Journal: Oncology Reports

Article Title: RORβ suppresses the stemness of gastric cancer cells by downregulating the activity of the Wnt signaling pathway

doi: 10.3892/or.2021.8131

Figure Lengend Snippet: RORβ exerts anti-proliferative and pro-apoptotic effects on GC cells. (A) MKN45 and AGS cells were transfected with control, RORβ overexpression vector or RORβ shRNA and confirmed by western blotting. The viability of GC cells was evaluated using an CCK-8 assay. Data were analyzed using one-way ANOVA. (B and C) Apoptosis of GC cells was detected using flow cytometry. GC cells were transfected with control or RORβ-overexpression vector for 24 h. Cell apoptosis was evaluated using the values in quadrants 2 and 4. Data were analyzed using a unpaired Student's t-test. (D) GeneChip was used to evaluate the gene expression profiles. The top differentially expressed genes are listed. (E) Protein expression levels of BCL2L11 were analyzed using western blotting. *P<0.05 and **P<0.01. RORβ, retinoic acid-related orphan receptor β; GC, gastric cancer; sh-, short hairpin; BCL2L11, Bcl-2 like protein 11; ov, overexpression.

Article Snippet: GC cells were transfected with 4 μg/μl RORβ-short hairpin (shRNA) and 4 μg/μl control shRNA (both from Santa Cruz Biotechnology, Inc.) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 days and then the cells were screened with puromycin (5 μg/ml) for 14 days to obtain the stable RORβ-knockdown cells further verified by western blotting.

Techniques: Transfection, Control, Over Expression, Plasmid Preparation, shRNA, Western Blot, CCK-8 Assay, Flow Cytometry, Gene Expression, Expressing

Journal: Oncology Reports

Article Title: RORβ suppresses the stemness of gastric cancer cells by downregulating the activity of the Wnt signaling pathway

doi: 10.3892/or.2021.8131

Figure Lengend Snippet: RORβ inhibits stemness properties of GCSCs. GC cells were transfected with control, RORβ overexpression vector or RORβ shRNA vector. (A) Images of colospheres of GC cells. Self-renewal capacity of GCSCs was determined using a sphere formation assay. (B and C) mRNA expression levels of CSC markers in RORβ-overexpressing or RORβ-silenced AGS cells and MKN45 cells were analyzed using RT-qPCR and confirmed by western blotting. (D and E) Tumorigenicity of RORβ-overexpressing cells. Nude mice were inoculated with 1×105, 5×105 and 1×106 RORβ-overexpressing AGS cells (n=5). The numbers and volumes of the tumors were observed within 4 weeks. (F) RNA and proteins from the tumors were extracted. Expression levels of EMT markers in the aforementioned tumors were analyzed using RT-qPCR and western blotting. (G) RNA and proteins from the tumors were extracted. Expression levels of GCSC markers in the aforementioned tumors were analyzed using RT-qPCR and western blotting. Data were analyzed using a unpaired Student's t-test. *P<0.05 and **P<0.01. RORβ, retinoic acid-related orphan receptor β; GCSCs, GC stem cells; GC, gastric cancer; CSCs, cancer stem cells; RT-qPCR, reverse transcription-quantitative PCR; ov, overexpression; sh-, short hairpin.

Article Snippet: GC cells were transfected with 4 μg/μl RORβ-short hairpin (shRNA) and 4 μg/μl control shRNA (both from Santa Cruz Biotechnology, Inc.) using Lipofectamine ® 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 2 days and then the cells were screened with puromycin (5 μg/ml) for 14 days to obtain the stable RORβ-knockdown cells further verified by western blotting.

Techniques: Transfection, Control, Over Expression, Plasmid Preparation, shRNA, Tube Formation Assay, Expressing, Quantitative RT-PCR, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Human Mutation

Article Title: The intellectual disability‐associated CAMK2G p.Arg292Pro mutation acts as a pathogenic gain‐of‐function

doi: 10.1002/humu.23647

Figure Lengend Snippet: CAMK2G knockdown in primary hippocampal neurons results in increased arborization. (A) Expression levels of human CAMK2A, CAMK2B, CAMK2D, and CAMK2G in the hippocampus during gestation, showing that CAMK2G is the dominant isoform in the first trimester, and contributes substantially in the second and the third trimester. Data was extracted from the BrainSpan database ( https://www.brainspan.org/ ). (B) Representative confocal images of hippocampal neurons co‐transfected on DIV7 with combinations of control shRNA, with control vector or CAMK2G WT and an RFP plasmid or shRNA against Camk2g with control vector or CAMK2G WT and an RFP plasmid. Transfected neurons are identified by the RFP plasmid (red). See also Supplement 1 related to Figure . (C) Sholl analysis of neurite complexity. (D) Summary bar graphs of total neurite length and arborization measured for each condition and normalized to the control shRNA (total neurite length: one‐way ANOVA, F [3,82] = 8.42, P = 6.05E‐05; control vector+control shRNA vs. control vector+ Camk2g ‐shRNA, P = 0.0004; CAMK2G WT +control shRNA, P = 0.99; CAMK2G WT + Camk2g ‐shRNA, P = 0.99; control vector+ Camk2g ‐shRNA vs. CAMK2G WT +control shRNA, P = 0.005; CAMK2G WT + Camk2g ‐shRNA, P = 0.0002; CAMK2G WT +control shRNA vs. CAMK2G WT + Camk2g ‐shRNA, P = 0.99; arborization: one‐way ANOVA, F [3,82] = 10.08, P = 1.00E‐05; control vector+control shRNA vs. control vector+ Camk2g ‐shRNA, P = 0.0001; CAMK2G WT +control shRNA, P = 0.99; CAMK2G WT + Camk2g ‐shRNA, P = 0.99; control vector+ Camk2g ‐shRNA vs. CAMK2G WT +control shRNA, P = 0.0003; CAMK2G WT + Camk2g ‐shRNA, P = 0.0005; CAMK2G WT +control shRNA vs. CAMK2G WT + Camk2g ‐shRNA, P = 0.99)

Article Snippet: Neurons were transfected after 7 days in vitro (DIV) with the following DNA constructs: control vector (1.8 ug per coverslip), CAMK2G WT , CAMK2G Arg292Pro , CAMK2G Lys43Arg , CAMK2G Lys43Arg/Arg292Pro , CAMK2G Thr287Ala , CAMK2G Thr287D/Thr306Val/Thr307Ala , CAMK2G Ala303Arg , CAMK2G‐NLS WT , CAMK2G‐NLS Arg292Pro , CAMK2G‐NLS Ser334Ala , CAMK2G‐NLS Ser334Ala/Arg292Pro , CAMK2G‐NLS Lys43Arg/Arg292Pro , CAMK2A WT , CAMK2A Lys291Pro , CAMK2B WT , and CAMK2B Lys292Pro (all 2.5 ug per coverslip) or for knockdown experiments with a pool of the CAMK2G shRNAs with an RFP plasmid (Addgene) or the control shRNA with an RFP plasmid (all in total 4 ug per coverslip).

Techniques: Knockdown, Expressing, Transfection, Control, shRNA, Plasmid Preparation

Journal: Human Mutation

Article Title: The intellectual disability‐associated CAMK2G p.Arg292Pro mutation acts as a pathogenic gain‐of‐function

doi: 10.1002/humu.23647

Figure Lengend Snippet: CAMK2A Lys291Pro and CAMK2B Lys292Pro exhibit similar neurodevelopmental pathogenicity as CAMK2G Arg292Pro . (A) Alignment of the protein sequence of CAMK2A, CAMK2B, and CAMK2G showing that the Arginine (R) at 292 in CAMK2G is a Lysine (K) in CAMK2A and CAMK2B. (B) Representative western blot of HEK‐293T cells transfected with control vector, CAMK2A WT , CAMK2A Lys291Pro (K291P), CAMK2B WT , or CAMK2B Lys292Pro (K292P). (C) Quantification of the levels of CAMK2–Thr286/Thr287 phosphorylation, normalized against total CAMK2 in the different conditions (CAMK2A WT vs. CAMK2A Lys291Pro , t [12] = 2.7, P = 0.01; CAMK2B WT vs. CAMK2B Lys292Pro , t (8) = 2.18, P = 0.03, one‐tailed unpaired t ‐test). (D) Representative confocal images of hippocampal neurons transfected on DIV7 with control vector, CAMK2A WT , CAMK2A Lys291Pro , CAMK2B WT , or CAMK2B Lys292Pro . Transfected neurons are identified by the tdTOMATO (red). (E) Summary bar graphs of total neurite length and arborization measured for each condition and normalized to the control vector (CAMK2A total neurite length: one‐way ANOVA, F [2,56] = 3.38, P = 0.04; control vector vs. CAMK2A WT , P = 0.9; CAMK2A Lys291Pro , P = 0.1, CAMK2A WT vs. CAMK2A Lys291Pro , P = 0.06; CAMK2A arborization: one‐way ANOVA F [2,56] = 6.37, P = 0.003; control vector vs. CAMK2A WT , P = 0.6; CAMK2A Lys291Pro , P = 0.04, CAMK2A WT vs. CAMK2A Lys291Pro , P = 0.003; CAMK2B total neurite length: one‐way ANOVA, F [2,55] = 27.26, P = 5,9E‐09; control vector vs. CAMK2B WT , P = 0.5; CAMK2B Lys292Pro , P = 0.0001, CAMK2B WT vs. CAMK2B Lys292Pro , P = 0.0001; CAMK2B arborization: one‐way ANOVA F [2,55] = 29.07, P = 2.4E‐09; control vector vs. CAMK2B WT , P = 0.02; CAMK2B Lys292Pro , P = 0.0001, CAMK2B WT vs. CAMK2B Lys292Pro , P = 0.0001). (F) Representative images of P20–P22 pups in utero electroporated at E14.5 with control vector, CAMK2A WT , CAMK2A Lys291Pro , CAMK2B WT , or CAMK2B Lys292Pro . tdTOMATO positive cells represent neurons successfully targeted. Arrowheads indicate layer 2/3 of the somatosensory cortex, whereas the arrowhead indicates the subventricular zone (SVZ). Right: quantification of the neuronal migration pattern from the Layer 1 (L1) to the intermediate zone (IZ); insets represent the percentage of targeted cells that reach the outer layers of the cortex measured as the sum of bin 1–4 (CAMK2A WT vs. CAMK2A Lys291Pro , t (29) = 12.97, P = 1.34E‐13, unpaired two‐tailed t ‐test; CAMK2B WT vs. CAMK2B Lys292Pro , t (23) = 13.33, P = 2.64E‐12, unpaired two‐tailed t ‐test). Data in (C) and (E) are presented as mean ± SEM. Numbers ( X / Y ) depicted in the bar graphs represent the number of samples (C) or the total number of cells ( X ) and number of independent cultures ( Y ) (E) analyzed

Article Snippet: Neurons were transfected after 7 days in vitro (DIV) with the following DNA constructs: control vector (1.8 ug per coverslip), CAMK2G WT , CAMK2G Arg292Pro , CAMK2G Lys43Arg , CAMK2G Lys43Arg/Arg292Pro , CAMK2G Thr287Ala , CAMK2G Thr287D/Thr306Val/Thr307Ala , CAMK2G Ala303Arg , CAMK2G‐NLS WT , CAMK2G‐NLS Arg292Pro , CAMK2G‐NLS Ser334Ala , CAMK2G‐NLS Ser334Ala/Arg292Pro , CAMK2G‐NLS Lys43Arg/Arg292Pro , CAMK2A WT , CAMK2A Lys291Pro , CAMK2B WT , and CAMK2B Lys292Pro (all 2.5 ug per coverslip) or for knockdown experiments with a pool of the CAMK2G shRNAs with an RFP plasmid (Addgene) or the control shRNA with an RFP plasmid (all in total 4 ug per coverslip).

Techniques: Sequencing, Western Blot, Transfection, Control, Plasmid Preparation, Phospho-proteomics, One-tailed Test, In Utero, Migration, Two Tailed Test

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Chromosomally Encoded mcr-5 in Colistin-Nonsusceptible Pseudomonas aeruginosa

doi: 10.1128/AAC.00679-18

Figure Lengend Snippet: GenBank submissions with mcr-5

Article Snippet: Notably, the colistin MICs for the control strains of E. coli MRSN 388734 and P. aeruginosa ATCC 27298 also increased from 8 to 16 μg/ml and from 0.25 to 1 μg/ml, respectively, in CE-MH medium, although the interpretations did not change.

Techniques: Plasmid Preparation