Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental scheme. B. Spearman correlation between expression profiles of Mbd3f/- system Calculated over all differential genes (n=8,042), showing an average correlation of R=0.93 between consecutive samples. C. As in B, but between Mbd3flox/- and Gatad2a-/- systems. D. Overlap between targets of OSKM in promoters and enhancers. Pixel shade indicates Jaccard Index. E. Correlation between consecutive samples in Mbd3f/- system (MEF-day1, day1-day2, day2..day8-iPS), measured over all ESPGs promoters (promoters with differential chromatin pattern, n=3,593, top), or all differential enhancers (n=40,174, bottom), for each chromatin mark. Negative controls were calculated between MEF and IPS, are marked with solid border. F. Overlap between binding targets of Oct4, Sox2, Klf4 or Myc, and previously published binding data of the same factors, calculated in ES and iPS samples. Percentage out of our measured binding targets is presented, along Fisher exact test p-values. G. Global transcriptional pattern of 8,042 differential genes (FC>4 & maximal FPKM value>1), sorted by their temporal pattern in Mbd3f/-system (the same gene order was applied for the other reprogramming systems). Heatmap represents unit-transformation of FPKM values. H. PCA analysis of all samples, alongside samples from previous publications (Polo et al., 2012). PCA was calculated on the same set of genes and normalization as in G. I. GO categories enriched among the genes that are active in each day. Gene is defined to be active in samples where RPKM is above 0.5 of the gene max value. P-values were calculated with Fisher exact test, and FDR corrected. Categories with corrected p-value<0.01 in at least two-time points are presented. Gray Shades represent FDR corrected p-values

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Expressing, Binding Assay, Transformation Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. ChIP-seq landscape of two examples. Promoters are marked in red, enhancers are marked in green. Signals are normalized to sample size (RPM). B. Overlap between binding of OSKM and active enhancers, in each day of reprogramming. Enhancer is defined as active in a specific day if its ATAC-seq z-score is above 1.5 STD in that day. Gray shades indicate Fisher exact test p-value for overlap between compared samples. Note that OSKM do not bind the enhancers that are active in MEF (D0, marked in red); these enhancers are not significantly bound by OSKM at any day during reprogramming. C. Number of enhancers bound by each of OSKM factors in each day of reprogramming. Upper row: out of enhancers that are bound by the factor in late stages (day8, iPS, ESC). Bottom row: out of enhancers that are bound by the factor in early stages (day1-day3). D. Probability to observe co-localized binding of transcription factors in promoters (gray) and enhancers (black). Calculated in days 1,8 and iPS (Error bars indicate S.E.M). Right – Myc binds 32% of promoters, and 8% of active enhancers. E. Significant motifs enriched in promoters and enhancers that are bound by each of Oct4, Sox2, Klf4 and c-Myc at different days of reprogramming, as detected by Homer/4.7 software. P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-30) in at least one time point are presented. F. a. Motifs enriched in differential enhancers that are active in each day of reprogramming (ATAC-seq z-score >1.5). P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-50) in at least one-time point are presented. G. Motifs found in “closed” vs. “open” binding targets of the indicated transcription factor. Accessibility of targets was calculated based on ATAC-seq. Motifs found in OSK binding targets calculated in Mbd3f/- day1. Motifs that are different between open and closed binding targets are marked in black line. Complementary motifs to canonical motif appear in reverse order. H. Spearman correlation between ATAC-seq profiles of the two efficient reprogramming systems: Mbd3f/- MEF and C/EBPaTg B cell systems calculated over 40,174 differential enhancers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: ChIP-sequencing, Binding Assay, Software

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Distribution of low (<0.02), mid (0.02-0.98) and high (>0.98) methylated CpG sites, along reprogramming. Average and SEM are indicated in red plot. B. Methylation level measured in covered enhancers (n=18,072), in Mbd3f/-, Gatad2a-/- and WT-2 systems. Enhancers are clustered into eight clusters using k-means. Cluster 8 consists of enhancers that undergo fast demethylation, compared to clusters 3 and 7. C. Average methylation measured in promoters of genes that were highly methylated (>80%) in day0. Genes that change their expression level (red) are compared to genes that do not change their expression level (gray). Wilcoxon p-value indicates places where methylation of differential genes is significantly lower than methylation of non-differential genes. D. Left: Enrichment of enhancer clusters, as shown in panel B, for OSK binding, DNA accessibility, and super enhancers, showing that cluster 8 is highly enriched for OSK binding and overlaps with super enhancers. Color shades represent FDR corrected enrichment p-value. Right: Enrichment of the same enhancer clusters to transcription factor binding, taken from hmChip database. Cluster size is indicated on the right. E. Experimental scheme summary. Reprogramming efficiency was measured by Oct4-GFP+ cells percentage in Tet1/2/3 null(Δ) and Tet1/2/3fl/fl with and without Gatad2a expression, after 8 days. **p<0.01, ***p<0.001 (Student’s t-test), n=6, error bars indicate SD. F. Secondary MEF harboring Mir290-RGM and Nanog GFP-reporter were sorted after reprogramming to 3 different populations: RGM-SE-Mir290-tdTomato positive cells (sorted at day 5), Nanog-GFP and Mir290-RGM positive cells (sorted at d10-14), and "double negative" cells (sorted at d5). The cells were seeded as single cell-per-well, and were treated with medium either supplemented with Dox or lacking Dox. On day 14 colonies were inspected for GFP and mCherry (RGM) markers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Methylation, Expressing, Binding Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental flow describing three experimental perturbation settings: (i) Mbd3f/- MEFs were virally infected with cMyc over-expression (OE) cassette, OSK-OE cassette or both cassettes. Gene expression was measured on day4 following infection. (ii) Mbd3f/- MEFs carrying OSK Dox-dependent cassette were treated for knockdown of c-Myc, n-Myc and l-Myc. Gene expression was measured on days 3 and 7, and colony formation was measured on day 11. (iii) Mbd3f/- MEFs carrying OSKM Dox-dependent cassette were treated with inhibitor of cMyc (10058-F4) and with Dox. Gene expression and colony formation were measured on day 3. B. Distribution of Expression fold change (FC) compared to WT MEF of up/down regulated ESPGs (down regulated ESPGs are enriched for somatic genes), and CAPGs. Presented perturbations are over-expression of OSK cassette, over-expression of c-Myc cassette, or over-expression of the two cassettes together. (*p<10-5, **p<10-20, Wilcoxon test). C-D. Reprogrammed colony formation in Myc knockdown ort small molecule inhibition, measured 11-14 days after Dox. E. Distribution of expression fold change (FC, in log2 scale) compared to MEF of up/down regulated ESPGs and CAPGs. Presented perturbations are Myc knockdown, inhibition of Myc activity with small molecular inhibitor (10058-F4). (*p<10-5, **p<10-20, Wilcoxon test). F. Experimental scheme. G. IPSC Reprogramming efficiency in different cells expressing both endogenous and/or exogenous cMyc and nMyc. H. FACS analysis for surface expression of fibroblast surface marker Thy1 on the indicated Mbd3flox/- cell types. Dotted line indicates positive threshold for detection. I. Representative pictures of Mbd3fl/- cells harboring mCherry-NLS and ΔPE-GOF18 Oct4-GFP cassettes after 13 days of reprogramming in the presence of MYCi. Scale = 100μM. J. Left panel - iPSC reprogramming efficiency by applying highly efficient mouse B cell and WT CMP reprogramming protocols by OSKM in the presence or absence of MYC small molecule inhibitor (MYCi). Right panel – Human iPSC reprogramming efficiency by applying OKS lentiviral transduction in the presence of absence of MYCi. K. Expression fold-change distribution (log2 scale) of selected GO categories in Myc over-expression or Myc knockdown, showing that upon over-expression of Myc, processes such as ribosomal biogenesis and chromosome segregation are induced. L. Fraction of Myc targets in significantly induced and repressed GO categories, compared to what is expected by random (dashed line). M. Overlap between differential genes detected in Myc perturbation experiments, and differential genes detected in previous published perturbations (Scognamiglio et al., 2016). Fisher exact test p-values are presented. N. Expression fold change of selected chromatin modifiers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Infection, Over Expression, Expressing, Inhibition, Activity Assay, Marker, Transduction

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Recombinant, Protease Inhibitor, Sample Prep, Methylation, Transgenic Assay, Negative Control, Software

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 1 Vector construction, validation and assessment of EphA2 expression. (A) Schematic of the C170 and C172 EphA2 expression viruses. (B) Southern blot confirming the anticipated NcoI fragments in our new recombinants (1.2 and 3.0 kb, C170; 2.3 and 3.0 kb, C172), (C) Immunofluorescent imaging shows different cellular distributions of the full length and secreted forms of the EphA2. (D) Western blots of CT2A infected cells and supernatants show that C170-expressed EphA2 remains cell associated, whereas C172 expressing the extracellular form of EphA2 secretes the protein into the supernatant. TAA: tumor- associated antigen. UTR: untranslated region. TGN: Trans Golgi Network. DAPI: 4′,6-diamidino-2-phenylindole

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Plasmid Preparation, Biomarker Discovery, Expressing, Southern Blot, Imaging, Western Blot, Infection

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 2 Viral replication and cytopathic effect in C57BL/6 murine CT2A MG panels show viral replication kinetics, cytopathic effect and EphA2 surface expression in infected CT2A C57BL/6-based MG cells. MG, malignant glioma.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Expressing, Infection

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 4 CT2A tumor infiltrate immunophenotypic analysis. (A). Representative summary of TILs and population changes 6 days post saline, C134, and C170 (C134 +Epha2) treatment. Numbers below pie chart represent TILs/ml brain. (B–D). T-cell tumor infiltrate and subset analysis from saline (green), C134 (salmon), and C170 (blue)-treated mice. (E) Representative gating summary of CD8 subsets and CD62L/CD44 staining in treated mice. CD8 subset analysis of (F) CD8, CD25+, (G) CD8+, CD44+, CD62L effector-like population changes, and (H) CD8+, CD44+, CD62L+central memory-like population changes in C170- treated mice. Data were analyzed by one-way analysis of variance; p values were adjusted by Holm’s procedure. TIL: tumor- infiltrating leukocyte, MDSC: myeloid derived suppressor cells.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Saline, Staining, Derivative Assay

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 7 Summary of 67C-4-oHSV antitumor response studies. C170 was evaluated in vitro and showed equivalent (A) replication and (B) cytopathic activity as the parent virus C134. (C) 67 C-4 expresses EphA2 on the cell surface in mock and C170 infected cells. (D) Schematic summary of experimental approach. (E&F) Independent studies show C170 virotherapy treatment of established 67 C-4 flank tumors shows that C170 significantly suppresses tumor growth when compared with saline or C134-treated cohorts. Data were analyzed by using analysis of variance with repeated measures. P values were adjusted by Holm’s procedure.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: In Vitro, Activity Assay, Virus, Infection, Saline

Journal: Journal for immunotherapy of cancer

Article Title: Eliciting an immune-mediated antitumor response through oncolytic herpes simplex virus-based shared antigen expression in tumors resistant to viroimmunotherapy.

doi: 10.1136/jitc-2021-002939

Figure Lengend Snippet: Figure 8 T-cell function studies from saline and oHSV treated mice shows that C170 treatment induces an antigen-specific T- cell response in the periphery of long-term survivors. Splenocytes from saline (blue column) or oHSV-treated mice (red column, C134; green column, C170) were analyzed. (A) At the start, there was no difference in the populations that used peptide pulsing; however, after pulsing with 10 µm EphA2 or 10µM OVA peptide (negative control), C170-treated mice significantly increase their activated (B) CD25(+), (C) GZMB(+), and (D) CD25+, GZMB + dual staining CD8 + populations indicative of an EphA2-specific population response. (E) Representative flow plot showingCD8(+) GZMB(+) population and gating, and the (F) GZMB CD25 dual-positive populations. oHSV, oncolytic herpes simplex virus; OVA, ovalbumin.

Article Snippet: Cells were labeled with the following antibody staining panels for analysis of the adaptive immune cells: CD11b- Violet 421 (M1/70), CD4- BV785 (GK1.5), CD25- PE (7D4/CD25), CD8aBV510 (53–6.7), CD3ε-BV 711 (145–2 C11), CD44- APC (IM7), CD45- BV605 (30- F11), NKp46–PE- Cy7 (29A1.4) and B220- AF488 (RA3- 6B2), and H2Kb/H2Db (28- 8- 6) from Bio- Legend (San Diego, California, USA) and mouse EphA2 (REA579) from Miltenyi Biotec (Auburn, California, USA).

Techniques: Cell Function Assay, Saline, Negative Control, Staining, Virus

Journal: Oncotarget

Article Title: Graded expression of microRNA-371a-3p in tumor tissues, contralateral testes, and in serum of patients with testicular germ cell tumor

doi: 10.18632/oncotarget.27565

Figure Lengend Snippet: ( A ) In situ hybridization with a probe against miR-371a-3p causes blue staining in cells. ( B ) Section from A. ( C ) Immunohistochemical staining of the same area with an OCT4 antibody for identification of EC cells. ( D ) H&E staining of the same area.

Article Snippet: OCT4 staining for identification of embryonal carcinomas (EC), PLAP staining for seminomas (SE) and glypican 3 for yolk-sac tumors (YST) (Diagnostic BioSystems, Pleasanton, CA, USA) were then conducted according to institutional standard operating procedures [ , ].

Techniques: In Situ Hybridization, Staining, Immunohistochemical staining

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: Cell mechanics regulate Wnt signaling and self-renewal through APC–β-catenin interaction. (A and B) Colocalization between APC and β-catenin stained by immunofluorescence in softened and stiffened tumor cells. The representative images were shown in (A), and the Pearson correlation coefficient was calculated in (B). n = 30 cells per condition. Scale bar, 25 μm. (C) Coimmunoprecipitation analysis of the interaction between APC and β-catenin. Representative of 2 independent experiments. (D and E) Immunoblotting of APC in softened and stiffened tumor cells. n = 3. (F) Immunoblotting of the total and phosphorylated β-catenin when APC was silenced or overexpressed. Representative of 2 independent experiments. (G) Expressions of Wnt downstream genes and stemness genes measured by qRT-PCR. n = 3. (H) Expressions of Oct4 and Bmi1 measured by Western blotting. Representative of 2 independent experiments. (I and J) Formation and growth of tumor spheroids in soft agar and fibrin when stiffened Huh-7 cells were transfected with APC siRNA. n = 3. (K and L) Formation and growth of tumor spheroids in soft agar and fibrin when softened cells were transfected with APC plasmids. n = 3. (M and N) Formation of tumor spheroids in soft agar when Huh-7 cells were transfected with APC siRNA or plasmids and then treated with IWR or LiCl. n = 3. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

Techniques: Staining, Immunofluorescence, Western Blot, Quantitative RT-PCR, Transfection

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: Cell mechanics-mediated Wnt signaling regulates self-renewal through β-catenin-induced Oct4 transcription. (A) Immunoblotting of Oct4 in softened and stiffened tumor cells. n = 3. (B and C) Formation and growth of tumor spheroids when softened Huh-7 cells were transfected with Oct4 siRNA. n = 3. (D and E) Formation and growth of tumor spheroids when stiffened Huh-7 cells were transfected with Oct4 plasmids. n = 3. (F and G) Formation and growth of tumor spheroids when Huh-7 cells were treated with LiCl and then transfected with Oct4 siRNA. n = 3. (H and I) Formation and growth of tumor spheroids when Huh-7 cells were treated with IWR and then transfected with Oct4 plasmids. n = 3. (J and K) mRNA expression of Oct4 analyzed by qRT-PCR in softened/stiffened Huh-7 cells that were treated with IWR/LiCl. n = 3. (L and M) Interaction between nuclear β-catenin and the promoter of Oct4 analyzed by the cleavage under targets and tagmentation (CUT&Tag) assay. n = 3. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

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

Journal: Research

Article Title: The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-Catenin Signaling

doi: 10.34133/research.0224

Figure Lengend Snippet: The cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling is correlated with tumor differentiation and patient survival. (A to E) Expressions of MLCK, APC, and Oct4 and Pearson correlation coefficient between β-catenin and DAPI in clinical HCC samples with different differentiation levels analyzed by immunofluorescence staining. Patient samples were histologically graded into low, medium, and high differentiation level according to the Edmondson–Steiner grading system. The expressions of the indicated genes and the correlation between β-catenin and DAPI in (A) were quantified in (B) to (E) accordingly based on their differentiation state. Scale bar, 80 μm. n = 10, 25, and 14 for low, medium, and high differentiation level. ANOVA was used for the statistical analysis together with the post hoc Bonferroni test. (F to K) Correlation of the cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling in clinical samples analyzed by linear regression. (L to R) Correlation between the expressions of the cytoskeleton–APC–Wnt/β-catenin–Oct4 signaling and patient survival. The Cancer Genome Atlas (TCGA) databases were utilized to analyze the relationship between the expressions of the indicated genes and patient survival. The statistics were conducted using multivariate Cox regression analysis.

Article Snippet: The following primary antibodies were used: mouse anti-Oct4 at 1:200 (Zenbio, catalog no. 220487), mouse anti-β-catenin at 1:200 (Proteintech, catalog no. 66379-1-Ig), rabbit anti-APC at 1:200 (ABclonal, catalog no. A2818), and rabbit anti-MYLK at 1:200 (Proteintech, catalog no. 21642-1-AP).

Techniques: Immunofluorescence, Staining