Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: PLK2 is significantly down-regulated in TMZ resistant GBM. a , Microarray analysis for differentially expressed kinases (DEKs) in TMZ resistant cells versus Naïve cells using GEO database (GSE68029). b , Microarray analysis for DEKs in GBM versus non-tumor tissues in GEO database (GSE16011). c , Differential kinase expression analysis in GBM versus non-tumor tissues using TCGA GBM dataset. d , Venn diagram for down-regulated kinases in both three datasets of comparisons, indicating PLK2 was one of the most significantly down-regulated kinases in TMZ resistant cells as well as GBM tissues

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Microarray, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: Decreased PLK2 expression is closely associated with poor outcomes in glioma patients. a , Gene expression analysis using TCGA GBM database (* P < .05, with student’s t-test). b , Gene expression analysis with Rembrandt database in different types of glioma versus non-tumor (* P < .05, ** P < .01, *** P < .001, with one-way ANOVA followed by Dunnett’s posttest). c , Gene expression analysis with TCGA GBM dataset in different subtypes of GBM versus non-tumor (*** P < .001, with one-way ANOVA followed by Dunnett’s posttest). d . Kaplan-Meier analysis for PLK2 expression using GBM patient samples ( P < .0001, with log-rank test). e , Kaplan-Meier analysis for PLK2 expression in Rembrandt dataset by dividing glioma samples into 3 groups including PLK2 high , PLK2 mid , PLK2 low (with log-rank test within each comparison)

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Expressing, Gene Expression, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: PLK2 overexpression reduces the malignancy of GBM cells both in vitro and in vivo . a , Representative images of immunofluorescence showing the transduction efficiency of U87 and U251 cell lines after lentiviral PLK2 transduction. b , qRT-PCR analysis for measuring the mRNA expression of PLK2 in U87 and U251 cells after lentiviral PLK2 transduction (** P < .001, with student’s t-test). c , Western blot analysis for detecting the PLK2 protein expression in U87 glioma cell line transduced with lentiviral PLK2, lentiviral vector and blank control. β-actin was used as an internal control. d , Time survival curve of U87 cell line transduced with lentiviral PLK2 and negative control (*** P < .001, with one-way ANOVA followed by Dunnett’s post-test). e and f , the colony formation ability of U87 cells pre-treated with either lentiviral vector or PLK2 (*** P < .001, with student t-test). g , Flow cytometry analyses using Annexin V and Propidium Iodide for apoptotic ratio analysis in U87 cells pretreated with indicated interventions. h and i the migratory ability of U87 cells pre-treated with either lentiviral vector or PLK2. j , Representative images of H&E-stained mouse brain sections after the intracranial transplantation of indicated U87 cell lines. Scale bars: 3 mm H, Kaplan-Meier curve comparing the overall survival of xenograft mice with U87 cells pre-treated with either lentiviral vector or PLK2 ( P = 0.0256, with log-rank test). All data were presented as the mean ± SD of triplicate independent experiments

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Over Expression, In Vitro, In Vivo, Immunofluorescence, Transduction, Quantitative RT-PCR, Expressing, Western Blot, Plasmid Preparation, Control, Negative Control, Flow Cytometry, Staining, Transplantation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: Elevated PLK2 promotes chemosensitivity in GBM. a , Concentration survival curve of U87 and U251 TMZ-resistant cell lines and non-treated cell lines ( P < 0.01, with one-way ANOVA followed by Dunnett’s post-test). b , qRT-PCR analysis for measuring the mRNA expression of PLK2 in glioma TMZ-resistant cell lines versus non-treated cell lines. c , Western blot assays to compare the PLK2 protein expression in TMZ resistant glioma cell lines with non-treated cell lines. β-actin was used as an internal control. d , qRT-PCR assays to measure the mRNA expression level of PLK2 in TMZ resistant cell lines when transduced with lentiviral PLK2 and negative control (** P < 0.01, with student’s t-test). e , Western blot analysis to detect the overexpression efficiency of lentiviral PLK2 in U87 resistant cell liens. β-actin was used as an internal control. f , In vitro cell proliferation assays were performed by using different interventions as indicated in U87 cell line. (** P < .01, with one-way ANOVA followed by Dunnett’s post-test). g , Upper panel: Flow cytometry analyses using Annexin V and Propidium Iodide for apoptotic ratio analysis in U87 resistant cells pretreated with indicated interventions. Lower panel: Representative images of H&E-stained mouse brain sections after the intracranial transplantation with indicated U87 cell lines. h , Kaplan-Meier analysis for in vivo intracranial xenograft mice using U87 cells pre-transduced with PLK2 lentivirus and negative control ( P = 0.0026, with log-rank test). All data were presented as the mean ± SD of triplicate independent experiments

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Concentration Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Transduction, Negative Control, Over Expression, In Vitro, Flow Cytometry, Staining, Transplantation Assay, In Vivo

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: Loss of PLK2 enhances TMZ resistance of GBM via activation of Notch signaling. a and b , Hierarchical bi-clustering analysis was performed by using GEO database (GSE16011) or TCGA GBM database, indicating the significant gene signature in PLK2 high GBM compared with PLK2 low GBM. c and d , Bubble plots were carried out to illustrate the results of GSEA analyses using the transcriptome profiles of GSE16011 and TCGA GBM, indicating that PLK2 was negatively correlated with these pathways. e , Venn diagram of all significantly enriched pathways that are negatively correlated with PLK2 from GSEA analyses, showing Notch signaling pathway was the only common pathway that is negatively correlated with PLK2. f , GSEA plot showed the negative correlation between PLK2 and Notch signaling pathway

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Activation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Loss of PLK2 induces acquired resistance to temozolomide in GBM via activation of notch signaling

doi: 10.1186/s13046-020-01750-4

Figure Lengend Snippet: Loss of PLK2 enhances TMZ resistance of GBM via activation of Notch signaling. a , qRT-PCR assays were performed to measure the mRNA expression levels of the downstream targets of Notch signaling when cells were pre-transduced with lentiviral PLK2 and its negative control. b , Western blot analyses were conducted to detect the protein levels of downstream targets of Notch signaling. β-actin was used as an internal control. c , qRT-PCR assays were carried out to measure the mRNA expression levels of Notch downstream targets when treated with lentiviral shPLK2. d , Western blot assays was used to detect the protein level of HES1 in PLK2 OE U87 cells when transduced with HES1 lentivirus. e , Time survival curve of U87 cell pretreated with indicating interventions. f and g , Colony formation ability of U87 cells pretreated with different lentiviruses as indicated ((*** P < .001, with student’s t-test). h , representative images of wound healing assays to indicate the migratory ability of U87 cells pretreated with different lentiviruses. i , the effect of PLK2 OE on activated Notch1 and Notch2 protein was detect by western blot, β-actin was used as an internal control. j , the effect of PLK2 knockdown on activated Notch1 and Notch2 protein was detected by western blot, β-actin was used as an internal control. k , the effect of proteasome inhibitor MG132 on protein level of Notch1 pretreated with or without PLK2 OE lentivirus, β-actin was used as an internal control. l , GST-pulldown assay showed that the ubiquitination level of GST-Notch1 increased when PLK2 was overexpressed compare with control group in U87 cell line. All data were presented as the mean ± SD of triplicate independent experiments

Article Snippet: The lentiviral overexpression/knockdown PLK2 was designed and synthesized by Genechem (Shanghai, China).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Transduction, Negative Control, Western Blot, Control, Knockdown, GST Pulldown Assay, Ubiquitin Proteomics

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: TLR4 mRNA and protein levels were examined via real-time PCR and Western blotting after low gucose or high glucose treatment. (A and B) TLR4 mRNA upregulation in the high glucose medium. (C) TLR4 protein levels increased after the high glucose treatment. (D) Experiments were analyzed via densitometry and normalized to the control (0 h). (E) MCP-1 mRNA upregulation in high glucose medium for 24 h. (F) Immunofluorescence analysis of TLR4 expression. PRTC cells were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium.The cells were fixed for immunofluorescent staining of TLR4 (red). Data are expressed as the mean ± S.D. (n≥3). *, p<0.05 versus the low glucose group; **, p<0.05 versus the control (0 h).

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Expressing, Cell Culture, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were treated for 24 hours with low glucose (5.5 mM), high glucose (30 mM), or the PKC inhibitor staurosporine (10 nM, added 1 hour before high glucose treatment), and the cell lysates were collected, TLR4 mRNA, phospho-PKC (pan), p-p38, p38 levels were examined via real-time PCR and Western blotting. (A) PKC phosphorylation and p-p38 upregulation after high glucose treatment. The PKC inhibitor staurosporine decreased phospho-PKC and p-p38 levels but not total PKC levels. (B) Staurosporine reduced TLR4 mRNA levels under the high glucose condition. (C) RPTC were pretreated with staurosporine (10 nM), then treated with high glucose (30mM) for 6, 12, 24 h, and cell lysates were collected at different time points. phospho-PKC (pan), p-p38 levels were examined via Western blotting. (D) Effect of staurosporine on scratch wound healing in low glucose and high glucose. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without staurosporine treatment.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were cultured for 2 days in low glucose (5.5 mM) or high glucose (30 mM) medium, and then used for following experiments. (A) Titration of TAK-242 concentration. RPTC cells were cultured for 12 h in low glucose or high glucose medium with or without TAK-242. (B) MyD88 upregulation was also inhibited by TAK-242 under high glucose conditions by Western blotting. (C) TLR4 mRNA upregulation was also inhibited by TAK-242 under high glucose conditions by real-time PCR analysis. (D) Enhanced wound healing in the high glucose condition with the TLR4 inhibitor. RPTC cells were scratch-wounded and incubated in low glucose or high glucose medium with or without 100 nM TAK-242 for 18 h and then the healing distance was measured. (E) Enhanced cell migration under the high glucose condition with the TLR4 inhibitor. A total of 3x10 5 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of medium with or without 100 nM TAK-242 in low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface of the insert were stained with PI and counted. In C, D and E, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus the high glucose group without TAK-242 treatment.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Cell Culture, Titration, Concentration Assay, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were infected with lentiviruses containing a scrambled control sequence (Scr) or the TLR4 shRNA sequence (shRNA) and then cultured for 2 days in low glucose or high glucose medium for the following experiments. (A) TLR4 knockdown caused by the TLR4 shRNA lentivirus. After infection and a high glucose treatment, whole-cell lysates were collected for an immunoblot analysis of TLR4 and MyD88. (B) Scratch-wound healing. After lentiviral infection, RPTC were scratch-wounded and incubated in low glucose or high glucose medium for 18 h to measure the distance over which healing occurred. (C) Transwell cell migration. After lentiviral infection, a total of 3x10 5 RPTC were seeded in a transwell insert, which was put in a 24-well plate containing 600 μL of low glucose or high glucose medium for 6 h. The cells that migrated to the undersurface were stained with PI and counted. In B and C, data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; **, p<0.05 versus high glucose group infected with the scrambled sequence.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Infection, Sequencing, shRNA, Cell Culture, Western Blot, Incubation, Migration, Staining

Journal: PLoS ONE

Article Title: Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

doi: 10.1371/journal.pone.0178147

Figure Lengend Snippet: RPTC were infected with lentiviruses containing TLR4 or control lentivirus and then subjected to low glucose and high glucose treatment for 2 days, followed by a scratch-wound healing assay. (A) Increased TLR4 levels were detected in the low-glucose- and high-glucose-treated RPTC. (B) MCP-1 mRNA level in low-glucose-treated and high-glucose-treated RPTC cells after TLR4 overexpression. (C) Overexpression of TLR4 in low-glucose-treated cells also suppressed wound healing, mimicking the effect of high glucose levels. (D) RPTC cells were infected with lentivirus containing TLR4 or control lentivirus, then cultured for 18 h in low glucose or high glucose medium with or without TAK-242, followed by a scratch-wound healing assay. Data are expressed as the mean ± S.D. (n = 4). *, p<0.05 versus the low glucose group; #, p<0.05 versus the group without TAK-242.

Article Snippet: Lentiviral shRNA plasmids targeting TLR4 and a scrambled non-targeting control plasmid were made using the pLV-mU6-EF1a-GFP vector (Biosettia, San Diego, CA).

Techniques: Infection, Wound Healing Assay, Over Expression, Cell Culture

Journal: Nutrients

Article Title: Transcriptional Activation of Chac1 and Other Atf4-Target Genes Induced by Extracellular l -Serine Depletion is negated with Glycine Consumption in Hepa1-6 Hepatocarcinoma Cells

doi: 10.3390/nu12103018

Figure Lengend Snippet: Extracellular L-Ser depletion induces cation transport regulator-like protein 1 ( Chac1 ) and Atf4-target genes mRNA level in Hepa1-6.Hepa1-6 were cultured for 6 h in L-Ser-depleted (4 μM) or -supplemented (50, 100, 400 μM) Eagle’s Minimal Essential Medium (EMEM), and Chac1 ( a ) mRNA levels were measured. Dunnett’s post hoc test, ** p < 0.005, *** p < 0.0005. Hepa1-6 were cultured under the L-Ser-depleted or -supplemented condition for 6 h, and Atf4 , Atf3 , Ddit3 ( b – d ), and Atf4 targeted genes Asns , Eif4ebp1 , Mthfd2 , Gadd45a and Cdkn1a ( e – i ) mRNA were measured. Student’s t -test, ** p < 0.005, *** p < 0.0005. Hepa1-6 were cultured under the L-Ser-depleted or -supplemented condition for 6 h, and non-targeting siRNA and siAtf4 were transfected for 24 h, and Atf4 and Chac1 ( j , k ) mRNA were measured. Tukey’s test, * p < 0.05, ** p < 0.005, *** p < 0.0005.

Article Snippet: Hepa1-6 were transfected with Atf4 siRNA (30 nmol/L) (SASI_Mm02_00316863, Merck KGaA, Darmstadt, Germany) and Non-Targeting siRNA (scramble) (MISSION ® siRNA Universal Negative Control, Merck) in EMEM, using ScreenFect™siRNA (FUJIFILM Wako Chemicals, Ltd. Osaka, Japan) according to the manufacturer’s protocol.

Techniques: Cell Culture, Transfection

Journal: Cell Proliferation

Article Title: lnc‐RHL, a novel long non‐coding RNA required for the differentiation of hepatocytes from human bipotent progenitor cells

doi: 10.1111/cpr.12978

Figure Lengend Snippet: Inducible shRNA knockdown of lnc‐RHL in HepaRG cells. A, Experimental design. Normal HepaRG cells and HepaRG cells transduced with lentiviral constructs encoding Dox‐inducible shRNAs encoding scrambled shRNA or lnc‐RHL shRNAs sh‐B and sh‐C were used to initiate cultures. These were then grown under 3 experimental conditions: (1) no‐Dox control group (14 days of expansion followed by 19 days of differentiation); (2) terminal Dox induction (14 days of expansion, followed by 19 days of differentiation, with Dox induction during the final 3 days of culture); (3) concurrent Dox induction (14 days of expansion followed by 19 days of differentiation in the presence of Dox. Periods of Dox induction are indicated with red bars. B, Representative images of control HepaRG cells (untransduced or transduced with scrambled shRNA) then imaged at 40X magnification (phase contrast and GFP). HepaRG cells without virus or expressing scrambled shRNA contained numerous hepatocyte colonies on a monolayer background of cholangiocytes. Induction of the lentiviral construct with Dox yielded GFP expression cells. C, lnc‐RHL knockdown in differentiated HepaRG cells transduced with anti‐lnc‐RHL shRNAs sh‐B and sh‐C. Induction of both shRNAs yielded cultures with reduced content of hepatocyte colonies

Article Snippet: Packaged lentivirus containing 3 shRNAs designed to target lnc‐RHL (based on our complete lnc‐RHL sequence) was procured from transOMIC Inc, and expressed from a pZIP‐inducible lentiviral vector as well as a non‐targeting scrambled control shRNA.

Techniques: shRNA, Transduction, Construct, Expressing