Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A ACE2 expression was measured for different Namalwa Syndecan cell lines in comparison to Huh7.5 cells and ACE2‐transfected 293T cells. B Huh7.5 cells were left untreated or treated with heparinase III for 1 h at 37°C and heparan sulfate or digested heparan sulfate expression was determined by flow cytometry. Cells were stained for HS and diHS respectively or an isotype control mAb, directly corresponding to the specific antibody (mouse IgM and IgG2b). One representative donor out of 3 is depicted. C Cell viability of infected Huh7.5 cells with SARS‐CoV‐2 pseudovirus in presence of different concentrations of UF heparin and LMWH enoxaparin ( n = 4 in duplicate). D Cell surface expression of ACE2 on 293T cells (control and ACE2‐transfected) was determined by quantitative real‐time PCR. E SARS‐CoV‐2 pseudovirus infection on 293T cells (control vs ACE2‐transfected cells) was measured by luciferase reporter activity. F SARS‐CoV‐2 isolate binding capacity to hACE2 was measured by quantitative real‐time PCR (ORFb1). SARS‐CoV‐2 isolate (10,000 TCID/ml) was pre‐incubated in presence or absence of different LMWH enoxaparin concentrations (250 IU/ml and 5 IU/ml) and added to a high binding ELISA plate coated with recombinant hACE2 (2 µg/ml). LMWH enoxaparin ( n = 3 measured in triplicate). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (D) unpaired, parametric, Student's t ‐test. * P ≤ 0.05, ** P ≤ 0.01, (E) 2‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3 in triplicate), HS: Heparan sulfate, diHS: digested Heparan sulfate, FI: fluorescent intensity, RLU: relative light units, ND: not determined.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Expressing, Transfection, Flow Cytometry, Staining, Infection, Real-time Polymerase Chain Reaction, Luciferase, Activity Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Huh7.5 cells were pre‐incubated with neutralizing antibody to ACE2 and SARS‐CoV‐2 pseudovirus was pre‐incubated with patient isolated mAb COVA1‐18, COVA1‐21 and COVA2‐15 (10 µg/ml) or UF heparin (250 IU/ml) for 30 min at 37°C. SARS‐CoV‐2 pseudovirus alone or with blocks was added to the cells for 4 h at 4°C and binding was determined by ELISA. B Heparan sulfates were removed from Huh7.5 cells by enzymatic treatment with heparinase III for 1 h at 37°C, then washed, and exposed to SARS‐CoV‐2 pseudovirus for 4 h at 4°C. Treated and untreated cells were subsequently lysed and binding was determined by ELISA. C Flow cytometry analysis of cell surface expression of heparan sulfates (HS) in control transduced cells or upon CRISPR/Cas9‐mediated EXT1 KO (EXT1 −/− ). D Control and EXT1 −/− XG1 cells were exposed to SARS‐CoV‐2 pseudovirus or SARS‐CoV‐2 pseudovirus pre‐treated with 250 IU/ml UF heparin for 30 min at 37°C. After incubation for 4 h at 4°C, cells were lysed and binding was measured by ELISA. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one‐way ANOVA with Tukey multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 ( n = 3), (B) unpaired Student’s t ‐test with Welch’s correction. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3), (D) two‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3).

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Incubation, Isolation, Binding Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, CRISPR

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Huh7.5 cells were exposed to SARS‐CoV‐2 pseudovirus directly or after pre‐treatment with different concentrations (0.0001, 0.001, 0.1, 0.5, 1, 5, 50, 100, 250 IU/ml) of UF heparin or LMWH enoxaparin for 30 min at 37°C. Infection was determined by luciferase reporter activity after 5 days. B SARS‐CoV‐2 pseudovirus was pre‐incubated for 30 min at 37°C with UF heparin (250 IU/ml) or LMWH tinzaparin (250 IU/ml) or dalteparin (250 IU/ml) or enoxaparin (250 IU/ml) or nadroparin (250 IU/ml). Huh7.5 was exposed to the SARS‐CoV‐2 pseudovirus, alone or treated with different LMWH for 4 h at 4°C, washed, lysed and binding was determined by ELISA. C SARS‐CoV‐2 pseudovirus was pre‐incubated for 30 min at 37°C with UF heparin (250 IU/ml) or LMWH tinzaparin (250 IU/ml) or dalteparin (250 IU/ml) or enoxaparin (250 IU/ml) or nadroparin (250 IU/ml). Huh7.5 cells were infected with SARS‐CoV‐2 pseudovirus in presence or absence of different LMWH and infection was determined after 5 days by luciferase reporter activity. D 293T cells expressing ACE2 were infected with SARS‐CoV‐2 pseudovirus in presence or absence of antibodies against ACE2, UF heparin (250 IU/ml), or LMWH enoxaparin (250 IU/ml), and infection was determined after 3 days by luciferase reporter activity. E VeroE6 cells were infected with SARS‐CoV‐2 isolate (hCoV‐19/Italy; 100 TCID/ml) previously treated with serial dilutions of LMWH enoxaparin. Cell viability was determined using an MTT assay ( n = 3 donors measured in triplicate). Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (A) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 donors measured in triplicate). (B) Ordinary one‐way ANOVA with Tukey’s multiple‐comparison test ( n = 3 donors measured in monoplo), (C) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 donors measured in triplicate), (D) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 3 measured in triplicate). RLU: relative light units. Source data are available online for this figure.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Infection, Luciferase, Activity Assay, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Expressing, MTT Assay

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A Syndecan 1 and Syndecan 4 expression by Namalwa cell lines, 293T cells, and Huh7.5 cells was detected by quantitative real‐time PCR. Representative data for an experiment repeated more than three times with similar results. B, C Different Namalwa Syndecan cell lines expressing either Syndecan 1 (B) or Syndecan 4 (C), determined by quantitative real‐time PCR. D Huh7.5 cells and Namalwa cells ectopically expressing Syndecan 1 were exposed to SARS‐CoV‐2 pseudovirus or control pseudovirus lacking S protein, in presence or absence of LMWH enoxaparin (250 IU/ml). Binding was measured after 4 h at 4°C by ELISA. E DCs were stained with antibodies against the surface markers CD209, CD14, and CD11b and analyzed by flow cytometry. F LCs were stained with antibodies against CD207 and CD1a and analyzed by flow cytometry. The histogram shows the cell surface expression of the receptor. Data information: Data show the mean values and error bars are the SEM. Statistical analysis was performed using (D) 2‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 2 in triplicate). DCs: Dendritic cells, LCs: Langerhans cells, ND: not determined, FI: fluorescent intensity.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A, B SARS‐CoV‐2 binding to monocyte‐derived DCs (A) or primary LCs (B) in absence or presence of UF heparin (250 IU/ml) or LMWH enoxaparin (250 IU/ml). C DCs and LCs were infected with SARS‐CoV‐2 pseudovirus and infection was determined after 5 days by measuring luciferase reporter activity. As positive controls, Huh7.5 cells were infected. D ACE2 cell surface expression on DCs, LCs, and Huh7.5 cells. Representative data for an experiment repeated more than three times with similar results ( n = 3 in duplicate). E Graphical overview of the cell‐to‐cell viral transmission assay. F, G DCs (F) and LCs (G) were pre‐incubated with SARS‐CoV‐2 pseudovirus for 4 h at 37°C in presence or absence of UF heparin (250 IU/ml) or LMWH enoxaparin (250 IU/ml), extensively washed, and co‐cultured with Huh7.5 cells. Transmission by DCs or LCs to Huh7.5 cells was determined by luciferase reporter activity. H, I SARS‐CoV‐2 isolate (hCoV‐19/Italy) was pre‐treated with LMWH enoxaparin (250 IU/ml) for 30 min at 37°C. DCs (H) and LCs (I) were exposed to either the untreated or pre‐treated SARS‐CoV‐2 isolate (100 TCID/ml) for 24 h, washed thoroughly, and co‐cultured with Huh7.5 cells. Quantification of viral RNA was measured by quantitative real‐time PCR. Data information: Data show the mean values and error bars are the SEM. (A, B) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (A) ( n = 4), (B) ( n = 4), (C) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 4 measured in triplicate), (F) ordinary one‐way ANOVA with Dunnett’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01 ( n = 4 measured in triplicate), (G) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 ( n = 3 measured in triplicate). (H, I) ordinary one‐way ANOVA with Tukey’s multiple‐comparison test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 (H) ( n = 3 in duplicate), (I) ( n = 4 in duplicate). DCs: Dendritic cells, LCs: Langerhans cells, RLU: relative light units, ND: not determined. Source data are available online for this figure.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Binding Assay, Derivative Assay, Infection, Luciferase, Activity Assay, Expressing, Transmission Assay, Incubation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

doi: 10.15252/embj.2020106765

Figure Lengend Snippet: A, B SARS‐CoV‐2 isolate (hCoV‐19/Italy, 100 TCID/ml) was either pre‐treated with LMWH enoxaparin (250 IU/ml) for 30 min at 37°C. DCs ( n = 3 independent donors) (A) and LCs (B) were exposed to untreated or pre‐treated SARS‐CoV‐2 (isolated for 24 h, washed thoroughly, and subsequently co‐cultured with Huh7.5 cells for another 24 h). Quantification of viral RNA was measured by quantitative real‐time PCR of Huh7.5 cells after removal of DCs or LCs.

Article Snippet: HuH7.5 cells were treated in D‐PBS/0.25% BSA with 46 milliunits heparinase III (Amsbio) for 1 h at 37°C, washed, and used in subsequent experiments.

Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: KDM5C is highly expressed in LIHC and indicates unfavorable patient prognosis. (A) Expression profiling of KDM5C in different cancer types predicted in the SangerBox system; (B) relevance of KDM5C expression to the overall survival rate of patients with LIHC; (C–D), mRNA (C) and protein (D) expression of KDM5C in LIHC cell lines (Hep3B, SNU‐387, HuH‐7, and SK‐HEP‐1) and liver epithelial cells THLE‐3 examined by RT‐qPCR and western blot analysis, respectively. Data were presented as mean ± SD from three independent experiments. In panels (C) and (D), differences were analyzed by one‐way ANOVA, * p < 0.05 versus THLE‐3 cells

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: Downregulation of KDM5C weakens proliferation, migration, and invasion, and increases apoptosis of LIHC cells. (A) interference efficiency of sh‐KDM5C 1, 2, 3# in SNU‐387 and SK‐HEP‐1 cells examined by RT‐qPCR; (B) proliferation of cells examined by the CCK‐8 assay; (C) colony formation ability of cells determined by the colony formation assay; (D) apoptosis rate in cells examined by TUNEL assay; (E) migration ability of cells examined by the wound‐healing assay; (F) invasion ability of cells determined by the Transwell assay. Data were presented as mean ± SD from three independent experiments. In all panels, differences were analyzed by two‐way ANOVA, * p < 0.05 versus the sh‐NC group

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Migration, Quantitative RT-PCR, CCK-8 Assay, Colony Assay, TUNEL Assay, Wound Healing Assay, Transwell Assay

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: ITIH1 level is negatively correlated with the KDM5C level in LIHC. (A) Genes showing a negative correlation with KDM5C in LIHC predicted in the Ualcan database (ranked in order of correlation; color depth of the blocks indicates the basic expression level of gene in LIHC); (B) expression profiling of ITIH1 in human cancers predicted in the SangerBox system; (C) correlation between ITIH1 and the survival of patients with LIHC predicted in the GEPIA database; (D) correlation between ITIH1 and KDM5C in patients with LIHC predicted in the Starbase system; (E and F) mRNA (E) and protein (F) levels of ITIH1 in LIHC cell lines (Hep3B, SNU‐387, HuH‐7, and SK‐HEP‐1) and liver epithelial cells THLE‐3 examined by RT‐qPCR and western blot analysis, respectively. Data were presented as mean ± SD from three independent experiments. In panels (E) and (F), differences were analyzed by one‐way ANOVA, * p < 0.05 versus the THLE‐3 cells

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: KDM5C removes H3K4me1 to reduce ITIH1 expression in LIHC cells. (A) Methylation modification at the promoter region of ITIH1 predicted in the UCSC database; (B) mRNA level of ITIH1 in cells transfected with sh‐KDM5C examined by RT‐qPCR; (C and D) H3K4me1 (C) and KDM5C (D) modification levels at ITIH1 promoter region in LIHC cell lines (Hep3B, SNU‐387, HuH‐7 and SK‐HEP‐1) and liver epithelial cells THLE‐3 examined by ChIP‐qPCR; E, protein levels of ITIH1 and H3K4me1 in cells transfected sh‐KDM5C examined by western blot analysis. Data were presented as mean ± SD from three independent experiments. In panels (C) and (D), differences were analyzed by one‐way ANOVA; in panels (B)–(E), differences were analyzed by two‐way ANOVA, * p < 0.05 versus the sh‐NC group; # p < 0.01 versus THLE‐3 cells

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Methylation, Modification, Transfection, Quantitative RT-PCR, ChIP-qPCR, Western Blot

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: KDM5C mediates ITIH1 expression and the PI3K/AKT signaling pathway to affect LIHC progression. (A) transfection efficacy of sh‐ITIH1 1, 2, 3# in SNU‐387 and SK‐HEP‐1 cells examined by RT‐qPCR; (B) phosphorylation of PI3K/AKT in cells examined by western blot analysis; (C) proliferation of SNU‐387 and SK‐HEP‐1 cells determined by the CCK‐8 assay; (D), colony formation ability of SNU‐387 and SK‐HEP‐1 cells examined by the colony formation assay; (E) apoptosis rate in SNU‐387 and SK‐HEP‐1 cells examined by the TUNEL assay; (F–G) migration (F) and invasion (G) abilities of SNU‐387 and SK‐HEP‐1 cells determined by the wound‐healing and Transwell assays, respectively. Data were presented as mean ± SD from three independent experiments. In all panels, differences were analyzed by two‐way ANOVA, * p < 0.05 versus the sh‐KDM5C + sh‐NC group; # p < 0.01 versus the sh‐NC group

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: Expressing, Transfection, Quantitative RT-PCR, Phospho-proteomics, Western Blot, CCK-8 Assay, Colony Assay, TUNEL Assay, Migration

Journal: The Kaohsiung Journal of Medical Sciences

Article Title: Lysine demethylase 5C epigenetically reduces transcription of ITIH1 that results in augmented progression of liver hepatocellular carcinoma

doi: 10.1002/kjm2.12501

Figure Lengend Snippet: Downregulation of KDM5C inhibits growth of LIHC xenograft tumors in vivo. (A) Growth rate of the xenograft tumors in nude mice; (B) weight of the xenograft tumors on the 4th week; (C) protein levels of ITIH1 and Ki67 in tumor tissues examined by IHC. There were five mice in each group. Data were presented as mean ± SD from three independent experiments. Differences were analyzed by the unpaired t ‐test (B) or two‐way ANOVA (A and C), * p < 0.05 versus the sh‐NC group

Article Snippet: Another two LIHC cell lines HuH‐7 (CL‐0120) and SK‐HEP‐1 (CL‐0212) were procured from Procell Life Science & Technology Co., Ltd. (Wuhan, Hubei, China).

Techniques: In Vivo

Journal: Cell Proliferation

Article Title: Rational engineering of adeno‐associated virus capsid enhances human hepatocyte tropism and reduces immunogenicity

doi: 10.1111/cpr.13339

Figure Lengend Snippet: Selection of AAV8 and AAVS3 variants in the Huh7 cell line with a CMV‐Luciferase‐GFP cassette at an MOI of 100,000. (A) Workflow of multi‐round selections. (B) Huh7 cells were transduced with single‐amino‐acid mutants of AAV8. (C) The second‐round selection of AAV8 mutants. (D) The third‐round selection of AAV8 mutants. (E) Huh7 cells were transduced with single‐amino‐acid mutants of AAVS3. (F) The second‐round selection of AAVS3 mutants. (G) Repeat experiments for AAVS3 candidates. The Y‐axis showed the intensity of the luminescence by relative luminescence units (RLU). Points represented the mean of 3 replicates and error bars represented the SD (standard deviation). Experimental values were analyzed via one‐way ANOVA using Dunnett's multiple comparison test and only statistically significant differences were indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: HEK293T, Huh7 (Procell, CL‐0120), HepG2 (ATCC, HB‐8065), Hep3B (Procell, CL‐0102), MRC‐5 (Procell, CL‐0161), HL‐1 (Procell, CL‐0605), AC16 (BeNa Culture Collection, BNCC337712), Hepa 1–6 (Procell, CL‐0105), and Renca (Procell, CL‐0568) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, 11,965,084) with 10% fetal bovine serum (FBS) (Gibco, 10,099,141), 1% GlutaMAX supplement (Gibco, 35,050,061), and 1% Antibiotic‐Antimycotic (Gibco, 15,240,062).

Techniques: Selection, Luciferase, Transduction, Standard Deviation, Comparison

Journal: Cell Proliferation

Article Title: Rational engineering of adeno‐associated virus capsid enhances human hepatocyte tropism and reduces immunogenicity

doi: 10.1111/cpr.13339

Figure Lengend Snippet: Evaluation of AAV mutants for multiple human liver carcinoma cell lines and human primary hepatocytes. (A, B) Huh7, HepG2, Hep3B, and human primary hepatocytes were transduced with the different AAV8 variants. (C, D) Cells were transduced with the different AAVS3 variants. The y‐axis showed the intensity of luminescence by relative luminescence units. Points represented the mean of 3 replicates and error bars represented the SD. Experimental values were analyzed via two‐way ANOVA using Dunnett's multiple comparison test and only statistically significant differences were indicated. (E, F) The variant v8‐1‐15 contained a CMV‐GFP cassette as well as the parental AAV8 at an MOI of 100,000 and the quantification of transduction efficiency was calculated. (G, H) Immunofluorescence for the variant vS3‐1‐8 and AAVS3 at an MOI of 100,000. (I–L) Cells were transduced at an MOI of 10,000 with different variants. Nuclei were stained with DAPI (blue) and transduction efficiency was assessed by GFP (green). Scale bar: 50 μM. The numbers of whole cells and GFP‐positive cells were quantified by ImageJ. Each data point represented an area for each sample. At least three areas were analyzed for each sample. Points represent the mean of replicates and error bars represent the SD. Experimental values were analyzed via two‐way ANOVA using Sidak's multiple comparison test and only statistically significant differences were indicated. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: HEK293T, Huh7 (Procell, CL‐0120), HepG2 (ATCC, HB‐8065), Hep3B (Procell, CL‐0102), MRC‐5 (Procell, CL‐0161), HL‐1 (Procell, CL‐0605), AC16 (BeNa Culture Collection, BNCC337712), Hepa 1–6 (Procell, CL‐0105), and Renca (Procell, CL‐0568) cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, 11,965,084) with 10% fetal bovine serum (FBS) (Gibco, 10,099,141), 1% GlutaMAX supplement (Gibco, 35,050,061), and 1% Antibiotic‐Antimycotic (Gibco, 15,240,062).

Techniques: Transduction, Comparison, Variant Assay, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance

doi: 10.1038/s41467-022-35022-w

Figure Lengend Snippet: a CLSM images of intracellular localization of FITC-labelled NMPNs and FITC-labelled PNPs after incubated for 6 h under different pH conditions. Scale bar: 20 μm. Arrows indicate NMPNs in the cytoplasm after the escape from endosomes in cisplatin-resistant Huh7 cells. b CLSM images of intracellular localization of FITC-labelled NMPNs and FITC-labelled PNPs after incubated for 12 h under different pH conditions. Scale bar: 40 μm. Asterisks indicate NMPNs in the nucleus of cisplatin-resistant Huh7 cells. c Quantitative analysis of the colocalization between lysotracker and FITC-labelled NMPNs or PNPs. d Quantitative analysis of the colocalization between DAPI and FITC-labelled NMPNs or PNPs. e Bio-TEM images of cells after incubation for 12 h with NMPNs under different pH conditions. Arrowheads indicate NMPNs accumulated in the nucleus of Huh7 cells. Black asterisks indicate NMPNs in nucleus. Scale bar: 1 μm. f Schematic diagram of the NMPNs to target the tumour cell nucleus. All data are presented as means ± SEM, n = 3 independent experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test ( c , d ). Source data are provided as a Source Data file.

Article Snippet: L02 cells (CL-0111) and Huh7 (CL-0120) cells were obtained from iCell Bioscience Inc. (Shanghai, China).

Techniques: Incubation

Journal: Nature Communications

Article Title: A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance

doi: 10.1038/s41467-022-35022-w

Figure Lengend Snippet: a The level of Pt-DNA adducts in the nucleus of cisplatin-resistant Huh7 cells after treatment with NMPNs at pH 6.5. Scale bar: 40 μm. Scale bar: 40 μm. b ROS levels in the nucleus of cisplatin-resistant Huh7 cells after treatment with PNPs or NMPNs at pH 6.5. Scale bar: 40 μm. c Immunofluorescence of the γ-H2AX in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. d Immunofluorescence of the XPA and Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. e Quantitative analysis of the colocalization between XPA and Pt-DNA adducts. f Immunofluorescence of the XPF and Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. g Quantitative analysis of the colocalization between XPF and Pt-DNA adducts. h Western blot analysis of XPF expression in the nucleus of cisplatin-resistant Huh7 cells after different treatments. i Quantitative analysis of XPF expression in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. j The schematic illustration of the mechanism underlying NMPNs to induce DNA platination and oxidative cleavage to combat Pt resistance in tumour cells. In comparison to Pt compounds and PNPs that cannot effectively generate ROS in the nucleus, NMPNs can readily accumulate in the nucleus by acidity-induced exposure of TAT peptides, and induce in situ ROS generation to induce DNA oxidative cleavage, thus destroying the DNA conformation required for NER. It hampers the recruitment of XPA and XPF and thus inhibiting NER pathway. All data are presented as means ± SEM, n = 3 independent experiments. Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: L02 cells (CL-0111) and Huh7 (CL-0120) cells were obtained from iCell Bioscience Inc. (Shanghai, China).

Techniques: Immunofluorescence, Western Blot, Expressing, Comparison, In Situ

Journal: Nature Communications

Article Title: A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance

doi: 10.1038/s41467-022-35022-w

Figure Lengend Snippet: a Immunofluorescence of the Pt-DNA adducts in cisplatin-resistant Huh7 cells after different treatments at pH 6.5. Scale bar: 40 μm. b Immunofluorescence of the Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with NMPNs or NMPNs+NAC at different time points. Scale bar: 40 μm. c Quantitative analysis of Pt-DNA adducts in cisplatin-resistant Huh7 cells after treatments with NMPNs or NMPNs+NAC at different time points. n = 6 independent experiments. d Quantitative analysis of DNA damage of cisplatin-resistant Huh7 cells after treatment with cisplatin or NMPNs. Cisplatin: n = 46; NMPNs: n = 44. e Flow cytometry analysis of cell apoptosis after different treatments at pH 6.5. f and corresponding quantitative results. n = 5 independent experiments. g The inhibition effect of cisplatin and NMPNs on cisplatin-resistant Huh7 cells growth at different incubation times. h The cell viabilities of cisplatin-resistant Huh7 cells or si XPA -transfected cisplatin-resistant Huh7 cells after treatment with NMPNs, PNPs or cisplatin. n = 4 independent experiments. All the data are presented as means ± SEM, Statistical significance was analyzed by one-way ANOVA with multiple comparisons test. Source data are provided as a Source Data file.

Article Snippet: L02 cells (CL-0111) and Huh7 (CL-0120) cells were obtained from iCell Bioscience Inc. (Shanghai, China).

Techniques: Immunofluorescence, Flow Cytometry, Inhibition, Incubation, Transfection