sgrna expressing vector cloning  (Addgene inc)

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 1. YY2 negatively correlated with CSC and HCC progression. A) mRNA expression levels of CD44, Nanog, EpCAM, and OCT4 in adherent and stem-like tumor spheres formed by HCC-LM3 cells, as determined using qRT-PCR. B) Volcano plot of log2 fold-change versus adjusted p-value for gene expression changes in adherent and tumor spheres formed by HCC-LM3 cells, as analyzed by RNA-seq. C) Fold-change of top 20 differentially expressed transcription factors in adherent and stem-like tumor spheres formed by HCC-LM3 cells. D) Heatmap showing the expression of YY2 and differently expressed stemness-related genes significantly enriched in tumor spheres formed by HCC-LM3 cells. E) KEGG analysis of differentially expressed genes in YY2-overexpressed cells using GSE184138 database. The top 20 pathways enriched in YY2-overexpressed cells are shown. F,G) YY2 mRNA (F) and protein (G) expression levels in adherent and stem-like tumor spheres formed by HCC-LM3, MHCC-97H, and HepG2 cells, as determined using qRT- PCR and western blotting, respectively. H,I) YY2 and CD44 expression level in clinical HCC tissues and the corresponding normal adjacent tissue, as analyzed using immunohistochemical staining (H) and western blotting (I); Scale bars: 50 μm. J) Kaplan–Meier plot of overall survival (OS) in clinical HCC patients with low and high YY2 expression as obtained from the TCGA dataset (n = 311; p < 0.01). 𝛽-actin was used for qRT-PCR normalization and as western blotting loading control. Ad: adherent cells; Sp: stem-like tumor spheres; LIHC: liver hepatocellular carcinoma. Quantification data are shown as mean ± SD (n = 3); p values were calculated using two-tailed unpaired Student’s t-test. For experiments using clinical samples, p values were calculated using one-way ANOVA. **p < 0.01.

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, RNA Sequencing, Western Blot, Immunohistochemical staining, Staining, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 2. YY2 modulates tumor-initiating capacities by regulating CSCs. A,B) Protein expression levels of CSC markers in YY2-overexpressed (A) and YY2 knock-out (B) HCC cells, as determined by western blotting. C,D) Tumor sphere formation potential (C) and CSC frequency (D) in YY2-overexpressed HCC cells, as determined using in vitro LDA. Representative images (left; scale bars: 200 μm) and quantification results (right; n = 6) are shown. E,F) Tumor sphere formation potential (E) and CSC frequency in YY2 knock-out HCC cells (F), as determined using in vitro LDA. Representative images (left; scale bars: 200 μm) and quantification results (right; n = 6) are shown. G,H) Invasion capacity of YY2-overexpressed (G) and YY2 knock-out (H) HCC cells. Representative images (scale bars: 100 μm) and quantification results from three independent experiments (n = 6 per experiment) are shown. I–K) Tumor-initiating potential of YY2-overexpressed HCC-LM3 cells, as examined by in vivo LDA using xenograft experiment. Tumor volume (I), morphological images (J), and CSC frequencies (K) are shown. The ratio of the number of mice with tumor to the number of total mice transplanted with indicated cells is shown. L) Immunohistochemical staining images against YY2, CD44, and EpCAM in the tissue sections of xenografted tumors formed by YY2-overexpressed HCC-LM3 cells (scale bars: 50 μm). Cells transfected with pcCon or corresponding wild-type cells were used as controls. 𝛽-actin was used as western blotting loading control. Quantification data are shown as mean ± SD. p values were calculated using two-tailed unpaired Student’s t-test. For xenograft experiments, p values were calculated using one-way ANOVA. pcCon: pcEF9-Puro; **p < 0.01.

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Expressing, Knock-Out, Western Blot, In Vitro, In Vivo, Immunohistochemical staining, Staining, Transfection, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 3. YY2 suppresses CSC asymmetric division. A,B) Percentage of CD44High cells in YY2-overexpressed (A) and YY2 knock-out (B) HCC-LM3 cells, as evaluated using flow cytometry. C,D) CD44 fluorescence intensity in YY2-overexpressed (C) and YY2 knock-out (D) HCC-LM3 cells. Representative images (scale bars: 10 μm) and quantification results (n = 10) are shown. E,F) Percentages of EGFP-positive cells in YY2-overexpressed (E) and YY2 knock- out (F) HCC-LM3 cells transfected with PCMV-Numb-EGFP, as analyzed using flow cytometry (n = 3). G) Schematic diagram of cell-cycle synchronization in M phase using nocodazole (final concentration: 100 ng mL−1). H,I) Sphere cell division types in YY2-overexpressed (H) and YY2 knock-out (I) HCC-LM3 cells. Immunofluorescence of Numb (red) and DAPI (blue) representing three division types: CSC/CSC (C/C; Numb−/Numb−), CSC/non-CSC (C/D; Numb−/Numb+), and non-CSC/non-CSC (D/D; Numb+/Numb+). Representative images (left; scale bars: 10 μm) and quantification results from three independent experiments (right, each dot represents 24 to 32-pairs of daughter cells) are shown. Cells transfected with pcCon or wild-type cells were used as controls. Quantification data are shown as mean ± SD. p values were calculated using two-tailed unpaired Student’s t-test. pcCon: pcEF9-Puro; *p < 0.05; **p < 0.01; ns: not significant.

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Knock-Out, Cytometry, Transfection, Concentration Assay, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 4. YY2 suppresses CSC stemness by inhibiting mitochondrial fission. A) Heatmap showing differentially expressed genes in MHCC-97HYY2KO

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques:

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 5. YY2 directly binds to DRP1 promoter and regulates its transcriptional activity. A,B) mRNA expression levels of mitochondrial fission-related genes in stem-like tumor spheres formed by YY2-overexpressed HCC-LM3 (A) and MHCC-97H (B) cells, as determined using qRT-PCR. C,D) DRP1 protein expression levels in stem-like tumor spheres formed by YY2-overexpressed (C) and YY2 knock-out (D) HCC cells, as examined using western blotting. E) Schematic diagram of mitochondrial fission. F,G) Distribution of DRP1 on mitochondria in YY2-overexpressed (F) and YY2 knock-out (G) HCC-LM3 stem-like tumor spheres, as examined using mitochondrial membrane marker DsRed2-Tom20. Representative images (left; scale bars: 10 μm) and quantification results (right; n = 10) are shown. H) DNA-binding motif of YY2 on DRP1 promoter, as predicted using JASPAR. I) Relative activities of DRP1 promoter reporter vectors (DR-Lucs) in HCC-LM3YY2KO cells, as analyzed using dual luciferase reporter assay. J) Binding capacity of YY2 to the predicted region in the DRP1 promoter region, as determined using ChIP assay followed by PCR. The location of the primer pair used for PCR is shown.

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Activity Assay, Expressing, Quantitative RT-PCR, Knock-Out, Western Blot, Membrane, Marker, Binding Assay, Luciferase, Reporter Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 6. DRP1 is crucial for YY2 regulation on mitochondrial fission. A,B) Distribution of DRP1 in mitochondria in YY2-overexpressed, DRP1- overexpressed HCC-LM3 cells (A) and DRP1 knock-down HCC-LM3YY2KO cells (B) was analyzed using DsRed-Tom20. Representative images (scale bars: 10 μm) and quantification results (right; n = 10) are shown. C) Transmission electron microscopy images of mitochondria in YY2-overexpressed, DRP1- overexpressed HCC-LM3 stem-like tumor spheres. Representative images (scale bars: 200 nm) and quantification results (right; n = 30) are shown. D) OCR of YY2-overexpressed, DRP1-overexpressed HCC-LM3 stem-like tumor spheres (n = 3). E) ΔΨm in YY2-overexpressed, DRP1-overexpressed HCC-LM3 stem-like tumor spheres, as determined using MitoTracker Red/MitoTracker Green. Representative images (scale bars: 10 μm) and quantifi-

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Knockdown, Transmission Assay, Electron Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 7. YY2/DRP1 axis is crucial for CSC asymmetric division. A) Protein levels of CSC markers in YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells, as determined using western blotting. B) CD44 fluorescence intensity in YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells. Representative images (scale bars: 10 μm) and quantification results (n = 10) are shown. C) Protein levels of CSC markers in DRP1 knock-down HCC-LM3YY2KO cells, as determined using western blotting. D) CD44 fluorescence intensity in DRP1 knock-down HCC-LM3YY2KO cells. Representative images (scale bars: 10 μm) and quantification results (n = 10) are shown. E,F) CSC frequency in YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells (E) and DRP1 knock-down HCC-LM3YY2KO cells (F), as determined using in vitro LDA. G,H) Sphere cell division types in YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells (G), and DRP1 knock-down HCC-LM3YY2KO cells (H). Immunofluorescence of Numb (red) and DAPI (blue) representing three division types: CSC/CSC (C/C; Numb−/Numb−), CCSC/non-CSC (C/D; Numb−/Numb+), and non-CSC/non-CSC (D/D; Numb+/Numb+). Representative images (left; scale bars: 10 μm) and quantification results from three independent experiments (right, each dot represents 24 to 32-pairs of daughter cells) are shown. I,J) Percentages of EGFP-positive cells in YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells (I) and DRP1 knock-down HCC-LM3YY2KO cells (J) transfected with PCMV-Numb-EGFP vector, as analyzed using flow cytometry (n = 3). 𝛽-actin was used as western blotting loading control. Cells transfected with pcCon or wild-type cells transfected with shCon were used as controls. Quantification data are shown as mean ± SD. p values were calculated using two-tailed unpaired Student’s t-test. pcCon: pcEF9-Puro; **p < 0.01; ns: not significant.

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: Western Blot, Knockdown, In Vitro, Transfection, Plasmid Preparation, Cytometry, Control, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: YY2-DRP1 Axis Regulates Mitochondrial Fission and Determines Cancer Stem Cell Asymmetric Division.

doi: 10.1002/advs.202207349

Figure Lengend Snippet: Figure 8. YY2/DRP1 is crucial for mitochondrial fission-regulated CSC homeostasis and HCC tumorigenic potential. A–C) Tumor-initiating potential of YY2-overexpressed HCC-LM3 cells, as examined by in vivo LDA using xenograft experiment. Tumor volume (A), morphological images (B), and CSC frequencies (C) are shown. Ratio of the number of mice with tumor to the number of total mice transplanted with indicated cells is shown. D) Immuno- histochemical staining of YY2, DRP1, CD44, and Numb in the tissue section of xenografted tumors formed by YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells (scale bars: 50 μm). E) Numb fluorescence intensity in the xenografted tumor lesions formed by YY2-overexpressed, DRP1-overexpressed HCC-LM3 cells, as examined using immunofluorescence staining. Representative images (left; scale bars: 10 μm) and quantification results (right; n

Article Snippet: Briefly, cells were transfected with vectors targeting YY2 (HCP301990-CG04-3-10-a, target site: 5′′-GAT GGC AAT TGG ATC TACGG-3′′; HCP301990-CG04-3-10-b, target site: 3′′-TAG CCC GTG TTC GTGAAG AG-5′′; HCP301990-CG04-3-10-c, target site: 3′′-TCC GTC GGA ATGTCC TCC AT-5′′; Gene Copoiea, Rockville, MD).

Techniques: In Vivo, Staining