Journal: Cell Death & Disease

Article Title: Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

doi: 10.1038/s41419-022-04729-5

Figure Lengend Snippet: a Lamin A/C (green) and β3-tubulin (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE); magnification 100x, scale bar 10 μm; b Lamin A/C (green) and neurofilament-H (NEF-H) (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE); magnification 100x, scale bar 10 μm; c qRT-PCR analysis of LMNA , NEF-H , β3-tubulin, nestin and SOX2 genes in untreated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). Data are shown as 2- ΔΔ Ct . GAPDH was used as a housekeeping gene. Data are shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01, *** p < 0.001; d Lamin A/C (green) and YAP (red) localization in non-treated TC-71 cells (CTRL) and in mevinolin treated (5 μM) EWS cells (MEV). DNA was counterstained with DAPI (DAPI). Merge of fluorescence signals are shown (MERGE). Graphs indicate the fluorescence intensity profile along the white arrows. Representative graphs of at least 30 nuclei analyzed for each sample were shown; magnification 100x, scale bar 10 μm; e qRT-PCR analysis of CTGF and CYR61 in non-treated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). Data are shown as 2- ΔΔ Ct . GAPDH was used as a housekeeping gene. Data are shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01; f Western blotting analysis of YAP, p(Ser127) YAP, MYC and ROCK2 protein expression in non-treated TC-71 cells (CTRL) and in mevinolin treated EWS cells (2.5 μM or 5μM MEV). GAPDH was used as loading control. Densitometric analysis is shown as mean values ± SD of three different experiments. Asterisks indicate statistically significant differences with respect to CTRL cells; one-way ANOVA test, * p < 0.05, ** p < 0.01.

Article Snippet: Gene expression was assessed using the TaqMan® Gene Expression Master Mix, and predesigned TaqMan probes (Thermo Fisher) LMNA (Hs.PT.58.24496716), nestin gene (Hs.PT.58.1185097), SOX2 (Hs.PT.58.237897.g), Connective Tissue Growth Factor ( CTGF ) (Hs00170014), Cysteine Rich Angiogenic Inducer 61 (CYR61 ) (Hs00155479), Neurofilament H ( NEF-H ) (Hs00606024) and β3-tubulin gene (Hs.PT.58.20385221) were employed, using the 7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA).

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

Journal: Investigative Ophthalmology & Visual Science

Article Title: Downregulation of microRNA-224-3p Hampers Retinoblastoma Progression via Activation of the Hippo-YAP Signaling Pathway by Increasing LATS2

doi: 10.1167/iovs.61.3.32

Figure Lengend Snippet: Primer Sequences for RT-qPCR

Article Snippet: The primary antibodies, including LATS2 (1 µg/mL, ab110780), B-cell lymphoma 2 (Bcl-2) (1:1000, ab32124), Bcl-2 associated X protein (Bax) (1:1000, ab3250), vascular endothelial growth factor (VEGF) (1:1000, ab32152), tafazzin (TAZ) (1 µg/mL, ab84927), Yes associated protein (YAP) (1:5000, ab52771), p-YAP (1:10000, ab76252), connective tissue growth factor (CTGF) (1:1000, ab6992), and cysteine rich angiogenic inducer 61 (CYR61) (1 µg/mL, ab24448), were purchased from Abcam, Inc. (Cambridge, UK) except p-TAZ (1:1000, sc-17610-R; Santa Cruz, USA).

Techniques: Sequencing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Downregulation of microRNA-224-3p Hampers Retinoblastoma Progression via Activation of the Hippo-YAP Signaling Pathway by Increasing LATS2

doi: 10.1167/iovs.61.3.32

Figure Lengend Snippet: miR-224-3p inhibits the Hippo-YAP signaling pathway by downregulating expression of LATS2. ( A ) Involvement of LATS2 in the Hippo-YAP signaling pathway analyzed on KEGG. ( B ) mRNA expression of TAZ and YAP in cells determined by PT-qPCR. ( C ) Protein expression of TAZ, YAP, p-TAZ, and p-YAP in cells normalized to GAPDH determined by Western blot analysis. ( D ) mRNA expression of TAZ, YAP, CTGF, and CYR61 in cells determined by PT-qPCR. ( E ) Protein expression of TAZ, YAP, CTGF, CYR61, p-TAZ, and p-YAP in cells normalized to GAPDH determined by Western blot analysis. * P < 0.05 versus Y79 cells transfected with NC mimic or oe-NC; # P < 0.05 versus Y79 cells transfected with NC inhibitor or cotransfected with miR-224-3p mimic and oe-NC. Data expressed by mean ± standard deviation among multiple groups were analyzed using 1-way ANOVA, followed by Tukey's post hoc test. The experiment was repeated three times independently.

Article Snippet: The primary antibodies, including LATS2 (1 µg/mL, ab110780), B-cell lymphoma 2 (Bcl-2) (1:1000, ab32124), Bcl-2 associated X protein (Bax) (1:1000, ab3250), vascular endothelial growth factor (VEGF) (1:1000, ab32152), tafazzin (TAZ) (1 µg/mL, ab84927), Yes associated protein (YAP) (1:5000, ab52771), p-YAP (1:10000, ab76252), connective tissue growth factor (CTGF) (1:1000, ab6992), and cysteine rich angiogenic inducer 61 (CYR61) (1 µg/mL, ab24448), were purchased from Abcam, Inc. (Cambridge, UK) except p-TAZ (1:1000, sc-17610-R; Santa Cruz, USA).

Techniques: Expressing, Western Blot, Transfection, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Downregulation of microRNA-224-3p Hampers Retinoblastoma Progression via Activation of the Hippo-YAP Signaling Pathway by Increasing LATS2

doi: 10.1167/iovs.61.3.32

Figure Lengend Snippet: A mechanism map depicting the role of the miR-224-3p/LATS2/Hippo-YAP axis in the progression of retinoblastoma. miR-224-3p targets and negatively regulates LATS2 to inhibit the YAP/TAZ phosphorylation, whereby the Hippo signaling pathway activation is inhibited; following that, the downstream genes CTGF and CYR61 are upregulated, by which the apoptosis of retinoblastoma cells is suppressed while proliferation and angiogenesis are promoted.

Article Snippet: The primary antibodies, including LATS2 (1 µg/mL, ab110780), B-cell lymphoma 2 (Bcl-2) (1:1000, ab32124), Bcl-2 associated X protein (Bax) (1:1000, ab3250), vascular endothelial growth factor (VEGF) (1:1000, ab32152), tafazzin (TAZ) (1 µg/mL, ab84927), Yes associated protein (YAP) (1:5000, ab52771), p-YAP (1:10000, ab76252), connective tissue growth factor (CTGF) (1:1000, ab6992), and cysteine rich angiogenic inducer 61 (CYR61) (1 µg/mL, ab24448), were purchased from Abcam, Inc. (Cambridge, UK) except p-TAZ (1:1000, sc-17610-R; Santa Cruz, USA).

Techniques: Activation Assay

Journal: Oncology Letters

Article Title: Loss of miR-873 contributes to gemcitabine resistance in triple-negative breast cancer via targeting ZEB1

doi: 10.3892/ol.2019.10697

Figure Lengend Snippet: Sequences of primers for reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Western blot analysis Antibodies for E-cadherin (catalog no. 14472; 1:2,000), AXL receptor tyrosine kinase (AXL; catalog no. 8661; 1:2,000), connective tissue growth factor (CTGF; catalog no. 86641; 1:2,000), cysteine rich angiogenic inducer 61 (CYR61; catalog no. 14479; 1:2,000) and ZEB1 (catalog no. 3396; 1:2,000) were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: Loss of miR-873 contributes to gemcitabine resistance in triple-negative breast cancer via targeting ZEB1

doi: 10.3892/ol.2019.10697

Figure Lengend Snippet: miR-873 represses ZEB1 expression in triple-negative breast cancer cells. (A) Overexpression of miR-873 decreased ZEB1 mRNA level in MDA-MB-231 and BT549 cells. Overexpression of miR-873 decreased ZEB1 protein level and elevated E-cadherin protein level in (B) MDA-MB-231 and (C) BT549 cells. Overexpression of miR-873 decreased AXL, CTGF and CYR61 mRNA levels in (D) MDA-MB-231 and (E) BT549 cells. Overexpression of miR-873 decreased AXL, CTGF and CYR61 protein levels in (F) MDA-MB-231 and (G) BT549 cells. *P<0.05, **P<0.01 and ***P<0.0001 vs. miR-NC mimics. ZEB1, zinc finger E-box binding homeobox 1; NC, negative control; AXL, AXL receptor tyrosine kinase; CTGF, connective tissue growth factor; CYR61, cysteine rich angiogenic inducer 61; miR, microRNA.

Article Snippet: Western blot analysis Antibodies for E-cadherin (catalog no. 14472; 1:2,000), AXL receptor tyrosine kinase (AXL; catalog no. 8661; 1:2,000), connective tissue growth factor (CTGF; catalog no. 86641; 1:2,000), cysteine rich angiogenic inducer 61 (CYR61; catalog no. 14479; 1:2,000) and ZEB1 (catalog no. 3396; 1:2,000) were purchased from Cell Signaling Technology (Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Over Expression, Binding Assay, Negative Control