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: Frontiers in Pharmacology

Article Title: HEXA-018, a Novel Inducer of Autophagy, Rescues TDP-43 Toxicity in Neuronal Cells

doi: 10.3389/fphar.2021.747975

Figure Lengend Snippet: HEXA-018 induces autophagy in neuronal cells. (A) Chemical structure of HEXA-018. (B,C) N2a cells were treated with HEXA-018 (5 µM) for 24 h. (B) Western blot analysis was performed to determine the amount of LC3-I/II or p62 protein. HEXA-018 significantly increased the LC3-II levels in N2a cells but did not affect total p62 protein levels. Quantification of the immunoblots was performed from 3 independent experiments. ** p < 0.005; n. s. , not significant (unpaired Student’s t -test). (C) RT-PCR for lc3a and lc3b mRNA expression in HEXA-018-treated N2a cells. The quantification of lc3a and lc3b mRNA transcript levels is presented as the mean ± SEM from 3 independent real-time RT-PCR experiments. 18S rRNA was used for normalization. * p < 0.05; ** p < 0.005 (unpaired Student’s t -test). (D,E) Primary neurons were treated with HEXA-018 (0.5 µM) for 24 h. (D) Western blot analysis was performed to determine LC3-I/II or p62 protein expression. Quantification of the immunoblots was performed from 3 independent experiments. * p < 0.05; n. s., not significant (unpaired Student’s t -test). (E) RT-PCR for lc3a and lc3b mRNA expression in HEXA-018-treated primary neurons. The quantification of lc3a and lc3b mRNA transcription levels are presented as the mean ± SEM from 3 independent real-time RT-PCR experiments. 18S rRNA was used for normalization. * p < 0.05; ** p < 0.005 (unpaired Student’s t -test). (F,G) N2a cells (F) and primary neurons (G) were treated with HEXA-018 (5 µM/0.5 µM) for 24 h. Rapamycin (200 nM) was used as a positive control. Cyto-ID fluorescence analysis was performed thereafter. The level of Cyto-ID fluorescence was significantly increased in the HEXA-018-treated cells, which indicates autophagic activation. The fluorescence signal intensity of Cyto-ID was normalized to that of Hoechst 33342. Data are presented as the mean ± SD from 3 independent experiments. ** p < 0.005; *** p < 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

Article Snippet: Quantitative RT-PCR was performed using the one-step SYBR ® PrimeScript™ RT-PCR kit (Takara Bio Inc, RR420A) according to the manufacturer’s instructions, followed by detection using an Applied Biosystems 7500 Real-Time PCR system (Applied Biosystems).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Positive Control, Fluorescence, Activation Assay