Journal: Cancers

Article Title: YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer

doi: 10.3390/cancers13246262

Figure Lengend Snippet: ( A ) Quantification of Yb1 mRNA expression levels based on log2 RSEM values batch normalized from Illumina HiSeq RNASeq data by breast cancer subtype in the breast cancer BRCA patient data from TCGA PanCancer Atlas. Expression levels of YB1 are significantly higher (*** p < 0.0001, Wilcoxon) in the basal (TNBC) subtype when compared to other BC subtypes. ( B ) Survival analysis based on YB1 expression levels (median values) in the basal subtype of the TCGA BRCA PanCancer Atlas cohort. YB1 activation in TNBCs is associated with poorer overall survival ( p = 0.049). ( C ) KM plot correlating survival of 4929 BC patients with YB1 mRNA expression levels. High YB1 expression levels correlate with poor survival probability in BC patients ( p < 1.9 × 10 −8 ). ( D ) KM plot correlating the survival of BC patients with YB1 protein expression levels. High YB1 expression levels correlate with poor survival probability in BC patients ( p = 0.047). ( E ) KM plot correlating the survival of TNBC patients with YB1 mRNA expression levels. High YB1 expression levels correlate with poor survival probability in TNBC patients ( p = 0.03).

Article Snippet: The sgRNA pool of 3 guide RNAs for mouse Yb1 (A*C*G*GGCAGCGGCGCGGGUAG, G*G*C*GGGGACAAGAAGGUCAU, and G*G*C*CCGAGCCACGGACUACG) and 2 guide RNAs for human Yb1 (U*U*U*UCCAGCAACGAAGGUUU and U*U*C*AUCAACAGGUGAGCUGC), with their respective Synthego modified EZ scaffolds targeting Yb1, were obtained from Synthego.

Techniques: Expressing, Activation Assay

Journal: Cancers

Article Title: YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer

doi: 10.3390/cancers13246262

Figure Lengend Snippet: ( A ) YB1 mRNA transcript quantification in AA and CA TNBC cell lines was determined by qt-RT-PCR and data was plotted as the fold change in HCC1806. ( B ) Average YB1 mRNA transcripts in AA and CA TNBC cell lines was plotted as the fold change in AA cell lines. YB1 transcript expression levels are significantly higher (* p < 0.001, Student ’ t test) in AA TNBC cell lines when compared to their CA counterparts. ( C ) Comparison of YB1 expression levels, levels based on log2 RSEM values batch normalized from Illumina HiSeq RNASeq data, between AA and CA BC patients from the TCGA BRCA PanCancer Atlas cohort. YB1 expression levels are significantly higher (* p < 0.01, Wilcoxon) in AA when compared to CA BC tumors. ( D ) Comparison of YB1 expression levels between CA and AA TNBC from the MetroHealth TNBC cohort, as determined by qt-RT-PCR. Data was plotted as the fold change to AA tumors. YB1 expression levels are significantly higher (* p < 0.01, Student ’ t test) in AA when compared to CA BC tumors.

Article Snippet: The sgRNA pool of 3 guide RNAs for mouse Yb1 (A*C*G*GGCAGCGGCGCGGGUAG, G*G*C*GGGGACAAGAAGGUCAU, and G*G*C*CCGAGCCACGGACUACG) and 2 guide RNAs for human Yb1 (U*U*U*UCCAGCAACGAAGGUUU and U*U*C*AUCAACAGGUGAGCUGC), with their respective Synthego modified EZ scaffolds targeting Yb1, were obtained from Synthego.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Comparison

Journal: Cancers

Article Title: YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer

doi: 10.3390/cancers13246262

Figure Lengend Snippet: ( A ) Western Blot analysis of protein lysates from parental (Ctrl) AA (black font) and CA (red font) TNBC cell lines and their YB1-KO derivatives, that were probed with the indicated antibodies. β-Actin was used a loading control. ( B , C ) Colony formation assay. The numbers under the bands represent the fold change of the Western Blot signal normalized to that of the parental cell line, as determined by densitometry analyses from one representative of 3 replicate blots. ( B ) Representative images of colony formation of parental and YB1-KO AA (black font) and CA (red font) TNBC cell lines. ( C ) Quantification of the number of colonies in each plate. Data is plotted as the percentage change from the control cells. Data shown are representative of 3 replicates (***, p < 0.0001, Student ’ t test).

Article Snippet: The sgRNA pool of 3 guide RNAs for mouse Yb1 (A*C*G*GGCAGCGGCGCGGGUAG, G*G*C*GGGGACAAGAAGGUCAU, and G*G*C*CCGAGCCACGGACUACG) and 2 guide RNAs for human Yb1 (U*U*U*UCCAGCAACGAAGGUUU and U*U*C*AUCAACAGGUGAGCUGC), with their respective Synthego modified EZ scaffolds targeting Yb1, were obtained from Synthego.

Techniques: Western Blot, Control, Colony Assay

Journal: Cancers

Article Title: YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer

doi: 10.3390/cancers13246262

Figure Lengend Snippet: ( A ) Incidence (top) and latency (bottom) of tumor growth of prenatal 4T1 or its YB1-KO derivative cells that were inoculated in the mammary fat pads of female Balb/C mice ( n = 5 mice per group). ( B ) Quantification of tumor volume of tumors generated from the inoculation of control 4T1 cells or their YB1-KO derivatives into the mammary fat pads of female Balb/C mice. ( C , top panel ) Quantification of lung metastasis nodules from the animal experiment described in ( B ). The bottom panel shows representative lungs from each mouse group. ( D ) Quantification of the tumor volume of tumors generated from the inoculation of control MDA-MB-231 (grey graph), MDA-MB-468 (black graph) cells or their respective YB1-KO derivatives (yellow and red graphs, respectively) into the mammary fat pads of female NSG mice (n = 5 mice per group). ( E , F ) Quantification of lung metastasis nodules from the MDA-MB-468 ( E ) and the MDA-MB-231 animal experiment described in ( D ) (**, p < 0.001, Student ’ t -test).

Article Snippet: The sgRNA pool of 3 guide RNAs for mouse Yb1 (A*C*G*GGCAGCGGCGCGGGUAG, G*G*C*GGGGACAAGAAGGUCAU, and G*G*C*CCGAGCCACGGACUACG) and 2 guide RNAs for human Yb1 (U*U*U*UCCAGCAACGAAGGUUU and U*U*C*AUCAACAGGUGAGCUGC), with their respective Synthego modified EZ scaffolds targeting Yb1, were obtained from Synthego.

Techniques: Generated, Control

Journal: Cancers

Article Title: YB1 Is a Major Contributor to Health Disparities in Triple Negative Breast Cancer

doi: 10.3390/cancers13246262

Figure Lengend Snippet: ( A – E ) Comparisons of the IC50 values of cisplatin ( A , B ), doxorubicin ( C , D ) and docetaxel ( E , F ) between AA (black bars) and CA (red bars) TNBC cell lines. The IC50 values were derived from the CCLE database. Average IC50 in the AA and CA TNBC cell lines was plotted as fold change to AA cell lines for cisplatin ( B ), doxorubicin ( D ) and docetaxel ( F ), (*, p < 0.01, Fisher’s Exact). ( G ) Gene set enrichment analysis of the TCGA BRCA PanCancer Atlas dataset shows that the gene signature associated with docetaxel resistance is enriched in the BC tumors that express high levels of YB1. ( H ) Western Blots with the indicated antibodies of protein lysates from parental MDA-MB-MB-468 cells or those that were treated with doxorubicin for 48 h (transient treatment), or MDA-MB-468 cells that were generated to be permanently resistant (chronic) to doxorubicin (468-Dox-res). The numbers under the bands represent the fold change of the Western Blot signal normalized to that of the parental cell line. ( I ) Western Blots with the indicated antibodies of protein lysates from parental MDA-MB-468, their cisplatin resistant (Cis-R) or doxorubicin (Dox-R) derivatives. β-Actin was used as a loading control. The numbers under the bands represent the fold change of the Western Blot signal normalized to that of the parental cell line. ( J ) Quantification of the tumor volume of tumors generated from the inoculation of parental MDA-MB-468 cells (black graph), or their cisplatin resistant (Cis-R) derivatives into the mammary fat pads of female NSG mice (n = 5 mice per group). Treatment with cisplatin started 21 days post inoculation. (***, p < 0.0001, Student ’ t test).

Article Snippet: The sgRNA pool of 3 guide RNAs for mouse Yb1 (A*C*G*GGCAGCGGCGCGGGUAG, G*G*C*GGGGACAAGAAGGUCAU, and G*G*C*CCGAGCCACGGACUACG) and 2 guide RNAs for human Yb1 (U*U*U*UCCAGCAACGAAGGUUU and U*U*C*AUCAACAGGUGAGCUGC), with their respective Synthego modified EZ scaffolds targeting Yb1, were obtained from Synthego.

Techniques: Derivative Assay, Western Blot, Generated, Control

Journal: Genes & Development

Article Title: A C9orf72–CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress

doi: 10.1101/gad.315564.118

Figure Lengend Snippet: Loss of C9orf72 increases FA flux and biogenesis. ( A , B ) Wild-type and C9KO MEFs were starved with glucose-free medium for 16 h, and then the association of autophagy with LDs was monitored by immunostaining of BODIPY and lysosomal marker LAMP1. The number of BODIPY and LAMP1 double-positive puncta per cell was counted, and 30–40 cells in each group from three independent experiments were statistically analyzed. ( C ) Wild-type and C9KO MEFs were grown with or without glucose starvation for 16 h, and then LDs were isolated and subjected to SDS-PAGE and immunoblot against anti-LC3 and P62. ADRP served as a loading control. The ratios of LC3II/I and the amounts of P62 were calculated and statistically analyzed. n = 3. ( D – F ) Wild-type, C9KO, and C9/Atg5 double-knockout MEFs were starved with glucose-free medium for 16 h. The LDs were labeled with BODIPY and counted, and the association of autophagy and LDs was monitored as in A . Twenty-eight to 30 cells in each group from three independent experiments were statistically analyzed. ( G ) Wild-type and C9KO MEFs were cultured in CM or glucose-free medium with [1,2- 14 C] acetate at a final concentration of 0.3 µCi for 6 h. The incorporation of [1,2- 14 C] in total lipids was measured by a scintillation counter. Counts per minute (CPM) were normalized to the total amount of protein. n = 6. ( H – J ) Wild-type and C9KO MEFs were grown in glucose-free medium, and the protein levels of acetyl-CoA carboxylase (ACC) and NOX2 were determined at the indicated time points by immunoblotting. n = 3. (GS) Glucose starvation. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01.

Article Snippet: The guide RNAs (gRNAs) for CRISPR–Cas9-mediated knockout of CARM1 or Atg5 were designed with Benchling as follows: CARM1 (oligo1: 5′-CACCGCTCACTATCGGCGACGCGAA-3′; oligo2: 5′-AAACTTCGCGTCGCCGATAGTGAGC-3′) and atg5 (oligo1: 5′-CACCGAAGAGTCAGCTATTTGACGT-3′; oligo2: 5′-AAACACGTCAAATAGCTGACTCTTC-3′).

Techniques: Immunostaining, Marker, Isolation, SDS Page, Western Blot, Control, Double Knockout, Labeling, Cell Culture, Concentration Assay

Journal: eLife

Article Title: Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma

doi: 10.7554/eLife.57189

Figure Lengend Snippet: ( A ) Western blots of the indicated proteins or phosphorylated ( p ) sites in CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum free medium for 48 hr (lanes 1 and 2) or serum-free media and stimulated with complete media for 15 min (lanes 3 and 4). ( B ) Relative growth of CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum-free media for 6 days (n = 3; ** = p value<0.01, two-way ANOVA). ( C ) RT-qPCR analysis of the indicated genes (normalised to GAPDH ) in serum starved CP-A cells stably transfected with empty or ERBB2 expressing vectors (n = 3; p-values, *=<0.05, **=<0.01). ( D ) ChIP-qPCR of KLF5 binding to regulatory regions associated with the indicated genes in CP-A cells stably transfected with empty or ERBB2 expressing vectors grown in serum-free media for 48 hr. Non-specific IgG is provided as a control (n = 3; ns = non-significant increases).

Article Snippet: Transfected construct (human) , Full length guide RNAs targeting KLF5 TSS , Synthego , , 5’-GUGCGCUCGCGGUUCUCUCG-3’ 5’-AGGACGUUGGCGUUUACGUG-3’ 5’-GCGUCAAGUGUCAGUAGUCG-3’.

Techniques: Western Blot, Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, ChIP-qPCR, Binding Assay, Control

Journal: eLife

Article Title: Repurposing of KLF5 activates a cell cycle signature during the progression from a precursor state to oesophageal adenocarcinoma

doi: 10.7554/eLife.57189

Figure Lengend Snippet:

Article Snippet: Transfected construct (human) , Full length guide RNAs targeting KLF5 TSS , Synthego , , 5’-GUGCGCUCGCGGUUCUCUCG-3’ 5’-AGGACGUUGGCGUUUACGUG-3’ 5’-GCGUCAAGUGUCAGUAGUCG-3’.

Techniques: Transfection, Plasmid Preparation, Control, Stable Transfection, Isolation, Construct, Western Blot, Recombinant, Sequencing, SYBR Green Assay, Concentration Assay, Software