Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Flowchart overview of single-cell sequencing in human skin of actinic keratosis (AK), squamous cell carcinoma in situ (SCCIS), and cutaneous squamous cell carcinoma (cSCC) patients. ( B ) Hematoxylin and eosin (H&E) staining of skin biopsies from representative AK (100× and 250×), SCCIS (50× and 250×), and cSCC (50× and 250×). ( C ) Uniform manifold approximation and projection (UMAP) plot of human normal skin labeled by cell type and patient, respectively. ( D ) Heatmap showing gene expression signatures of each cell type. ( E ) Violin plot displaying the expression of representative genes to identify subpopulations for each cell type. ( F ) Representative gene ontology (GO) terms of signature genes in different cell subpopulations. The color keys from yellow to red indicate the range of p-value.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Sequencing, In Situ, Staining, Labeling, Gene Expression, Expressing

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Violin plots showing the expression levels of ALDH3A1 and IGDBP2 in actinic keratosis (AK), squamous cell carcinoma in situ (SCCIS), and matched normal samples (Wilcoxon test, p_val_adj <0.05). ( B ) Violin plots showing the expression levels of ALDH3A1 and IGDBP2 in cutaneous squamous cell carcinoma (cSCC) and matched normal samples. ( C ) The effect of UVB on ALDH3A1 expression in HaCaT cells. Left, the cells were collected at 24 hr, 48 hr, 72 hr, 96 hr, and 120 hr after UVB irradiation with a UVB dose of 10 mJ/cm 2 per 24 hr; right, the cells were collected at 24 hr after UVB irradiation with UVB doses of 10 mJ/cm 2 , 20 mJ/cm 2 , 30 mJ/cm 2 , 40 mJ/cm 2 , and 50 mJ/cm 2 . ( D ) The changes of cell proliferation after IGFBP2 overexpression in HaCaT and A431 cells. ( E ) The changes of cell invasion after IGFBP2 overexpression in HaCaT and A431 cells. * p<0.05; **p<0.01; ***p<0.001; ns, not significant.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Expressing, In Situ, Irradiation, Over Expression

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Representative gene ontology (GO) terms for genes with specific expression in basal cell of SCCIS, actinic keratosis (AK), and normal samples in P2 (upper) and up-regulated differentially expressed genes (DEGs) from AK versus normal, SCCIS versus normal, SCCIS versus AK, respectively (lower). ( B ) Heatmap showing copy number variation (CNV) levels of all keratinocytes from AK and SCCIS samples in P2. The keratinocytes from normal sample in P2 were defined as references. ( C ) Uniform manifold approximation and projection (UMAP) of subgroups generated from basal cells in SCCIS sample showing basal cells with higher CNV level enriched in one subgroup; red dot representing basal cells with higher CNV level (cnv.score>81.5). ( D ) DEGs detected between Basal-SCCIS-tumor and Basal-SCCIS-normal. ( E ) Representative enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) and GO terms in up-regulated genes (avg_log 2 FC >0.58 and p_val_adj <0.05). ( F ) Violin plots showing the expression level of representative DNA damage response marker genes in Basal-SCCIS-tumor and Basal-SCCIS-normal subgroups. ( G ) Chord plot showing the top up-regulated genes included in representative GO terms. ( H ) Violin plots showing the expression level of major members in HSP family across Basal-SCCIS-tumor and Basal-SCCIS-normal subgroups (Wilcoxon test, p_val_adj <0.05). ( I ) Violin plots showing the expression level of MAGEA4 and ITGA6 in Basal-SCCIS-tumor and Basal-SCCIS-normal subgroups (Wilcoxon test, p_val_adj <0.05). ( J ) Immunohistochemistry staining of MAGEA4 and ITGA6 in human skin of normal, SCCIS, and cutaneous squamous cell carcinoma (cSCC) samples.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Expressing, Generated, Marker, Immunohistochemistry, Staining

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Violin plots showing the different expression levels of candidate genes across all types of keratinocytes in P2, cutaneous squamous cell carcinoma (cSCC), and normal groups (Wilcoxon test, p_val_adj <0.05). ( B ) The statistical results of immunohistochemistry staining of MAGEA4 in different stages of cSCC including SCCIS, well-differentiated cSCC (WD cSCC), and moderately differentiated/poorly differentiated cSCC (MD/PD cSCC), respectively. ( C ) The statistical results of immunohistochemistry staining of ITGA6 in different stages of cSCC including SCCIS, WD cSCC, and MD/PD.cSCC, respectively.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) The mRNA expression of MAGEA4 and ITGA6 in human immortalized keratinocytes (HaCaT) and cutaneous squamous cell carcinoma (cSCC) cell lines (A431, SCL-I, SCL-II). *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( B ) Effect of small interfering RNA (siRNA) on the expression of MAGEA4 in A432 and ITGA6 in A431, SCL-I, and SCL-II determined by quantitative real-time PCR (qRT-PCR). ( C ) Functional experiment of MAGEA4 in A432. Upper, left, effect of MAGEA4 cSCC cell proliferation by CCK-8 proliferation in A431; upper, right, the effect of MAGEA4 on cSCC cell apoptosis was measured by staining with Annexin V-FITC/PI, followed by FACS analysis. Lower, left, the scratch experiment showed that MAGEA4 knockdown resulted in a shorter vertical migration distance compared with the control group after 72 hr; lower, right, transwell assay showed that the invasion abilities of the si-MAGEA4 groups significantly decreased compared with the si-NC group. **p<0.01. ( D ) Effect of ITGA6 on cSCC cell proliferation by CCK-8 proliferation assay in A431, SCL-I, and SCL-II. *p<0.05; **p<0.01. ( E ) The effect of ITGA6 on cSCC cell apoptosis. **p<0.01. ( F ) ITGA6 knockdown resulted in a shorter vertical migration distance compared with the control group after 72 hr. *p<0.05; **p<0.01. ( G ) The invasion abilities of the si-ITGA6 groups significantly decreased compared with the si-NC group. *p<0.05; **p<0.01.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Expressing, Small Interfering RNA, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Functional Assay, CCK-8 Assay, Staining, Knockdown, Migration, Control, Transwell Assay, Proliferation Assay

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Uniform manifold approximation and projection (UMAP) of all cells from cSCC patients labeled by sample and cell type, respectively. ( B ) Cell proportion of keratinocytes in cSCC and normal groups. ( C ) Expression of basal, Pro KC, and differentiated genes in all keratinocytes of cSCC and normal groups. ( D ) Left, immunohistochemical staining of LGALS1, IFITM3, and FTH1 in normal skin (200×), well-differentiated cSCC (WD cSCC) (50× and 250×), and moderately differentiated/poorly differentiated cSCC (MD/PD cSCC) (50× and 250×). Scale bar, 200 μm and 50 μm. Right, the immunoreactivity score (IRS) analyses of LGALS1, IFITM3, and FTH1 in normal skin, WD cSCC, and MD/PD cSCC. n=15 for each group. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( E ) The mRNA expression of LGALS1, IFITM3, and FTH1 in human immortalized keratinocytes (HaCaT) and cSCC cell lines (A431, SCL-I, SCL-II). *p<0.05; **p<0.01; ***p<0.001; ns, not significant.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Labeling, Expressing, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: The changes of cell proportion and significance test in all cutaneous squamous cell carcinoma (cSCC) samples and patient-matched normal skin samples (t test, p<0.05).

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques:

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Heatmap showing CNV levels of all keratinocytes from all cSCC samples. The keratinocytes from all patient-matched normal samples were defined as references. ( B ) Left, heatmap showing CNV levels of all keratinocytes from well-differentiated cSCC (WD cSCC) sample; right, the proportion of tumor and normal cells in WD cSCC sample defined by cnv.cut (probs = 0.99). ( C ) Left, heatmap showing CNV levels of all keratinocytes from poorly differentiated cSCC (PD cSCC) sample; right, the proportion of tumor and normal cells in PD cSCC sample defined by cnv.cut (probs = 0.99). ( D ) Left, heatmap showing CNV levels of all keratinocytes from moderately differentiated cSCC (MD cSCC) sample; right, the proportion of tumor and normal cells in MD cSCC sample defined by cnv.cut (probs = 0.99).

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques:

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) The enriched gene ontology (GO) terms of up-regulated differentially expressed genes (DEGs) in Basal1, Basal2, Pro KC, Follicular2, Spinous1 and Spinous2 between cSCC and normal groups. ( B ) Violin plots showing the different expression levels of candidate genes in cSCC and normal groups (Wilcoxon test, p_val_adj <0.05). ( C ) Left, immunohistochemical staining of BST2 and SAT1 in cSCC in normal skin (200×), well-differentiated cSCC (WD cSCC) (50× and 250×) and moderately differentiated/poorly differentiated cSCC (MD/PD cSCC) (50× and 250×). Scale bar, 200 μm and 50 μm. Right, the immunoreactivity score (IRS) analyses of BST2 and SAT1 in normal skin, WD cSCC and MD/PD cSCC. n=15 for each group. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( D ) The mRNA expression of BST2 and SAT1 in human immortalized keratinocytes (HaCaT) and cSCC cell lines (A431, SCL-I, SCL-II). *p<0.05; **p<0.01; ***p<0.001; ns, not significant.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Effect of small interfering RNA (siRNA) on the expression of LGALS1, IFITM3, and FTH1 in A431, SCL-I, and SCL-II determined by quantitative real-time PCR (qRT-PCR). ( B ) Effect of LGALS1, IFITM3, and FTH1 on cSCC cell proliferation. The CCK-8 proliferation assay demonstrated a significant decrease in the proliferation of the si-LGALS1, si-IFITM3, and si-FTH1 groups compared with the si-NC group. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( C ) The effect of LGALS1, IFITM3, and FTH1 on cSCC cell apoptosis. Significant increase in the apoptosis of the si-LGALS1, si-IFITM3, and si-FTH1 groups compared with the si-NC group. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( D ) The scratch experiment showed that LGALS1 and IFITM3 knockdown resulted in a shorter vertical migration distance compared with the control group after 72 hr, while there was no significant change in the si-FTH1 group. *p<0.05; **p<0.01; ***p<0.001; ns, not significant. ( E ) Transwell assay showed that the invasion abilities of the si-LGALS1, si-IFITM3, and si-FTH1 groups significantly decreased compared with the si-NC group. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CCK-8 Assay, Proliferation Assay, Knockdown, Migration, Control, Transwell Assay

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Effect of small interfering RNA (siRNA) on the expression of BST2 and SAT1 in A431, SCL-I, and SCL-II determined by quantitative real-time PCR (qRT-PCR). ( B ) Effect of BST2 and SAT1 on cutaneous squamous cell carcinoma (cSCC) cell proliferation by CCK-8 proliferation assay in A431, SCL-I, and SCL-II. **p<0.01. ( C ) The effect of BST2 and SAT1 on cSCC cell apoptosis. **p<0.01. ( D ) BST2 and SAT1 knockdown resulted in a shorter vertical migration distance compared with the control group after 72 hr. *p<0.05; **p<0.01. ( E ) The invasion abilities of the si-ITGA6 groups significantly decreased compared with the si-NC group. *p<0.05; **p<0.01.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Small Interfering RNA, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, CCK-8 Assay, Proliferation Assay, Knockdown, Migration, Control

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Identification of TME cell subpopulations based on their marker genes, including T cells, dendritic (DC) cells, and stromal cells. ( B ) Cell proportion of cell subpopulations in T cells, DC cells, and stromal cells, respectively. ( C ) The monotonically changed differentially expressed genes (DEGs) in TME cells in cSCC samples of all stages (Wilcoxon test, p_val_adj <0.05). ( D ) Comparison of cell interactions among the different clinical stages of cSCC (Wilcoxon test, ****p<0.0001). ( E ) Comparison of total incoming path weights vs total outgoing path weights across all cell populations in three cSCC samples, respectively (Wilcoxon test, p<0.1). ( F ) Circle plot showing the inferred intercellular communication signaling strength network of MHC-II pathway in three cSCC samples, respectively (Wilcoxon test, p<0.1).

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Marker, Comparison

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Uniform manifold approximation and projection (UMAP) of subpopulations generated from fibroblasts in all cutaneous squamous cell carcinoma (cSCC) samples. ( B ) Feature plots showing the expression of signature genes for . ( C ) Feature plots showing the expression of signature genes for Fib3. ( D ) Feature plots showing the expression of signature genes for Fib4.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Generated, Expressing

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Comparison of cell interactions between poorly differentiated cSCC and matched normal samples (Wilcoxon test, ****p<0.0001). ( B ) Circle plot showing the inferred intercellular communication signaling strength network of MHC-II, LAMININ, and TNF pathways in poorly differentiated cSCC and matched normal samples were predicted by CellPhoneDB approach (Wilcoxon test, p<0.1).

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: Comparison

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) Left, uniform manifold approximation and projection (UMAP) of single-cell RNA sequencing (scRNA-seq) dataset from poorly differentiated cSCC (PD cSCC) sample labeled by cell type; right, UMAP plot of single-cell ATAC sequencing (scATAC-seq) dataset from PD cSCC sample labeled by cell type after integration and label transfer with scRNA-seq data. ( B ) Bar plot of annotated differentially accessible region (DAR) location for each type. ( C ) Fragment coverage (frequency of Tn5 insertion) around the DAR on the gene KRT5 and CD83. ( D ) Left, heatmap of average chromVAR motif activity for each cell type. The color scale represents a z-score scaled by row. Right, UMAP plot displaying chromVAR motif activity, gene activity, and gene expression of TP63 (upper) and FOSL1 (lower). The color scale for each plot represents a normalized log-fold-change for the respectively assay. ( E ) Cell-specific mean chromVAR motif activity from the JASPAR database was plotted against cell-specific average expression for the corresponding transcription factor for all cell types and transcription factors. ( F ) Mean chromVAR activity was plotted against average expression for TP63 (left) and FOSL1 (right). Significant correlation was assessed with Pearson’s product moment correlation coefficient using the cor.test function in R.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: RNA Sequencing, Labeling, Sequencing, Activity Assay, Gene Expression, Expressing

Journal: eLife

Article Title: Single-cell sequencing highlights heterogeneity and malignant progression in actinic keratosis and cutaneous squamous cell carcinoma

doi: 10.7554/eLife.85270

Figure Lengend Snippet: ( A ) The attribution plot showing the similarity between Basal-SCCIS-tumor/normal with the cell populations defined by Ji et al. ( B ) The classification heatmap showing the similarity between Basal-SCCIS-tumor/normal with the cell populations defined by Ji et al. ( C ) The attribution plot showing the similarity between basal cells in cutaneous squamous cell carcinoma (cSCC) samples with the cell populations defined by Ji et al. ( D ) The classification heatmap showing the similarity between basal cells in cSCC samples with the cell populations defined by Ji et al. SCCIS, squamous cell carcinoma in situ.

Article Snippet: Human immortalized epidermal keratinocytes cell line (HaCaT cells) and human cSCC cell line A431, SCL-I, and SCL-II ( ; ) were used in this study and were obtained from the American Type Culture Collection (ATCC) and Free University of Berlin.

Techniques: In Situ

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: PFKFB3 is highly expressed in CSCC cells. (a) PFKFB3 mRNA expression and (b) PFKFB3 expression in CSCC cells and HaCaT cells was respectively examined using RT-qPCR and Western blot. ***P < 0.001 vs. HaCaT

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: PFKFB3 silencing suppresses the proliferation of CSCC cells. (a) PFKFB3 mRNA expression and (b) PFKFB3 protein expression in A431 cells after transfection with shRNA-PFKFB3#1/2 was respectively tested by RT-qPCR and Western blot. (c) The viability, (d) proliferation and (e) Ki-67 expression of A431 cells transfected with shRNA-PFKFB3#2 was in turn analyzed by MTT assay, clone formation assay and immunofluorescence assay. *P < 0.05 and ***P < 0.001 vs. Control. ## P < 0.01 and ### P < 0.001 vs. shRNA-NC. @@ P < 0.01 vs. shRNA-PFKFB3#1

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, MTT Assay, Tube Formation Assay, Immunofluorescence

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: PFKFB3 knockdown restrains the metastasis of CSCC cells. (a) The migration and (b) invasion of A431 cells transfected with shRNA-PFKFB3#2 were respectively evaluated with wound healing assay and transwell assay. The levels of (c) migration and invasion related proteins and (d) EMT related proteins in A431 cells transfected with shRNA-PFKFB3#2 were assessed using Western blot. ***P < 0.001 vs. Control. ### P < 0.001 vs. shRNA-NC

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Migration, Transfection, shRNA, Wound Healing Assay, Transwell Assay, Western Blot

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: PFKFB3 knockdown inhibits the aerobic glycolysis of CSCC cells. (a) The glucose consumption, (b) lactic acid production and (c) ATP production in A431 cells transfected with shRNA-PFKFB3#2 were in turn detected by Glucose Assay kit, L-Lactic Acid Colorimetric Assay kit and ATP Assay Kit. (d) The levels of aerobic glycolysis related proteins in A431 cells transfected with shRNA-PFKFB3#2 was analyzed by Western blot. ***P < 0.001 vs. Control. ### P < 0.001 vs. shRNA-NC

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Transfection, shRNA, Glucose Assay, Colorimetric Assay, ATP Assay, Western Blot

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: KLF9 negatively modulated PFKFB3 transcription in CSCC. (a) The binding sites between KLF9 and PFKFB3. (b) KLF9 mRNA expression and (c) KLF9 protein expression in several CSCC cells and HaCaT cells was respectively determined with RT-qPCR and Western blot. **P < 0.01 and ***P < 0.001 vs. HaCaT. (d) KLF9 mRNA expression and (e) KLF9 protein expression in A431 cells after transfection was measured with RT-qPCR and Western blot. ***P < 0.001 vs. Control. ### P < 0.001 vs. shRNA-NC. @@@ P < 0.001 vs. shRNA-KLF9#1. $$$ P < 0.001 vs. pcDNA3.1. (f)The interaction between KLF9 and PFKFB3 was analyzed by dual-luciferase reporter assay. ***P < 0.001 vs. pcDNA3.1. (g) The binding ability of KLF9 to PFKFB3 promoter was evaluated with ChIP. ***P < 0.001 vs. IgG

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Luciferase, Reporter Assay

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: KLF9 knockdown attenuated the impacts of PFKFB3 knockdown on the proliferation of CSCC cells. (a) PFKFB3 mRNA expression and (b) PFKFB3 protein expression in A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 was respectively detected using RT-qPCR and Western blot. (c) The viability, (d) proliferation and (e) Ki-67 expression of A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 was tested with MTT assay, clone formation assay and immunofluorescence analysis. *P < 0.05 and ***P < 0.001 vs. Control. # P < 0.05 and ### P < 0.001 vs. shRNA-NC. @ P < 0.05 and @@@ P < 0.001 vs. shRNA-PFKFB3. $ P < 0.05 and $$$ P < 0.001 vs. shRNA-PFKFB3 + shRNA-NC

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Expressing, Transfection, shRNA, Quantitative RT-PCR, Western Blot, MTT Assay, Tube Formation Assay, Immunofluorescence

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: KLF9 knockdown reversed the inhibitory effect of PFKFB3 knockdown on the metastasis of CSCC cells. (a) The migration and (b) invasion of A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 were assessed with wound healing assay and transwell assays. The expression of (c) migration and invasion related proteins and (d) EMT related proteins in A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 was examined by Western blot. ***P < 0.001 vs. Control. ### P < 0.001 vs. shRNA-NC. @ P < 0.05, @@ P < 0.01 and @@@ P < 0.001 vs. shRNA-PFKFB3. $ P < 0.05, $$ P < 0.01 and $$$ P < 0.001 vs. shRNA-PFKFB3 + shRNA-NC

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Migration, Transfection, shRNA, Wound Healing Assay, Expressing, Western Blot

Journal: Bioengineered

Article Title: KLF9 (Kruppel Like Factor 9) induced PFKFB3 (6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3) downregulation inhibits the proliferation, metastasis and aerobic glycolysis of cutaneous squamous cell carcinoma cells

doi: 10.1080/21655979.2021.1980644

Figure Lengend Snippet: KLF9 silencing reversed the inhibitory effect of PFKFB3 knockdown on the aerobic glycolysis of CSCC cells. (a) The glucose consumption, (b) lactic acid production and (c) ATP production in A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 were examined using Glucose Assay kit, L-Lactic Acid Colorimetric Assay kit and ATP Assay Kit. (d) The levels of aerobic glycolysis related proteins in A431 cells transfected with shRNA-PFKFB3#2 and shRNA-KLF9#2 was tested with Western blot. ***P < 0.001 vs. Control. ### P < 0.001 vs. shRNA-NC. @@@ P < 0.001 vs. shRNA-PFKFB3. $$$ P < 0.001 vs. shRNA-PFKFB3 + shRNA-NC

Article Snippet: Human immortalized keratinocytes (HaCaT) and human CSCC cells (SCL-1, SCC13, HSC-5 and A431) were purchased from BioVector NTCC Inc. (Beijing, China).

Techniques: Transfection, shRNA, Glucose Assay, Colorimetric Assay, ATP Assay, Western Blot

Journal: PLoS ONE

Article Title: miR-365 Promotes Cutaneous Squamous Cell Carcinoma (CSCC) through Targeting Nuclear Factor I/B (NFIB)

doi: 10.1371/journal.pone.0100620

Figure Lengend Snippet: (A) The expression of miR-365 and NFIB in CSCC cell lines (Tca8113, HSC-1 and A431) compared with normal HaCaT cells was detected by qRT-PCR and/or western blotting. The expression of NFIB is inversely correlated with miR-365 levels. (B) The expression of miR-365 in CSCC primary tumors was detected by microRNA-FISH using normal skin tissue as control. Bars = 50 µm. (C) Correlation between miR-365 expression and NFIB RNA/protein expression in CSCC primary tumors. The expression of NFIB is inversely correlated with miR-365 levels. In this figure, the expression of miR-365 was examined by qRT-PCR and normalized to U6 snRNA expression. The expression of NFIB protein or mRNA in CSCC tumors compared with normal skin tissue was detected by western blot using as a loading control or qRT-PCR normalized to GAPDH expression. The P value (<0.01) shows significant inverse correlation between the levels of miR-365 and NFIB.

Article Snippet: CSCC lines A431, Tca8113 (China Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HSC-1 (Dongguang Biojet Biotech.

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

Journal: PLoS ONE

Article Title: miR-365 Promotes Cutaneous Squamous Cell Carcinoma (CSCC) through Targeting Nuclear Factor I/B (NFIB)

doi: 10.1371/journal.pone.0100620

Figure Lengend Snippet: (A) Two WT luciferase reporter plasmids were generated by fusing miR-365 binding sites of the NFIB 3′UTR downstream of the luciferase reporter gene. Two mutant plasmids were generated by mutating the binding sites. The mutated sequences were underlined. WT or mutant reporter constructs were then transfected into HaCaT cells with NC or miR-365 mimics. Dual luciferase assay were performed 48 h post transfection and normalized to Renila luciferase activities. Data represent the average of three independent experiments ± SD. (B) NFIB mRNA (Left panel) and miR-365 (Right panel) expression was measured in NC, miR-365, NC inhibitor or miR-365 inhibitor transfected HaCaT cells respectively by qRT-PCR normalized to GAPDH for NFIB or to U6 for miR-365. Expression folds are shown with respect to NC mimic or NC inhibitor transfected cells where normalized copy number was set to 1. (C) qRT-PCR and western blot showing NFIB mRNA and NFIB protein expressed after hsa-miR-365 inhibitors were transferred into A431 and HSC-1 cells. Representative experiments are shown. Means ± SD, n = 4.

Article Snippet: CSCC lines A431, Tca8113 (China Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HSC-1 (Dongguang Biojet Biotech.

Techniques: Luciferase, Generated, Binding Assay, Mutagenesis, Construct, Transfection, Expressing, Quantitative RT-PCR, Western Blot

Journal: PLoS ONE

Article Title: miR-365 Promotes Cutaneous Squamous Cell Carcinoma (CSCC) through Targeting Nuclear Factor I/B (NFIB)

doi: 10.1371/journal.pone.0100620

Figure Lengend Snippet: (A) The expression of NFIB, p53, CDK6 and Bcl-2 proteins in CSCC cells transfected with antagomiR NC and antagomiR-365 was detected by western blot using GAPDH as a loading control. A representative result is shown here. (B) A representative picture shows the change in tumor volume in xenograft model of BALB/c-nu mice after antagomir-365 treatment 3 weeks with intratumoural injection. The right back flank of BALB/c-nu mice was injected subcutaneously with A431 cells in vivo with a volume of more than 150 mm 3 (n = 5) in comparison with PBS treatment (n = 5). Red arrow head shows the tumor formation from representative mice 21 days after treatment (controls were treated with PBS). (C) Tumor volumes (mm 3 ) were recorded in time points as indicated in the growth curve. Relative tumor volumes are shown with respect to day 7. Data are plotted as mean ± S.E. (D) AntagomiR-365 injection drastically decreased the expression levels of the miR-365 in xenografts and thus led to the up-regulation of NFIB and p53 and down-regulation of CDK6 and Bcl-2. Each bar represents the average expression from 5 individual xenografts. Data are plotted as mean ± S.E. (E) IHC staining of NFIB, p53, CDK6 and Bcl-2 on sections of xenograft tumors. Representative fields are shown here and index of positive signal was calculated (n = 10).

Article Snippet: CSCC lines A431, Tca8113 (China Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HSC-1 (Dongguang Biojet Biotech.

Techniques: Expressing, Transfection, Western Blot, Control, Injection, In Vivo, Comparison, Immunohistochemistry

Journal: PLoS ONE

Article Title: miR-365 Promotes Cutaneous Squamous Cell Carcinoma (CSCC) through Targeting Nuclear Factor I/B (NFIB)

doi: 10.1371/journal.pone.0100620

Figure Lengend Snippet: (A) NFIB protein levels were detected in A431 cells after siRNA oligos of NFIB was transferred and incubated for 24 h, 48 h, 72 h. (B) The expression levels of NFIB, p53, CDK6 and Bcl-2 proteins and mRNA in CSCC cells transfected with two distinct siRNA oligos against NFIB were detected by western blot using GAPDH as a loading control or by qRT-PCR normalized to GAPDH expression. (C) The expression miR-365 examined by qRT-PCR and normalized using U6 snRNA after siRNA NFIB_2, siRNA NFIB_3 and siRNA negative control were transfected into A431 and HSC-1 cells and incubated for 72 h. (D) A mechanism model of miR-365 in CSCC shows that the pro-carcinogenic role of miR-365 is functionally performed through targeting NFIB.

Article Snippet: CSCC lines A431, Tca8113 (China Center for Type Culture Collection and Cell Bank of the Chinese Academy of Sciences, Shanghai, China) and HSC-1 (Dongguang Biojet Biotech.

Techniques: Incubation, Expressing, Transfection, Western Blot, Control, Quantitative RT-PCR, Negative Control