Journal: PLoS ONE

Article Title: Ectopically Expressed Variant Form of Sperm Mitochondria-Associated Cysteine-Rich Protein Augments Tumorigenicity of the Stem Cell Population of Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0069095

Figure Lengend Snippet: A. SMCP expression profiles in normal human organs. SMCP mRNA expression in normal tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, leukocyte, colon, stomach and bone marrow) was investigated by RT-PCR. cDNAs were obtained from TAKARA BIO INC. (Human Multiple Tissue cDNA Panels I and II). GAPDH was used as an internal positive control. B. SMCP expression profiles in human cancer cells. SMCP mRNA expression in lung cancer line cells (Sq-1, Lc817, 86-2, Lu99, 1-87, A549), renal cancer line cells (Caki-1, ACHN, SMKTR1, SMKTR2, SMKTR3, SMKTR4) and pancreas cancer line cell (HPC3) was investigated by RT-PCR. cDNAs were generated by total RNAs derived from cell lines. GAPDH was used as an internal positive control. C. SMCP expression profiles in human lung cancer tissues. SMCP mRNA expression in human lung cancer tissues was investigated by RT-PCR. #1 and #2 are adenocarcinoma cases. #3 and #4 are squamous cell carcinoma cases. #5 is a large cell carcinoma case. T indicates lung cancer tissue. N indicates adjacent lung normal tissue. GAPDH was used as an internal positive control. D. Schema of novel variant form of SMCP. SMCP wild type (variant 1) is composed of exon 1 and exon 2. The novel isoform of SMCP (variant 2) has only one exon with a 5′ terminal additional extension. E. Expression profiles of SMCP variant 1 and variant 2 in the testis and cancer cells. SMCP mRNA expression was investigated by RT-PCR using an F1 and F2 mixture primer as a forward primer and R1 primer as a reverse primer in testis and cancer cells. Variant 1 was a 152-bp PCR product, and variant 2 was a 624-bp PCR product. F. Expression of SMCP protein. Detection of SMCP protein in HEK293T cells transfected with expression vectors of variant1 CDS and variant2 as assessed by Western blot analysis with polyclonal SMCP antibody. Beta-actin was used as a protein loading control.

Article Snippet: Human breast adenocarcinoma cell line MCF7, human lung adenocarcinoma cell line A549 and human embryonal kidney cell line HEK293T were purchased from ATCC (Manassas, VA, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Generated, Derivative Assay, Variant Assay, Transfection, Western Blot

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: Over-expression of STYK1/NOK causes mitotic abnormality. (A) The endogenous STYK1/NOK expressions in different cell lines by immunoblot analysis in PC-3 cells, U-87 MG cells, DLD-1 cells, HeLa cells, HEK293T cells, RPE-1 cells and HCT116 cells. GAPDH was used as a loading control. The band intensities of STYK1/NOK were quantified with FIJI software and then normalized to that of GAPDH. The relative expressions of all others were compared to that of DLD-1 cells of which was set as 1.00. The original blots are presented in Supplementary Figure 1A and 1B. (B) Establishment of the doxycycline-controlled STYK1/NOK stable cell line. HeLa cells were infected with lentiviral constructs containing doxycycline-inducible STYK1/NOK expression cassettes. The infected cells were selected with 1 μg/mL of puromycin for 5 days to obtain the stable cell line Tet-S/N. Tet-S/N cells were treated with doxycycline for 48 h before harvesting. The original blots are presented in Supplementary Figure 1C. (C) Representative immunofluorescent images of the established stable HeLa cells that were stained with α-tubulin (Green) and DAPI (Blue). Arrows indicated cells undergoing cytokinesis. Quantification of immunofluorescent results presented in (C) by calculating proportions of mitotic cells (D) and cells undergoing cytokinesis (E) to the total cells counted. The data were mean ± SEM from four independent experiments. (F) Immunoblot analysis on doxycycline-induced STYK1 silencing. DLD-1 cells were stably transfected with lentiviral doxycycline-controlled lentiviral constructs expressing shSTYK1/NOK to establish the Tet-shS/N cell line. The transfected cells were selected with 2 μg/mL of puromycin for 5 days. Cells were cultured in the presence and absence of doxycycline for 3 days before harvesting. The original blots are presented in Supplementary Figure 1D. (G) Representative immunofluorescent images of the established stable Tet-shS/N cells that were stained with α-tubulin (Green) and DAPI (Blue). Quantification of immunofluorescent results showed the proportions of mitotic cells (H) and cytokinesis (I) as mean ± SEM from three independent experiments. Mitotic cells were distinguished and counted by morphological evaluation based on the α-tubulin and DAPI staining. ∗ p < 0.05; ∗∗∗ p < 0.001.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Over Expression, Western Blot, Software, Stable Transfection, Infection, Construct, Expressing, Staining, Transfection, Cell Culture

Journal: Heliyon

Article Title: STYK1/NOK affects cell cycle late mitosis and directly interacts with anaphase-promoting complex activator CDH1

doi: 10.1016/j.heliyon.2022.e12058

Figure Lengend Snippet: CDH1 interacts with STYK1/NOK in a kinase domain dependent manner. (A) HeLa cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 or an empty vector. Cells were treated with 50 μg/mL of cycloheximide for the indicated time before harvesting. Immunoblot analysis (upper panel) was conducted to detect the expression of STYK1/NOK. The expression of GAPDH served as an internal control. The original blots are presented in Supplementary Figure 4A. Quantification analysis (lower panel) of the relative band intensity of STYK1/NOK was performed to determine the protein half-life of STYK1/NOK. The STYK1/NOK band intensities were normalized to that of GAPDH, then further normalized to that of t = 0 time point. (B) HEK293T cells were co-transfected with pcDNA3.0-STYK1/NOK and FLAG-CDH1 and treated with 10 μM of MG132 for 4 h before harvesting. Co-immunoprecipitation for detection of STYK1/NOK and CDH1 interaction was then performed. The original blots are presented in Supplementary Figure 4B. (C) Schematic representation of the wild type STYK1/NOK domain structure and its five D-boxes (D1 to D5). Red letters represent conserved residues and green letters represent preferred residues. (D) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its D-box deleted mutants (ΔD1 to ΔD5) and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation was carried out to detect the interaction between CDH1 and either STYK1/NOK or its mutants. The original blots are presented in Supplementary Figure 4C. (E) Schematic presentation of the full-length STYK1/NOK and its truncated forms. (F) HEK293T cells were co-transfected with FLAG-CDH1 and pcDNA3.0-STYK1/NOK or its truncated forms and treated with 10 μM of MG132 for 4 h before harvesting. Coimmunoprecipitation assay was used to detect the interaction between CDH1 and full-length STYK1/NOK or its truncated forms. The star ∗ represents non-specific proteins. The original blots are presented in Supplementary Figure 4D.

Article Snippet: Human prostatic adenocarcinoma cell line PC-3, human brain glioblastoma cell line U-87 MG, human colorectal adenocarcinoma cell line DLD-1, human cervical cancer cell line HeLa, human embryonic kidney 293 derived cell line HEK293T, hTERT-immortalized retinal pigment epithelial cell line RPE-1 and human colorectal cancer cell line HCT116 were from the American Type Culture Collection.

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: TRPC channels blockade abolishes endotoxemic cardiac dysfunction by hampering intracellular inflammation and Ca 2+ leakage

doi: 10.1038/s41467-022-35242-0

Figure Lengend Snippet: a The interacted domain of TLR4 (Myc-tagged) with CaM (FLAG-tagged) in co-transfected HEK293T cells measured using Co-IP ( n = 2 biological independent experiments). b Representative immunofluorescent photomicrographs (left panel) and traces of fluorescence intensity spatial profiles (right panel) of TLR4 (Myc-tagged) with CaM (FLAG-tagged) localization in co-transfected HEK293T cells ( n = 6 images from 3 biological independent experiments). c The CD spectroscopies of the synthesized peptides. d The interaction of CaM with the peptides encompassing nonclassical IQ motifs detected by a non-denaturing gel ( n = 2 biological independent experiments). e The dissociation constants of CaM and peptide TLIQ2 measured using MST (mean ± SEM, n = 3 biological independent experiments). f Structural model of signaling complex formed by TLR4 and CaM. Secondary structure elements and position of mutational sites in the human TLR4 sequence (upper panel) and a homologous modeling of TLR4 (TLR2, PDB 1Fyx) and TLIQ2 contacting with CaM (PDB 1QX5) (lower panel) are shown. Source data are provided as a Source Data file.

Article Snippet: Human embryonic kidney 293 T (HEK293T) cell line was obtained from ATCC (CRL-3216).

Techniques: Transfection, Co-Immunoprecipitation Assay, Fluorescence, Synthesized, Sequencing

Journal: PLoS ONE

Article Title: Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

doi: 10.1371/journal.pone.0028462

Figure Lengend Snippet: HEK 293T cells were transfected with indicated packaging and gene transfer vectors. One set of transfections received pCI-Neo (speckled bars) and a parallel set of transfections received pCI-HIV Rev (cross-hatched bars). All transfections received VSV-G envelope expression construct and a SEAP expression construct. The supernatants were harvested 72 h post-transfection and used for infection of naïve HEK 293T cells. The virus titers (top panel) were determined by flow cytometry of infected cells as described in and normalized for transfection efficiency against SEAP activity. The supernatants were also assayed for virus particle content by ELISA for HIV-1 capsid protein (p24). The SEAP-adjusted p24 values are shown in the bottom panel. Discordant results between the titers and the p24 levels are indicated by asterisks (*) above the corresponding bars. Each experiment was carried out in duplicate. Error bar = 1 SD.

Article Snippet: Human embryonic kidney (HEK 293T) cell line was obtained from the American Type Culture Collection (Manassas, VA) (catalog number CRL-11268) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 u/ml) and streptomycin (100 µg/ml).

Techniques: Transfection, Expressing, Construct, Infection, Flow Cytometry, Activity Assay, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Rev-Free HIV-1 Gene Delivery System for Targeting Rev-RRE-Crm1 Nucleocytoplasmic RNA Transport Pathway

doi: 10.1371/journal.pone.0028462

Figure Lengend Snippet: The vectors pN- EF1α-EGFP-WPRE, pN- EF1α-EGFP-1xCTE, pN- EF1α-EGFP-2xCTE, and pN- EF1α-EGFP-4xCTE (A and C) and their corresponding SIV RRE containing vectors (B and D) were individually packaged using pGP/4xCTE in HEK 293T cells as described in . One set of transfections received pCI-Neo (speckled bars) and a parallel set received pCI-HIV Rev (cross-hatched bars) The SEAP-adjusted titers are shown in panels A and B while the SEAP-adjusted p24 levels are shown in panels C and D. Each experiment was carried out in duplicate. Error bar = 1 SD.

Article Snippet: Human embryonic kidney (HEK 293T) cell line was obtained from the American Type Culture Collection (Manassas, VA) (catalog number CRL-11268) and maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (100 u/ml) and streptomycin (100 µg/ml).

Techniques: Transfection

Journal: Cell & Bioscience

Article Title: Aberrant methylation of Serpine1 mediates lung injury in neonatal mice prenatally exposed to intrauterine inflammation

doi: 10.1186/s13578-022-00901-8

Figure Lengend Snippet: Demethylation promoted Rela but inhibited Hdac2 binding to the Serpine1 promoter. A–C Serpine1 expression analysed using RT-qPCR and western blot in Rela-, Hif1a-, and Rest-overexpressing MLE-12 cells. D Transcriptional activity of Rela, Hif1a, and Rest at the Serpine1 promoter analysed using luciferase assay in MLE-12 cells. E The binding ability of Rela and Hdac2 to the biotin-labelled Serpine1 promoter probe analysed via DNA pull-down assay. F–H Serpine1 expression analysed using RT-qPCR and western blot in Hdac2-knockdown MLE-12 cells. I Transcriptional activity of Rela, Hif1a, and Rest at the methylated-Serpine1 promoter analysed using luciferase assay in HEK293T cells. J Transcriptional activity of Rela at the Serpine1 promoter analysed using luciferase assay in Hdac2-knockdown MLE-12 cells. K The enrichment of TFs at the Serpine1 promoter region determined using anti-Rela and anti-Hdac2 antibodies via ChIP-qPCR in 5-Aza-CdR-treated MLE-12 cells. Data show mean ± SEM; Data was analysed using unpaired t -tests

Article Snippet: MLE-12, A549, BEAS-2B and human epithelial kidney 293 T (HEK293T) cell lines were purchased from ATCC.

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Activity Assay, Luciferase, Pull Down Assay, Methylation