Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: The Cnot genes maintain self-renewal by repressing early trophectoderm (TE) transcription factors. (A): Cnot1, Cnot2, and Cnot3 knockdown did not immediately affect known self-renewal factors and pathways. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) in M15 medium. Cells were collected 48 hours after transfection, and total Stat3, Smad1, b-Catenin as well as phospho-Stat3, phospho-Smad1, phosphor-b-Catenin, Oct4, and Nanog levels were determined by Western blot. Starved: control-transfected ESCs cultured in serum-free and LIF-free medium for additional 4 hours. (B): Comparing gene expression changes caused by perturbations of known self-renewal factors: Cnot1, 2, and 3 silencing induced similar changes to those of Oct4 or Sox2 silencing. Pearson's correlation coefficients were calculated between microarray datasets and depicted in a heatmap. The self-renewal factors were clustered by unsupervised hierarchical clustering based on the correlation coefficients. Microarray datasets used for this plot are listed in Supporting Information Table 2. (C): Cnot2 or Cnot3 overexpression cannot rescue Oct4 or Sox2 silencing-induced differentiation. Oct4GiP cells and Oct4GiP cells overexpressing Cnot2 (Cnot2-Rescue, same as in Fig. 1C) or Cnot3 (Cnot3-Rescue, same as in Fig. 1C) were transfected with control, Oct4 (Oct4-KD), or Sox2 (Sox2-KD) siRNAs, and the % differentiation was determined by the Oct4GiP reporter assay. (D): Cnot1, Cnot2, and Cnot3 knockdown induced TE differentiation in the presence of sustained Oct4 expression. ZHBTc4 cells that constitu-tively express Oct4 at the normal level from a Tet-Off promoter were transfected with control or Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), Cnot3-siRNA2 (Cnot3-KD), and the expression of TE markers Cdx2 and Gata3 was determined by qRT-PCR after 4 days. (E): Cdx2 deletion partially rescued Cnot1, Cnot2, and Cnot3 silencing-induced differentiation. Oct4GiP (WT) or dKO23-5 (Cdx2-/- ) cells were transfected with Control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD), and the expression of lineage markers was determined by qRT-PCR 96-hour after transfection. Abbreviations: ESC, embryonic stem cell; KD, Knockdown; WT, wild type.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Transfection, Western Blot, Cell Culture, Expressing, Microarray, Over Expression, Reporter Assay, Quantitative RT-PCR

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Silencing Cnot1, Cnot2, or Cnot3 led to mouse embryonic stem cell (ESC) differentiation. (A): Silencing Cnot1, Cnot2, or Cnot3 resulted in ESC differentiation based on the Oct4GiP reporter assay. Oct4GiP ESCs were transfected with indicated siRNAs (two different siR NAs for each CCr4-Not complex gene) in M15 medium and cultured for 4 days. The percentage of differentiated cells (% differentiation) was determined by measuring the percentage of green fluorescent protein-negative cells by fluorescence-activated cell sorting (FACS) at the end of the culture. (B): Expression of siRNA-resistant Cnot2 or Cnot3 rescued the differentiation caused by Cnot2 or Cnot3 knockdown, respectively. Oct4GiP cells or Oct4GiP cells expressing siRNA-resistant Cnot2 (Cnot2-Rescue) or Cnot3 (Cnot3-Rescue) were transfected with Control, Cnot1-siRNA1, Cnot2-siRNA2, or Cnot3-siRNA2, and the percentage of differentiated cells was determined by the Oct4GiP reporter assays. Note that Cnot2-Rescue cells were not able to rescue the differentiation caused by Cnot1 or Cnot3 silencing, and Cnot3-Rescue cells were not able to rescue Cnot1 or Cnot2 silencing. ***, p < .001. (C): Silencing Cnot1, Cnot2, or Cnot3 resulted in morphological changes and loss of alkaline phosphatase (AP) staining in ESCs. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were stained with the AP staining kit and imaged 4 days after transfection. (D): Cnot1, Cnot2, or Cnot3 silencing led to downregulation of ESC marker and upregulation of differentiation markers. Oct4GiP cells were transfected with the indicated siRNAs and cultured in the M15 medium. Cells were harvested for quantitative real-time PCR (qRT-PCR) analysis 4 days after transfection. ESC marker: Oct4; differentiation markers: Cdx2, Eomes, Gata3, Hand1, and Krt8. (E): Cnot1, Cnot2, or Cnot3 silencing reduced cell proliferation or viability in 2i medium. Oct4GiP cells were transfected with control-siRNA (Control), Cnot1-siRNA1 (Cnot1-KD), Cnot2-siRNA2 (Cnot2-KD), or Cnot3-siRNA2 (Cnot3-KD) and cul tured in 2i medium. Cell numbers were counted by FACS 4 days after transfection and normalized to control-transfected cells. (F): Cnot1, Cnot2, or Cnot3 silencing led to differentiation in 2i medium. Oct4GiP cells were transfected with indicated siRNAs and cultured in 2i medium. Cells were harvested for qRT-PCR analysis 4 days after transfection. (G): Expression of C-terminally HA-tagged Cnot2 (Cnot2-HA) in E14Tg2a cells. Expression of the exogenous Cnot2-HA was detected in Western blot with the HA-antibody, and Ran was used as a loading control. Expression of total (endogenous and exogenous) Cnot2 was determined by qPCR in wild-type E14Tg2a (E14) and Cnot2-HA expressing cells. The expression of the Cnot2-HA was estimated to be ∼2-fold of the endogenous Cnot2 on the mRNA level. (H): Identification of Cnot1 and Cnot3 in Cnot2-HA immunoprecipitation. HA-pull-down was carried out in E14Tg2a cells expressing Cnot2-HA. The presence of Cnot1, Cnot2-HA, and Cnot3 in the total lysate and pull-down sample (HA-beads) were detected by Western blot. Note that Oct4 was not detected in the pull down sample. As a negative control, protein-A beads were used in an independent pull-down. Abbreviations: HA, hemagglutinin; IP, immunoprecipitation; KD, knockdown.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Reporter Assay, Transfection, Cell Culture, Fluorescence, FACS, Expressing, Staining, Marker, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Negative Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Cnot1, Cnot2, and Cnot3 Maintain Mouse and Human ESC Identity and Inhibit Extraembryonic Differentiation

doi: 10.1002/stem.1070

Figure Lengend Snippet: Cnot1, Cnot2, and Cnot3 are required for human embryonic stem cell (ESC) self-renewal. (A): Cnot1, Cnot2, and Cnot3 were down-regulated during human ESC differentiation. H1 human ESCs were differentiated for 7 days using 100 ng/ml human recombinant BMP4. The expression levels of Cnot1, Cnot2, and Cnot3 as well as Oct4 and differentiation markers Cdx2 and Hand1 were determined by quantitative realtime PCR (qRT-PCR). (B): Silencing of Cnot1, Cnot2, or Cnot3 led to morphological changes of human ESCs. H1 cells were imaged 6 days after transfection. Phase-contrast images highlight the undifferentiated morphology of human ESCs in the lipids-only transfected cells (mock) versus the differentiated phenotype in the Cnot1, Cnot2, or Cnot3 siRNA transfected cells. (C): Silencing of the Cnot genes led to upregulation of the Cdx2 and Gata3 proteins. H1 cells were transfected with lipids-only (mock), Oct4, Cnot2, or Cnot3 siRNAs. Cells were fixed and stained for Cdx2 or Gata3 expression by immunofluorescence staining 6 days after transfection. (D): Silencing of the Cnot genes led to downregulation of the ESC marker and upregulation of the extraembryonic markers. H1 cells were harvested 6 days after transfection and marker expression was determined by qRT-PCR. Abbreviations: BMP, bone morphogenetic protein; DAPI, 4′-6-diamidino-2-phenylindole.

Article Snippet: Human ESC Culture and siRNA Transfection Human ESC line H1 (WA01) and H9 (WA09) were received from WiCell Research Institute.

Techniques: Recombinant, Expressing, Quantitative RT-PCR, Transfection, Staining, Immunofluorescence, Marker

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: Relationships between lncRNA AC010761.9 expression (ΔCt value) and patient clinical pathologic factors and serum tumor markers

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Expressing

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: LncRNA AC010761.9 was over-expressed in GA tissues by lncRNA expression chip assay. T cancer tissues, N matched non-cancer tissues. Cluster analyses from the six GA and their paired non-GA tissues lncRNA chip results showed that LncRNA AC010761.9 was over-expressed in GA tissues compared with that in the paired non-GA tissues (mean increased fold = 2.01 times, p < 0.05)

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Expressing

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: LncRNA AC010761.9 was over-expressed in GA tissues by quantified RT-PCR measurement. T cancer tissues, N matched non-cancer tissues. T versus N, p < 0.01. The data were from 145 cases of GA. The higher the ΔCt values, the lower the lncRNA AC010761.9 expression. Data were obtained from three independent tests

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: LncRNA AC010761.9 was over-expressed in GA cell lines. The data were from three GA cell lines (MGC-803, BGC-823, and SGC-7901) and control cells (normal gastric cell line [GES-1]). GA cells versus control cells (all p < 0.05). The higher the ΔCt values, the lower the lncRNA AC010761.9 expression. Data were obtained from three independent tests

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Control, Expressing

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: Relationships between lncRNA AC010761.9 expression (ΔCt value) and patient clinical pathologic factors analyzed by univariate and multivariate

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Expressing

Journal: World Journal of Surgical Oncology

Article Title: Evaluation of the expression and clinical value of lncRNA AC010761.9 in human gastric adenocarcinoma

doi: 10.1186/s12957-017-1289-y

Figure Lengend Snippet: The expression of lncRNA AC010761.9 showed a positive correlation with the expression of TRAF4 mRNA. r = 0.385 and p < 0.01 were obtained by Pearson correlation analysis

Article Snippet: The results from the Human LncRNA Expression Profile Microarray V3.0 (Arraystar, Rockville, MD, USA) were obtained from the previous work and completed by Kang Chen Bio-tech (Shanghai, China) based on the manufacturer’s instructions [ ].

Techniques: Expressing

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: (A) Immunofluorescence of SKmel147 cells stably expressing AMIGO2-GFP (green), stained with AMIGO2 antibody (red) and Hoechst 33342 (blue). Scale bar, 20 μm. (B) Functional annotation of AMIGO2-interacting proteins detected by GFP pull-down followed by MS in SKmel147 cells stably expressing AMIGO2-GFP (see Table S4). (C) PTK7 and GFP immunoblots following GFP pull-down from 501MEL cells stably expressing AMIGO2-GFP. (D) Full-length PTK7 (FL-PTK7), C-terminal fragments CTF1- and CTF2-PTK7, and FOXM1 immunoblots of 501MEL cells 72 hr post-infection with shSCR or shPTK7 (shP7 #1 and #2). Actin was used as a loading control. (E) Relative growth curves of 501MEL (left) and SKmel147 (right) cells stably transduced with shSCR or shPTK7 (shP7 #1 and #2). Values are normalized to seeding control (n = 3). (F) Percent Annexin V-positive cells 6 days post-transduction for same cells as in (E). (G) FL-PTK7, CTF-PTK7, and FOXM1 immunoblots of 501MEL cells 48 hr post-transduction with shSCR or shAMIGO2 (shA2 #1 and #2). Actin was used as a loading control. (H) FL-PTK7, CTF-PTK7, FOXM1, and AMIGO2 immunoblots of 501MEL cells untreated or treated with JQ1 (JQ1[+]) for 72 hr. Tubulin was used as a loading control. (I) CTF2-PTK7 immunoblot of nuclear lysates from same cell as in (G) (left). Lamin B1 was used as loading control. Signal quantification (right), normalized to Lamin B1, relative to shSCR (n = 3). All values and error bars represent mean ± SD or ± SEM. See also Figures S3 and S4.

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Immunofluorescence, Stable Transfection, Expressing, Staining, Functional Assay, Western Blot, Infection, Control, Transduction

Journal: Molecular cell

Article Title: Harnessing BET Inhibitor Sensitivity Reveals AMIGO2 as a Melanoma Survival Gene

doi: 10.1016/j.molcel.2017.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: LAMIN B1 , Santa Cruz , SC-6217.

Techniques: Microarray, Derivative Assay, Recombinant, Magnetic Beads, Flow Cytometry, Caspase Activity Assay, DNA Library Preparation, Blocking Assay, Extraction, TA Cloning, Sequencing, RNA Sequencing, Western Blot, Mass Spectrometry, Expressing, Software

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 5 Involvement of miR-1247-3p in the Gallic acid (GA)-inhibited integrin/FAK cascade in DLD-1 cells. Cells were treated with 90 μM GA for 24 h, lysed for total RNA extraction, and subjected to (A) miRNA expression profiling assay via microarray analysis or (B) quantitative analysis of the miRNAs via qPCR. miRNAs with up- and downregulation in response to GA were labeled with red and green, respectively. (C) Cells were transfected with control vector (Ctl-miR) or anti-miR-1247-3p (anti-miR), treated with GA for 24 h, and subjected to the assessment of integrins, FAK, and paxillin by Western blot. Densitometric analysis was performed for semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: RNA Extraction, Expressing, Microarray, Labeling, Transfection, Control, Plasmid Preparation, Western Blot, Quantitation Assay

Journal: Environmental toxicology

Article Title: Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

doi: 10.1002/tox.24087

Figure Lengend Snippet: FIGURE 4 Gallic acid (GA) reduced integrin expression and inhibited FAK/Paxillin/Src and PI3K/AKT signaling in DLD-1 cells. Cells were treated with GA at the indicated concentrations for 24 h; collected; and lysed for the immunodetection of (A) integrins and the associated signaling proteins, (B) phosphor-paxillin and Src, and (C) PI3K, phosphor-AKT, and AKT via Western blot. Semi-quantitation of signals was conducted by densitometric analysis. DMSO treatment was used as control (C). Densitometric analysis was performed for the semi-quantitation of signals. The tubulin signal was used as internal control. Average signal ratios of phosphorylated protein/total protein compared with the control were indicated.

Article Snippet: Antibodies specifically recognizing human tubulin (SC-134237), integrin αV (SC-376156), integrin β3(SC-365679), focal adhesion kinase (FAK, SC-271126), phosphorylated FAK (pY397-FAK, SC-81493), paxillin (SC-136297), phosphorylated paxillin (pY118-Paxillin, SC-365020), Src (SC-32789), phosphorylated Src (pY416-Src, SC-24621, and CS#2101), and peroxidase-conjugated antibodies against mouse IgG or rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

Techniques: Expressing, Immunodetection, Western Blot, Quantitation Assay, Control

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: Leukemic progenitor assays replicate patterns of patient response to DRD2 antagonist TDZ (A) Leukemic blast counts were monitored before and after treatment with TDZ as a monotherapy in 11 relapsed or refractory AML patients (NCT02096289). Percentage change in blasts in the peripheral blood on day 5 versus day 1 is reported after treatment with TDZ. Percentage change in BM blast content is reported for trial patient 2T and 9T in the absence of circulating blast values. Partial response and progressive disease patterns are indicated as “response” and “no response” and are illustrated as gray versus black silhouettes, respectively. (B) Candidate trial patient samples from either response group were interrogated for progenitor content at baseline (day 1) and after clinical exposure to TDZ (day 5) using limiting dilution analysis (LDA). Leukemic progenitor frequency was estimated by LDA analysis and normalized to day 1. Baseline progenitor frequency of 1 in 75,000 cells was considered the progenitor frequency for trial patient 3T at day 1 since an absolute frequency was not achieved with the analysis of 75,000 cells for this patient. Dashed lines represent 95% confidence interval. Raw colony counts are shown in Figure S1 D. (C) Trial patient samples obtained at baseline were exposed to TDZ (“+TDZ”) versus DMSO control (“−TDZ”) for 24 h, followed by analysis of progenitor cell function in CFU assays. Data are normalized to DMSO control. Before normalization, the average DMSO control values were 79 and 2 colonies for trial patients 1T and 8T (non-responders) and 61, 28, 56, 2, 11, 28, and 14 colonies for trial patients 2T, 4T, 6T, 7T, 9T, 10T, and 11T, respectively (responders). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (D) Correlation between percentage change in leukemic blast levels versus percentage change in progenitor capacity (demonstrated in C). Patients 3T and 5T were not included in this analysis due to a lack of detectable progenitor function. (E) Schematic illustrating in vivo AML xenografts were treated with TDZ (22.5 mg/kg “+”) or 30% captisol (vehicle control “−”) in vivo , followed by analysis of leukemic chimerism levels (F), gene expression analysis (G), and progenitor CFU assays (H). (F) Leukemic chimerism levels (hCD45 + CD33 + ) after in vivo treatment with TDZ relative to vehicle control (“−“). Symbols represent individual recipient mice. ∗p = 0.05 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. (G) Gene set enrichment analysis (GSEA) plot of a gene set representing cellular pathways associated with AML (Kyoto Encyclopedia of Genes and Genomes [KEGG]; ), applied to transcription profiles from TDZ-treated versus vehicle control-treated AML xenografts derived from AMLs 1, 3, and 4. (H) Human AML grafts were recovered from mouse BM and evaluated in progenitor CFU assays. Symbols represent individual CFU wells, plated using cells recovered from a minimum of 2 individual mice per condition. Colony-forming capacity for AML 4 was not detectable with up to 150,000 human cells assayed. ∗∗∗p ≤ 0.0001 (2-way factorial ANOVA). There was no significant interaction effect between patient sample and treatment group. Data are summarized as means ± SEMs. See also and and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Control, Cell Function Assay, In Vivo, Gene Expression, Derivative Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: DRD2 expression profiles reliably predict functional response to DRD antagonism (A) DRD2 expression patterns within leukemic CD34 + cells. Dotted line represents FMO control (left). Comparison of DRD2 protein levels in CD34 + cells of AML patient versus healthy donor samples (right). Healthy donor samples consist of cord blood (n = 3), adult mobilized peripheral blood (n = 3), and adult non-mobilized peripheral blood (n = 5). Blue versus red shading indicates the threshold of normal versus aberrant DRD2 levels. ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test). (B) DRD2 protein expression within CD34 + subset of low versus intermediate-/high-risk AML patients based on ELN criteria. Dots represent individual AML patients. ∗∗p = 0.006 (Mann-Whitney U test). (C) Mononuclear cells (MNCs) isolated from healthy donors and AML patients were treated with TDZ or DMSO (vehicle control, “−”) for 24 h and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n = 3–10 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). Source data can be found in . (D) Proliferative capacity of leukemic versus healthy progenitor units was compared after in vitro exposure to TDZ for 24 h. Cell number output per colony was evaluated by custom scripts as a measure of proliferation. (E) Representative FACS plots demonstrate gating strategy to purify DRD2 + vs DRD2 − human AML cells (left) and human leukemic chimerism in mice transplanted with 1 million DRD2 + or DRD2 − human AML cells. (F) Western blot of DRD2, activated CREB (p-CREB at Ser-133), and histone H3 (loading control) in DRD2 + versus DRD2 − sorted fractions illustrated in (E). (G) Representative whole-well CFU images after treatment with dopamine (DA) at physiological levels (10 nM) versus DMSO control (-DA). (H) Progenitor cell activity was quantified in n = 6 distinct AML patients after treatment with physiological levels of DA (10–100 nM) relative to DMSO control. n = 2–3 CFU wells per AML sample. ∗p = 0.03 (unpaired t test). (I) Circulating DA levels in healthy individuals (n = 8 healthy adult peripheral blood (PB) and 11 cord blood (CB) samples, as hollow circles and squares, respectively) versus n = 11 AML patients (black circles). ∗p = 0.04 (unpaired t test). Data are summarized as means ± SEMs relative to vehicle control. See also Figure S3 and .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Expressing, Functional Assay, Control, Comparison, MANN-WHITNEY, Isolation, In Vitro, Western Blot, Activity Assay

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: cAMP elevation is associated with leukemic progenitor suppression (A) Trial patients (NCT02096289) were exposed to TDZ in vitro , followed by analysis of cAMP level changes. Trial patients with abundant cell numbers available were prioritized for this analysis, including patients 1T and 3T from non-responders, and patients 7T, 10T, and 11T for responders. n = 3–6 technical replicates per condition. ∗p ≤ 0.05 (unpaired t test). (B) cAMP levels in response to DRD1 agonist (SKF 38393) relative to DMSO control. n ≥ 4 replicates across OCI-AML3 and NB4 cell lines. ∗∗p = 0.008 (Mann-Whitney U test). Progenitor response was evaluated after treatment with DRD1 agonist (SKF 38393) relative to DMSO control. n = 2–3 CFU replicates per AML sample (n = 5 AML samples total). (C) cAMP levels in response to anti-DRD1 antibody alone or in combination with DRD1 antagonist (SCH 23390) in AML cell lines OCI-AML3 and NB4. n = 2–4 replicates per condition. (D) Western blot of activated CREB (p-CREB at Ser-133) after exposure to anti-DRD1 antibody in OCI-AML3 cell line (top). Western blot of activated CREB (p-CREB at Ser-133) exposure to TDZ in OCI-AML3 and NB4 cell lines (bottom). (E) MNCs isolated from healthy donors and AML patients were treated with anti-DRD1 antibody or immunoglobulin G (IgG) control (“−“) for 30 min, and evaluated in progenitor CFU assays. Distinct shapes or colors indicate individual samples. n =3–7 CFU wells per condition, ∗∗∗∗p ≤ 0.0001 (unpaired t test). (F) Cytospin preparations of AML cells from patient 2 after exposure to TDZ or vehicle control (DMSO). Yellow arrowheads indicate evidence of hematopoietic maturation (increased cell size, reduced nuclear:cytoplasmic ratio, increased cytoplasmic vacuolization). (G) FACS plot showing expression of granulocytic cell marker (CD15) after in vitro exposure to TDZ or DMSO control (“-TDZ“) in representative DRD2 lo and DRD2 + AML samples. CD15 frequencies were quantified for AMLs 1, 6, and 7 (n = 2 technical replicates per AML sample in each condition). ∗∗p = 0.002 (Mann-Whitney U test). (H) AML patient cells were treated with TDZ or DMSO for 24 h and evaluated in progenitor CFU assays, followed by analysis of re-plating capacity. ∗∗p = 0.004 (unpaired t test). (I) cAMP levels in response to TDZ relative to DMSO control. DRD2 + AML includes AML 1, 6, OCI-AML3, and NB4. DRD2 − AML and healthy controls include AML 12 and 3 CB samples, respectively. n ≥ 3 replicates per condition. ∗∗∗p = 0.007 (unpaired t test). (J) cAMP levels in response to forskolin (FSK) relative to DMSO control. n = 6 replicates per condition, across 1 AML cell line and n = 2 healthy donor cells. ∗∗∗p ≤ 0.0001 (unpaired t test). Data are summarized as means ± SEMs. See also Figure S4 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: In Vitro, Control, MANN-WHITNEY, Western Blot, Isolation, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet: TDZ + displays superior potency and reduced toxicity relative to TDZ (A) Chiral separation of TDZ using supercritical fluid chromatography. Chromatograms show the first and second peaks, indicating the (−) enantiomer “TDZ − ” and (+) enantiomer “TDZ + ,” respectively. Purified enantiomers were evaluated for effects on cAMP levels (B), and in progenitor CFU assays (C and D). (B) cAMP levels were evaluated after in vitro treatment with TDZ and its two enantiomers in AML cell lines (NB4 and OCI-AML3) and primary patient cells (AMLs 2, 9, and 27). Symbols represent individual CFU wells. ∗p ≤ 0.05 and ∗∗p ≤ 0.01 (unpaired t test). (C) AML patient cells were exposed to TDZ and its 2 enantiomers for 24 h in a dose-response assay in vitro , and subsequently evaluated in progenitor CFU assays. Bar graphs summarize half-maximal inhibitory concentration (IC 50 ) in progenitor CFU assays performed with AML patient cells. ∗∗p ≤ 0.01 and ∗∗∗p ≤ 0.001 (paired t test). (D) Comparison of TDZ and TDZ + IC 50 for individual AML patients in CFU assays (represented in C). ∗∗p = 0.004 (paired t test). (E) A 30-min monitoring of QTc level changes after intravenous injection of TDZ and TDZ + in a guinea pig assay (n = 5 animals per cohort). QTc increases over 5% were considered indicators of safety risks. No group averages were statistically different from baseline values (repeated-measures ANOVAs). (F) DRD2 transcript (Gene: 1813) was analyzed from TGCA (tumor and normal tissue) and GTEx (normal tissue) RNA-sequencing projects. Data points represent normalized gene expression levels (fragments per kilobase of transcript per million mapped reads [FPKM]) for DRD2 from individual cancer patients or healthy donors. ∗∗∗p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001 (Mann-Whitney U test), ∗∗p = 0.01 (Kolmogorov-Smirnov test). Data are summarized as means ± SEMs. See also Figure S5 .

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Supercritical Fluid Chromatography, Purification, In Vitro, Concentration Assay, Comparison, Injection, RNA Sequencing, Gene Expression, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Abnormal dopamine receptor signaling allows selective therapeutic targeting of neoplastic progenitors in AML patients

doi: 10.1016/j.xcrm.2021.100202

Figure Lengend Snippet:

Article Snippet: Mouse anti-human DRD2 , Santa Cruz , Cat#sc-5303; RRID: AB_668816.

Techniques: Recombinant, Binding Assay, Purification, Microarray, Software, Imaging

Journal: bioRxiv

Article Title: WNT11 is a novel ligand for ROR2 in human breast cancer

doi: 10.1101/2020.12.18.423402

Figure Lengend Snippet: a , Gene expression of the four non-canonical WNT co-receptors ROR1, ROR2, PTK7 and RYK in normal breast (green) or breast cancer (red) from the IST Online™ database (ist.medisapiens.com). b , Microarray gene expression data from 2,075 breast cancer primary tumors were correlated with either poor (≤ 5 years) or better (> 5 years) metastasis-free survival (MFS). Significance was calculated with a t-test. Boxes represent the 25-75 th percentiles with the line at the median. Outliers are marked as small dots.

Article Snippet: To analyse protein expression, cells were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors, incubated for 15 min on ice, and lysates cleared for 5 min at 20.000 g. Up to 75 μg of protein were separated by SDS-PAGE (8-12% gels), blotted onto nitrocellulose and incubated overnight at 4°C with primary antibodies against WNT11 (#ab31962, abcam), ROR2 (#sc-98486, #sc-80329), RHOA (#sc-418), ROCK1 (#sc-17794), ROCK2 (#sc-398519), PKC (#sc-10800), P-JNK (Thr183/Tyr185, #sc-6254), DAAM1 (#sc-100942), GAPDH (#sc-32233), HSP90 (#sc-13119, all from santa cruz), V5-Tag (#13202), total JNK (#9252, both from cell signaling), TUBA (#05-829, Millipore).

Techniques: Gene Expression, Microarray

Journal: bioRxiv

Article Title: WNT11 is a novel ligand for ROR2 in human breast cancer

doi: 10.1101/2020.12.18.423402

Figure Lengend Snippet: a , RNA-Seq of MCF-7 pROR2 cells: Network of differentially expressed genes associated with non-canonical WNT signaling grouped according to their cellular localization. b , MCF-7 cells were stimulated for 24 h with rWNT5A (100ng/ml) and WNT11 expression was analyzed by western Blot. c+d , Expression of the non-canonical WNT ligands was measured by qRT-PCR in MCF-7 (c) or the indicated ROR2-overexpressing human breast cancer cell lines (d) (mean±SD, n=3-9, *p<0.05, p<0.01, n.e.= not expressed). e , Co-immunoprecipitation (Co-IP) of V5-WNT11 in MCF-7 pROR2 cells detects ROR2 by western blot. f , Schematic representation of the ROR2 N-terminal deletion constructs. g , Co-IP of V5-Wnt11 in MCF-7 expressing either pROR2-FL or pROR2-ΔΔ. h , Cell invasion assays of MCF-7 expressing N-terminal ROR2 deletion constructs (mean±SD, n=3, *p<0.0001).

Article Snippet: To analyse protein expression, cells were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors, incubated for 15 min on ice, and lysates cleared for 5 min at 20.000 g. Up to 75 μg of protein were separated by SDS-PAGE (8-12% gels), blotted onto nitrocellulose and incubated overnight at 4°C with primary antibodies against WNT11 (#ab31962, abcam), ROR2 (#sc-98486, #sc-80329), RHOA (#sc-418), ROCK1 (#sc-17794), ROCK2 (#sc-398519), PKC (#sc-10800), P-JNK (Thr183/Tyr185, #sc-6254), DAAM1 (#sc-100942), GAPDH (#sc-32233), HSP90 (#sc-13119, all from santa cruz), V5-Tag (#13202), total JNK (#9252, both from cell signaling), TUBA (#05-829, Millipore).

Techniques: RNA Sequencing, Expressing, Western Blot, Quantitative RT-PCR, Immunoprecipitation, Co-Immunoprecipitation Assay, Construct

Journal: bioRxiv

Article Title: WNT11 is a novel ligand for ROR2 in human breast cancer

doi: 10.1101/2020.12.18.423402

Figure Lengend Snippet: a , Western blot: RHOA and ROCK2 in MCF-7 pROR2 cells treated with control siRNA (siCTL) or siRNA against WNT11 (siWNT11). b , Levels of active RHOA-GTP in the indicated cells were assessed by ELISA (mean±SD, n=5, *p<0.05). c , MCF-7 pcDNA and pROR2 siCTL/siWNT11 cells were characterized by RPPA for phospho-proteins associated with the WNT signaling pathway (n=3, *p<0.05). d , Schematic representation of the master regulator analysis of ROR2/WNT11 signaling. The illustration was created with BioRender.com.

Article Snippet: To analyse protein expression, cells were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors, incubated for 15 min on ice, and lysates cleared for 5 min at 20.000 g. Up to 75 μg of protein were separated by SDS-PAGE (8-12% gels), blotted onto nitrocellulose and incubated overnight at 4°C with primary antibodies against WNT11 (#ab31962, abcam), ROR2 (#sc-98486, #sc-80329), RHOA (#sc-418), ROCK1 (#sc-17794), ROCK2 (#sc-398519), PKC (#sc-10800), P-JNK (Thr183/Tyr185, #sc-6254), DAAM1 (#sc-100942), GAPDH (#sc-32233), HSP90 (#sc-13119, all from santa cruz), V5-Tag (#13202), total JNK (#9252, both from cell signaling), TUBA (#05-829, Millipore).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: WNT11 is a novel ligand for ROR2 in human breast cancer

doi: 10.1101/2020.12.18.423402

Figure Lengend Snippet: Pathway enrichment for RNA-Seq data from 31 patients with brain metastases given for different WNT subpathways. Significance was calculated with a log rank test. b , qRT-PCR: Expression of ROR2 and WNT11 in samples of human brain metastases (line at median, n.e.= not expressed). c , Kaplan-Meier survival curves showing the OS of metastatic patients based on their averaged WNT11 and ROR2 expression. The separation high/low was computed based on an optimal cutoff using the maxstat method . Significance was calculated with a log rank test.

Article Snippet: To analyse protein expression, cells were lysed in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 0.1 % SDS, 0.5 % sodium deoxycholate, 1 % Triton X-100, pH 7.2) supplemented with protease (Sigma) and phosphatase (Roche) inhibitors, incubated for 15 min on ice, and lysates cleared for 5 min at 20.000 g. Up to 75 μg of protein were separated by SDS-PAGE (8-12% gels), blotted onto nitrocellulose and incubated overnight at 4°C with primary antibodies against WNT11 (#ab31962, abcam), ROR2 (#sc-98486, #sc-80329), RHOA (#sc-418), ROCK1 (#sc-17794), ROCK2 (#sc-398519), PKC (#sc-10800), P-JNK (Thr183/Tyr185, #sc-6254), DAAM1 (#sc-100942), GAPDH (#sc-32233), HSP90 (#sc-13119, all from santa cruz), V5-Tag (#13202), total JNK (#9252, both from cell signaling), TUBA (#05-829, Millipore).

Techniques: RNA Sequencing, Quantitative RT-PCR, Expressing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: Antibodies used in this study include: CRABP-II mouse mAbs (Millipore, MAB5488), CRABP-II rabbit polyclonal antibody (Proteintech, 10,225–1-AP), HuR (3A2, Santa Cruz, sc-5261), Flotilin-2 (Santa Cruz, sc-28320), GAPDH (Santa Cruz, sc-365062), and Actin (Santa Cruz, sc-1615), anti-Flag M2 mAb (Sigma, F9291), anti-Flag agarose beads (Clontech, #635,686), Ki67 (SP6, ThermoFisher, RM-9106-S0), ADRP (Novus, NB110-40,877), Caspas3 (Cell Signaling, #9662), PARP (Cell Signaling, #9542), AKT (Cell Signaling, #4691), mTOR (Cell Signaling, #2983), S6 (Cell Signaling, #2217), pAKT (S473, Cell Signaling, #9018), pmTOR (Cell Signaling, #5536), pS6 (Cell Signaling, #4858), and pGSK3β (Cell Signaling, #5558).

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation