Journal: The Journal of Biological Chemistry

Article Title: Receptor for Activated C-Kinase (RACK1) Homolog Cpc2 Facilitates the General Amino Acid Control Response through Gcn2 Kinase in Fission Yeast *

doi: 10.1074/jbc.M112.445270

Figure Lengend Snippet: Cpc2, but not snoU24b RNA, is important for Gcn2 regulation. A, small nucleolar RNA U24b is encoded within the intron of cpc2 mRNA. Gray box, coding sequence; dashed line, intron; solid line, untranslated region. The position encoding Trp43 is indicated by an arrowhead. B, gene expression levels of cpc2, snoU24b, and cdk9+ (control) were analyzed by quantitative RT-PCR from wild-type (YT3033), cpc2Δ (YT2307), and cpc2_W43* (YT4299) cells. All data are normalized to the expression level of 18 S rRNA, and the average expression level of two independent experiments is shown as the relative fold to those in wild-type cells. C, 10-fold serial dilutions of wild-type (JK317), cpc2Δ (YT3540), and cpc2_W43* (YT4299) cells were spotted on EMM plates ± 10 mm 3AT. D, immunoblots of anti-FLAG-Gcn2 immunoprecipitates from Flag:gcn2 (YT3372) and Flag:gcn2 cpc2_W43* (YT4309) cells treated with 10 mm 3AT, probed with anti-phospho-Gcn2 and anti-FLAG (control) antibodies.

Article Snippet: To construct expression plasmids, cDNA of S. pombe cpc2 , S. cerevisiae asc1 , and human RACK1 ( GNB2L1 ) were cloned into the NdeI-SmaI site in the plasmid pREP2 ( 39 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain Genotype JK317 h − leu1-32 ura4-D18 YT2307 h − cpc2 :: ura4 + leu1-32 ura4-D18 YT2313 h − cpc2 : 2HA6His : ura4 + leu1-32 ura4-D18 YT2453 h − hri2 :: ura4 + leu1-32 ura4-D18 YT2459 h − hri2 :: ura4 + gcn2 :: Kan r leu1-32 ura4-D18 YT2465 h − rpS3 : Flag : ura4 + leu1-32 ura4-D18 YT2824 h − cpc2 :: ura4 + gcn2 :: Kan r leu1-32 ura4-D18 YT3033 h − leu1-32 YT3173 h − hri2 :: ura4 + cpc2 :: ura4 + leu1-32 ura4-D18 YT3360 h − gcn2 :: ura4 + leu1-32 ura4-D18 YT3372 h − 5Flag : gcn2 leu1-32 ura4-D18 YT3540 h − cpc2 :: Kan r leu1-32 ura4-D18 YT3542 h − cpc2 _ DE leu1-32 ura4-D18 YT3559 h − 5Flag : gcn2 cpc2 _ DE : ura4 + leu1-32 ura4-D18 YT3598 h − 5Flag : gcn2 cpc2 :: ura4 + leu1-32 ura4-D18 YT3648 h − 5Flag : gcn2 :: ura4 + leu1-32 ura4-D18 YT3656 h − 5Flag : gcn2 _ K585R leu1-32 ura4-D18 YT3657 h − 5Flag : gcn2 _ T818/823A leu1-32 ura4-D18 YT4251 h − atf1 :: ura4 + leu1-32 ura4-D18 YT4254 h − 5Flag : gcn2 atf1 :: ura4 + leu1-32 ura4-D18 YT4279 h + / h − 5Flag : gcn2 / 12myc : gcn2 ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4280 h + / h − 5Flag : gcn2 / 12myc : gcn2 cpc2 :: ura4 + / cpc2 :: ura4 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4281 h + / h − 12myc : gcn2 / gcn2 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4299 h − cpc2 _ W43 * leu1-32 ura4-D18 YT4309 h − 5Flag : gcn2 cpc2 _ W43 * leu1-32 ura4-D18 YT4376 h + / h − 5Flag : gcn2 / gcn2 + ade6-M210 / ade6-M216 leu1-32 / leu1-32 ura4-D18 / ura4-D18 YT4386 h − Flag : cpc2 leu1-32 ura4-D18 YT4388 h − Flag : cpc2 _ W43 * leu1-32 ura4-D18 YT4389 h − cpc2 :: Kan r :2HA6His : ura4 + leu1-32 ura4-D18 YT4390 h − cpc2 _ W43 *: 2HA6His : ura4 + leu1-32 ura4-D18 Open in a separate window S. pombe strains used in this study RT-PCR Total RNA was prepared as described previously ( 40 ). cDNA was synthesized using an RNA PCR kit (Takara) with random 9-mer primers.

Techniques: Sequencing, Expressing, Quantitative RT-PCR, Western Blot

Journal: BMC Cancer

Article Title: Effects of HMGA2 on the epithelial-mesenchymal transition-related genes in ACHN renal cell carcinoma cells-derived xenografts in nude mice

doi: 10.1186/s12885-022-09537-w

Figure Lengend Snippet: RT-PCR and western blotting results validated the efficacy of shRNAs to silence the expression of HMGA2 gene in ACHN cells. A RT-PCR analysis of the HMGA2 mRNA expression in ACHN cells receiving either a scrambled control shRNA or a shRNA (#1-#3) specific to HMGA2 gene (statistical significance with p = 0.002, p < 0.001, p = 0.002 for the three shRNA 1–3). B Western blotting analysis of the HMGA2 and GAPDH protein expression in ACHN cells receiving HMGA2 shRNA2. C Quantification was performed by normalizing the protein expression level of HMGA2 against the level of the house-keeping gene GAPDH ( p = 0.002**). D the mRNA expression level of HMGA2 in stable-transfected and puromycin-selected ACHN cells ( p = 0.013*). This is done before xenograft modeling. E-G The mRNA expression level of EMT markers E- cadherin, N-cadherin and Snail in cultured ACHN cells ( p = 0.0132 *, p = 0.0102 *, p = 0.0024 **, for the three groups). H Western blot results for protein expression of EMT-related genes in HGMA2-silenced ACHN cells before xenografting. Data were expressed as mean +/− standard deviation. For each group, data were obtained from three independent repeat experiments, with triplicate samples in each repeat. Statistical significance was calculated using Kruskal-Wallis test

Article Snippet: The Reverse Transcription kit and One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) were purchased from TaKaRa-Bio (Dalian, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, shRNA, Transfection, Cell Culture, Standard Deviation

Journal: BMC Cancer

Article Title: Effects of HMGA2 on the epithelial-mesenchymal transition-related genes in ACHN renal cell carcinoma cells-derived xenografts in nude mice

doi: 10.1186/s12885-022-09537-w

Figure Lengend Snippet: RT-PCR and western blotting analysis of E-cadherin, N-cadherin and Snail expression in xenograft tumors of untreated ACHN cells, ACHN cells treated with scrambled control shRNA and cells treated with HMGA2-specific shRNA. A-D mRNA expression of E-cadherin ( p = 0.002**), N-cadherin ( p = 0.013*), Snail ( p = 0.0117*) and HMGA2 ( p = 0.002**). E Western blot gel pictures of E-cadherin, N-cadherin, Snail and GAPDH. F-H Quantification of E- cadherin ( p = 0.007**), N-cadherin ( p = 0.011*) and Snail ( p = 0.013*) protein expression under three treatment conditions. Normalization was performed by comparing the protein expression level of the above describe genes against the level of the house-keeping gene GAPDH (statistically significance with p < 0.05). Data was present as mean value +/− standard deviation. Statistical significance was calculated using Kruskal-Wallis test

Article Snippet: The Reverse Transcription kit and One Step TB Green™ PrimeScript™ RT-PCR Kit II (SYBR Green) were purchased from TaKaRa-Bio (Dalian, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, shRNA, Standard Deviation

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: CC-induced dose-dependent apoptosis of HEK-293 and human renal cancer SW-839 cells. (A) HEK-293 and (B) SW-839 cells were treated with various concentrations of CC (0, 5 and 10 µM) for 24 h, and stained with annexin V-fluorescein isothiocyanate/propidium iodide. The percentage of early-stage apoptotic cells is shown in the lower right quadrant, while the percentage of late-stage apoptotic cells is shown in the upper right quadrant. (C) Treatment of HEK-293 and SW-839 cells with 5 and 10 µM CC for 24 h induced cell apoptosis. CC, chelerythrine chloride; FL-H, fluorescence line height.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Staining, Fluorescence

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: Tumor growth suppression in a human renal cancer SW-839 xenograft nude mouse model. (A and B) Tumor volume of CC and control tumors. (C) Tumor weight of CC and control tumors. (D) No significant toxicity to mice was observed following treatment with CC (5 mg/kg/day), according to the body weight of the mice. (E) Representative TUNEL staining (red fluorescence) of SW-839 renal cancer xenografts. (F) Quantification of TUNEL + SW-839 cells in tumor xenografts was performed using cellSens Standard software. The data are presented as the mean ± standard deviation of three experiments. **P<0.01 vs. control cells. (G) IHC staining for Bcl-2 and Bcl-2-associated X protein on tumor sections, and (H) quantification of IHC staining using cellSens Standard software. CC, chelerythrine chloride; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; IHC, immunohistochemistry.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Control, TUNEL Assay, Staining, Fluorescence, Software, Standard Deviation, Immunohistochemistry, End Labeling

Journal: Oncology Letters

Article Title: Chelerythrine chloride induces apoptosis in renal cancer HEK-293 and SW-839 cell lines

doi: 10.3892/ol.2016.4520

Figure Lengend Snippet: (Aa) Western blot analysis of the expression levels of apoptosis-associated proteins in HEK-293 and human renal cancer SW-839 cells following treatment with CC. Quantification of the expression of various proteins in HEK-293 and SW-839 cells, such as (Ab) p53, (Ac) Bax, (Ad) Bcl-2, (Ae) pro-caspase-3, (Af) cleaved caspase 3 and (Ag) PARP using GAPDH as a control. Multiple bands were observed in the cleaved caspase-3 lane due to non-specific binding of antibodies. (Ab-Ag) Quantification of western blotting (*P<0.05 and **P<0.01 vs. controls). (Ba) Western blot analysis of MAPK and Akt pathways after CC treatment in HEK-293 and SW-839 cells. The quantification of protein expression was performed for (Bb) pERK, (Bc) p-p38, (Bd) p-JNK and (Be) p-AKT, with GADPH as a control. The results are representative of ≥3 independent experiments. Multiple bands were observed in the p-ERK, p-JNK and JNK lanes due to non-specific binding of antibodies. *P<0.05 and **P<0.01 vs. controls. CC, chelerythrine chloride; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein; PARP, poly (adenosine diphosphate-ribose) polymerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; p-, phospho-; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Article Snippet: One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to stain the apoptotic tumor cells.

Techniques: Western Blot, Expressing, Control, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: DIM-3,5 analogs inhibit cancer cell growth and modulate expression of NR4A1-regulated gene products. RKO ( A ), SW480 ( B ) and HCT116 ( C ) cells were treated with 2.5–20 µmol/L DIM-3,5-CI 2 , DIM-3-CI-5-CF 3 and DIM-3,5-Br 2 for 24 h and effects on cell proliferation were determined as outlined in . RKO and SW480 cells were treated with DIM-3,5 analogs for 24 h and whole-cell lysates were analyzed by Western blots (in triplicate), G9a, β1-integrin and apoptosis ( D , E ) and Sp/NR4A gene products ( F , G ). Results are expressed as means ± SE for at least 3 determinations for each treatment group and significant ( p < 0.05) inhibition/induction compared to DMSO (control) is indicated (*) ( A – C ); quantitative results for ( D – G ) are summarized in ( H , I ) within the figure.

Article Snippet: A TUNEL assay was performed using a One-Step TUNEL In Situ Apoptosis Kit (Green, FITC) (E-CK-A320, Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Western Blot, Inhibition, Control

Journal: International Journal of Molecular Sciences

Article Title: Expression of Prooncogenic Nuclear Receptor 4A (NR4A)-Regulated Genes β1-Integrin and G9a Inhibited by Dual NR4A1/2 Ligands

doi: 10.3390/ijms26083909

Figure Lengend Snippet: NR4A1 and NR4A2 knockdown and DIM-3,5 analogs inhibit invasion and induce TUNEL staining. SW480 cells were transfected with siNR4A1 and siNR4A2 or treated with DIM-3,5 analogs, and migration ( A ) and TUNEL ( B ) staining were determined as outlined in the Results are expressed as means ± SE for at least 3 determinations per treatment group, and significant ( p < 0.05) differences from the control group are indicated (*).

Article Snippet: A TUNEL assay was performed using a One-Step TUNEL In Situ Apoptosis Kit (Green, FITC) (E-CK-A320, Elabscience, Houston, TX, USA) according to the manufacturer’s protocol.

Techniques: Knockdown, TUNEL Assay, Staining, Transfection, Migration, Control

Journal: iScience

Article Title: AXL cooperates with EGFR to mediate neutrophil elastase-induced migration of prostate cancer cells

doi: 10.1016/j.isci.2021.103270

Figure Lengend Snippet:

Article Snippet: All quantitative PCR reactions were carried out in the StepOne plus Real-Time PCR system (Applied Biosystems) using the qScript XLT one-Step RT-qPCR ToughMix Kit (#89236-672, QuantaBio) and TaqMan primers (Applied Biosystems) for the following: human epidermal growth factor (EGF) (Hs01099990_m1), heparin-binding EGF-like growth factor (HBEGF) (Hs00811813_m1), transforming growth factor-alpha (TGFA) (Hs00608187_m1), epigen (EPGN) (Hs02385424_m1), betacellulin (BTC) (Hs01101201_m1), amphiregulin (AREG) (Hs00950669_m1), and epiregulin (EREG) (Hs00914313_m1).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, BrdU Cell Proliferation Assay, Western Blot, Software, Membrane, Plasmid Preparation