, and deoxyribonucleosides ( GIBCO BRL , Gaithersburg, MD) containing 10% fetal bovine serum.

Techniques: Membrane, Cell Culture, Staining, Labeling, Western Blot, Marker, Centrifugation, Gradient Centrifugation, SDS Page, Clinical Proteomics, Fractionation

Journal: The Journal of Cell Biology

Article Title: A Novel Dynamin-like Protein Associates with Cytoplasmic Vesicles and Tubules of the Endoplasmic Reticulum in Mammalian Cells

doi:

Figure Lengend Snippet: Intracellular Distribution of DLP1 in Rat Liver Hepatocytes In Situ *

Article Snippet: Mouse hepatocytes (normal mouse liver cell line BNL CL.2; American Type Culture Collection [ATCC], Rockville, MD) and primary human foreskin fibroblasts were grown in MEM α medium with l-glutamine, ribonucleosides, and deoxyribonucleosides ( GIBCO BRL , Gaithersburg, MD) containing 10% fetal bovine serum.

Techniques: In Situ, Labeling, Clinical Proteomics