Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Identification of differentially expressed genes based on ImmuneScore and StromalScore. ( A-C ) Overall survival analysis of high and low Immune/Stromal/ESTIMATE Scores in patients with metastatic melanoma using the ESTIMATE algorithm. ( D , E ) Heatmap depicting the top 100 DEmRNAs with the most significant p-values between high and low Immune/Stromal Scores (|log Fold change|>1.9, p < 0.001). Blue represents downregulation, while red represents upregulation. ( F ) Venn diagram showing the upregulated and downregulated genes commonly intersecting between Stromal and Immune Scores, with green indicating immune-related DEmRNAs and purple indicating stromal-related DEmRNAs. ( G ) Construction of a protein-protein interaction network for overlapping genes using the STRING online tool (confidence score > 0.900) and Cytoscape software. ( H ) The top 15 genes ranked by nodes in the PPI network are shown in the graph. ( I ) Univariate Cox analysis was conducted on genes with a node count of 15 or more in the PPI network to identify differentially expressed genes significantly associated with prognosis

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Software

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: The expression of PTPN6 and its correlation analysis with survival. ( A ) Boxplot showing differential expression of PTPN6 in metastatic melanoma samples and normal skin tissue samples. ( B ) Correlation analysis of PTPN6 expression in metastatic melanoma samples with overall survival. ( C ) Correlation analysis of PTPN6 expression in metastatic melanoma samples with progression-free survival. ( D ) RT-qPCR assay analyzed the transcription level of PTPN6 in human keratinocyte cell line HaCaT and human metastatic melanoma cell lines A2058 and M14. The RT-qPCR results showed the relative mRNA expression of PTPN6 normalized to GAPDH. ( E ) Western blotting assay analyzed the expression of PTPN6 in human keratinocyte cell line HaCaT and human metastatic melanoma cell lines A2058 and M14. (** p < 0.01, *** p < 0.001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

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

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 inhibited the proliferation and viability of metastatic melanoma cells in vitro. ( A–C ) RT-qPCR and Western blotting assays were performed to evaluate the mRNA and protein expression of PTPN6 in A2058 and M14 cells after transfection with PTPN6. The RT-qPCR results showed the relative mRNA expression of PTPN6 normalized to GAPDH. ( D , E ) CCK8 assay was used to determine the impact of PTPN6 overexpression on the proliferation of A2058 and M14 cells. ( F , G ) EdU assay was conducted to assess the impact of PTPN6 overexpression on the viability of A2058 and M14 cells. Representative images were randomly selected from three independent experiments. Data are presented as the mean ± standard deviation (SD). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Western Blot, Expressing, Transfection, CCK-8 Assay, EdU Assay, Standard Deviation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 suppressed the migration and invasion of metastatic melanoma cells in vitro. ( A , B ) Wound healing assay was performed to determine the impact of PTPN6 overexpression on the migration of A2058 and M14 cells. ( C , D ) Transwell migration and Matrigel invasion assays were used to assess the impact of PTPN6 overexpression on the migration and invasion of A2058 and M14 cells. ( E , F ) Western blotting assay was conducted to verify the expression of migration and invasion-related genes, including MMP2, MMP9, E-cadherin, and Vimentin, in control, NC, and PTPN6 overexpression groups in A2058 and M14 cells. Representative images were randomly selected from three independent experiments. Data are presented as mean ± standard deviation (SD). (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, Migration, In Vitro, Wound Healing Assay, Western Blot, Expressing, Control, Standard Deviation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Overexpression of PTPN6 inhibited the activity of the JAK2-STAT3 signaling pathway in metastatic melanoma cells. ( A-D ) Western blotting assay was used to assess the expression of p-JAK2, JAK2, p-STAT3, and STAT3 after transfection with the PTPN6 overexpression plasmid. (**** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Over Expression, Activity Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: PTPN6 may affect immune cell infiltration and immune therapy response in metastatic melanoma. ( A ) TIMER 2.0 online analysis tool was used to examine the relationship between PTPN6 and the levels of infiltration of 14 immune cells and tumor purity in metastatic melanoma. ( B ) The correlation between PTPN6 and immune checkpoint therapy (alone or in combination with PD-1 or CTLA-4) response was analyzed using the TCIA database. ( C , D ) Western blotting assay was used to detect PD-L1 expression after PTPN6 overexpression (* p < 0.05, ** p < 0.01)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Western Blot, Expressing, Over Expression

Journal: Cancer Cell International

Article Title: Prognostic value and immune infiltration of a tumor microenvironment-related PTPN6 in metastatic melanoma

doi: 10.1186/s12935-024-03625-6

Figure Lengend Snippet: Knockdown of PTPN6 promoted the proliferation, migration, and invasion of metastatic melanoma cells in vitro. ( A , B ) Western blotting assay was performed to evaluate the protein expression of PTPN6 in A2058 and M14 cells after transfection with GV341-PTPN6-3FLAG-SV40-puromycin lentivirus. ( C , D ) Western blotting assay was performed to evaluate the protein expression of PTPN6 in A2058 and M14 cell lines stably overexpressing PTPN6 after transiently transfected siPTPN6 or siNC. ( E , F ) CCK8 assay was used to determine the impact of PTPN6 knockdown on the proliferation of cells. ( G , H ) Wound healing assay was performed to determine the impact of PTPN6 knockdown on the migration of cells. ( I , J ) Transwell migration and Matrigel invasion assays were used to assess the impact of PTPN6 knockdown on the migration and invasion of cells. Representative images were randomly selected from three independent experiments. Data are presented as mean ± standard deviation (SD). (** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Human immortalized epidermal keratinocyte cell line (HaCaT) and human metastatic melanoma cell lines (A2058, M14) were obtained from Procell Life Science & Technology Co., Ltd. (Wuhan, China).

Techniques: Knockdown, Migration, In Vitro, Western Blot, Expressing, Transfection, Stable Transfection, CCK-8 Assay, Wound Healing Assay, Standard Deviation

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside inhibits Srx expression, leading to the hyperoxidation of Prxs caused by reactive oxygen species (ROS) accumulation. ( A , B ) Melanoma M14 and A375 cells were treated with frugoside in ( A ) time-dependent (0.5 μg/mL) and ( B ) dose-dependent manners for 24 h, and the cells were subjected to a western blot analysis with antibodies against Srx, cytosolic Prx (Prx2), mitochondrial Prx (Prx3), and the loading control, tubulin. ( C , D ) M14 ( C ) and A375 ( D ) cells were treated with 200 μM H 2 O 2 for 10 min with 0.5 μg/mL frugoside. After 10 min, H 2 O 2 was washed out, and cells were incubated for the indicated times in a fresh medium. As a control of H 2 O 2 -stimulated samples, sample C was harvested without H 2 O 2 stimulation. The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( E , F ) M14 cells were treated with different doses of frugoside for 12 h ( E ) or 0.5 μg/mL of frugoside for different lengths of time ( F ). Dichlorofluorescein (DCF) fluorescence was analyzed with a fluorescence microscope after staining with 5 μM CM-H2DCFDA at 37 °C for 30 min. Representative fluorescent microscope images are shown, with quantified data given as a graph in the right panel. Data represent mean ± SD. p values were derived to assess statistical significance and are indicated as follows: ** p < 0.01; and *** p < 0.001. Original magfication, 100×.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Expressing, Western Blot, Control, Incubation, Fluorescence, Microscopy, Staining, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside induces mitochondria-mediated cell death in melanoma cells. ( A , B ) M14 ( A ) and A375 ( B ) cells were treated with frugoside at the indicated time and dose. The cytotoxicity level was measured by the CCK-8 assay. ( C – F ) M14 and A375 cells were treated with frugoside at the indicated dose for 24 h. The viability of frugoside-treated M14 ( C ) and A375 ( D ) cells was determined by a fluorescence-activated cell sorting (FACS) analysis after propidium iodide (PI)/Annexin V-fluorescein isothiocyanate (V-FITC) staining. The apoptotic cell population (Sub-G1) was measured by PI staining and a flow cytometry analysis in frugoside-treated M14 ( E ) and A375 ( F ) cells. ( G , H ) M14 and A375 cells were treated with various doses of frugoside for 24 h ( G ) or for the indicated periods ( H ; 0.5 μg/mL). The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( I , J ) M14 ( I ) and A375 ( J ) cells were treated with frugoside in a dose-dependent manner for 24 h. Cytosolic and mitochondrial fractions were prepared from the frugoside treated cells. The identity of the cytosolic and mitochondrial fractions was verified by the specific marker proteins, tubulin and Prx3. ( K ) M14 and A375 cells were treated with frugoside at the indicated doses for 24 h. The cells were analyzed by a western blot analysis with antibodies against Bcl2 and tubulin. p values were derived to assess statistical significance and are indicated as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: CCK-8 Assay, Fluorescence, FACS, Staining, Flow Cytometry, Western Blot, Marker, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside results in mitochondrial dysfunction via ROS overproduction. ( A–F ) Melanoma cells were treated with frugoside in dose- (24 h) and time- (0.5 μg/mL dosage) dependent manners. Stimulated cells were analyzed using flow cytometry for mitochondrial conditions, including the membrane potential, mitochondrial ROS, and calcium overload. The mitochondrial membrane potential was measured using a FACSCanto II apparatus after tetraethylrhodamine ethyl ester (TMRE) staining in M14 and A375 cells treated with various doses of frugoside ( A ) over time ( B ). Mitochondrial ROS was measured in frugoside-treated melanoma cells in dose- ( C ) and time- ( D ) dependent manners via MitoSOX staining. The mitochondrial calcium level was measured using Rhod2-AM fluorescent dye and quantified (dose: E , time: F ). Values are presented as mean ± SD. ( G–I ) Melanoma cells were treated with 0.5 μg/mL frugoside for 12 h and then stained with TMRE, MitoSOX, and Rhod2-AM dye. The membrane potential ( G ), mitochondrial ROS ( H ), and mitochondrial calcium level ( I ) were also analyzed using fluorescence microscopy. p values were derived to assess statistical significance and are indicated as follows: * p < 0.05; ** p < 0.01; and *** p < 0.001. Original magfication, 100×.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Flow Cytometry, Membrane, Staining, Fluorescence, Microscopy, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside-mediated ROS accumulation induces sustained p38 mitogen-activated protein kinase (MAPK) activation, and this activation contributes to apoptotic cell death. ( A ) M14 cells were treated with frugoside in time- and dose-dependent manners as described in . The cell lysates were subjected to western blotting with antibodies specific to phosphorylated MAPKs as indicated. The same membrane was re-probed with antibodies to the following MAPKs: c-Jun N-terminal kinases (JNK), p38 MAPK, and extracellular signal-regulated kinase (ERK). ( B ) M14 cells were treated with 0.5 μg/mL frugoside in the presence or absence of the p38 MAPK inhibitor, SB202190, or MAP/ERK kinase (MEK) 1/2 inhibitor, U0126, as indicated. The cells were analyzed for cell death using FACSCanto II after PI/Annexin V-FITC staining. ( C ) M14 cells were treated with 0.5 µg/mL frugoside for 24 h after a 1 h of SB202190 pretreatment. The cell lysates were subjected to western blotting with antibodies against poly (ADP-ribose) polymerase (PARP), caspase-3, Bcl2, and tubulin. ( D ) M14 cells were pretreated with 2 mM N-acetyl cysteine (NAC) or 10 μM diphenyleneiodonium (DPI) for 1 h, followed by 1 μg/mL frugoside for 24 h. The cells were subjected to a FACS analysis after PI/Annexin V-FITC staining. ( E ) M14 cells were treated with frugoside at the indicated doses for 24 h in the presence of 2 mM NAC or 10 μM DPI following a 1 h pretreatment, and the cell lysates were subjected to western blotting with the indicated antibodies. ( F ) M14 cells were pretreated with 2 mM NAC or 10 μM DPI for 1 h, followed by 1 μg/mL frugoside for 24 h, and the cells were analyzed with a FACSCanto II after TMRE staining. ( G ) As described in ( D ), frugoside- and antioxidant-treated M14 cells were subjected to western blotting with antibodies against phosphorylated and non-phosphorylated p38 MAPK. p values were derived to assess statistical significance and are indicated as follows: ** p < 0.01; and *** p < 0.001.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activation Assay, Western Blot, Membrane, Staining, Derivative Assay

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Srx is essential for ROS-mediated cell death by frugoside. ( A ) HA-Srx and Mock plasmids were transfected into M14 and A375 cells. After transfection for 24 h, the cells were treated with or without frugoside (0.5 μg/mL) for 12 h, followed by H 2 O 2 treatment (200 μM) for 10 min. H 2 O 2 was removed by replacement with a fresh medium, then the cells were incubated for the indicated times. The lysates were assessed by a western blot analysis using antibodies against the indicated proteins. ( B , C ) M14 ( B ) and A375 ( C ) cells were transfected with HA-Srx-overexpressing or Mock plasmids, and the cells were treated with various doses of frugoside. Cell death was measured by a FACS analysis after PI/Annexin V-FITC staining. ( D ) M14 cells were transfected as indicated in ( B ), and the cells were treated with frugoside in dose- and time-dependent manners. The cell lysates were subjected to western blotting with specific antibodies for caspase-3, HA, phospho-p38, p38, and tubulin. ( E ) Srx-overexpressed M14 cells were treated with frugoside and H 2 O 2 , as described in ( A ), and the cells were separated into cytosolic and mitochondrial fractions. The fractionated lysates were subjected to western blotting with antibodies specific to Prx-SO 2 , Prx3, and tubulin.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Transfection, Incubation, Western Blot, Staining

Journal: Cancers

Article Title: Frugoside Induces Mitochondria-Mediated Apoptotic Cell Death through Inhibition of Sulfiredoxin Expression in Melanoma Cells

doi: 10.3390/cancers11060854

Figure Lengend Snippet: Frugoside inhibits tumorigenic ability in vitro and in vivo. ( A ) A total of 6 × 10 4 M14 or A375 cells were seeded in a six-well plate. After three days, the cells were treated with frugoside at the indicated dose for one week. The cells were stained with crystal violet solution. Original magfication, 100×. ( B ) M14 and A375 cells were subjected to soft agar assay after 72 h in the medium containing frugoside at the indicated dose, which was changed every five days. The number of colonies generated per 10,000 cells was counted three weeks later. Original magfication, 100×. ( C , D ) Frugoside reduced the tumor volume in M14 and A375-inoculated nude mice. To generate the xenograft model, six-week-old BALB/c nude mice were inoculated with 1 × 10 6 M14 ( C ) and A375 ( D ) cells. After two weeks, frugoside (100 µg/kg in 10% DMSO and 90% PBS) or vehicle were injected once every two days for 17 days. The M14 and A375-inoculated nude mice were sacrificed, and tumor tissue was collected. The photograph shows tumor masses extracted from each group of mice after 17 days (upper panel). ( E ) Schematic illustration: The anti-cancer mechanism of frugoside.

Article Snippet: The human melanoma cell lines M14, A375, A2058, and LOXIMVI were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: In Vitro, In Vivo, Staining, Soft Agar Assay, Generated, Injection

Journal: Oncology Letters

Article Title: A conditionally replicating adenovirus expressing IL-24 acts synergistically with temozolomide to enhance apoptosis in melanoma cells in vitro

doi: 10.3892/ol.2017.6007

Figure Lengend Snippet: ZD55-IL-24 efficiently delivered IL-24 to (A) A375 and (B) M14 melanoma cells. Cells were infected with ZD55-IL-24 in the presence or absence of TMZ for 48 h. Cells treated with PBS were used as the negative control. Levels of IL-24 and the adenovirus E1A protein were analyzed by western blotting. β-actin was used as the loading control. Lanes: 1, PBS; 2, ZD55-IL-24 only; 3, ZD55-IL-24 + TMZ; and 4, TMZ only.

Article Snippet: The human melanoma A375 and M14 cell lines were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

Techniques: Infection, Negative Control, Western Blot, Control

Journal: Oncology Letters

Article Title: A conditionally replicating adenovirus expressing IL-24 acts synergistically with temozolomide to enhance apoptosis in melanoma cells in vitro

doi: 10.3892/ol.2017.6007

Figure Lengend Snippet: Combining ZD55-IL-24 with TMZ increased the expression of proteins associated with apoptosis induction. Melanoma cells (A) A375 and (B) M14 were treated with ZD55-IL-24 + TMZ, ZD55-IL-24 only or TMZ only. Cells treated with PBS were used as the negative control. The cells were lysed 72 h subsequent to treatment, and the protein expression levels of proapoptotic (Bax and γ-H2AX) and antiapoptotic (Bcl-2, Mcl-1 and NF-κB) proteins were assessed. β-actin was used as the loading control. The change in protein expression level relative to the PBS control was quantified by densitometry. TMZ, temozolomide; IL, interleukin; ZD55-IL-24, E1B-55 gene-deleted conditionally replicating adenovirus expressing the IL-24 gene; Bcl-2, B-cell lymphoma-2; Bax, Bcl-2-like protein 4; NF-κB, nuclear factor κB; γ-H2AX, γ-H2A histone family member X; Mcl-1, myeloid cell leukemia-1.

Article Snippet: The human melanoma A375 and M14 cell lines were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

Techniques: Expressing, Negative Control, Control

Journal: Oncology Letters

Article Title: A conditionally replicating adenovirus expressing IL-24 acts synergistically with temozolomide to enhance apoptosis in melanoma cells in vitro

doi: 10.3892/ol.2017.6007

Figure Lengend Snippet: Combination of ZD55-IL-24 and TMZ upregulated p53 protein expression. Melanoma cells (A) A375 and (B) M14 were treated with ZD55-IL-24 + TMZ, ZD55-IL-24 only or TMZ only. The cells were lysed at the time points indicated and the p53 levels were analyzed by western blotting. β-actin was used as the loading control. TMZ, temozolomide; IL, interleukin; ZD55-IL-24, E1B-55 gene-deleted conditionally replicating adenovirus expressing the IL-24 gene; Con, control.

Article Snippet: The human melanoma A375 and M14 cell lines were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

Techniques: Expressing, Western Blot, Control

Journal: Oncology Letters

Article Title: A conditionally replicating adenovirus expressing IL-24 acts synergistically with temozolomide to enhance apoptosis in melanoma cells in vitro

doi: 10.3892/ol.2017.6007

Figure Lengend Snippet: Combination of ZD55-IL-24 and TMZ increased the CPE of each treatment. Melanoma (A) A375 and (B) M14 cells were treated with different concentrations/doses of ZD55-IL-24 and TMZ in a systematic matrix to assess the CPE in melanoma cells. Cells were plated at a density of 10 5 cells/6-cm dish and stained with crystal violet 7 days subsequent to infection. The presence of staining indicated live cells. Representative results of a CPE assay in A375 and M14 cells are shown. TMZ, temozolomide; IL, interleukin; ZD55-IL-24, E1B-55 gene-deleted conditionally replicating adenovirus expressing the IL-24 gene; CPE, cytopathic effect; MOI, multiplicity of infection.

Article Snippet: The human melanoma A375 and M14 cell lines were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China).

Techniques: Staining, Infection, Expressing