Journal: Nature immunology

Article Title: Mapping Systemic Lupus Erythematosus heterogeneity at the single-cell level

doi: 10.1038/s41590-020-0743-0

Figure Lengend Snippet: (a). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on monocyte SCs (Mono-SCs, n=8). Each color represents a distinct SC. (b). Flow cytometry detection of ISG15 in PBMCs gated on CD14+ monocytes from 17 cSLE patients with different SLEDAI scores, as well as 14 cHD. (c). Percentage of ISG15+ CD14+ monocytes in cHD and cSLE as categorized based on SLEDAI (cHD cells ‘none’ in gray, SLEDAI <=4, in yellow; n=9, and SLEDAI >4, in red; n=8). T-test was used for statistical analysis. P-values are shown for the respective comparisons. (d). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on B cells SCs (B-SCs, n=7). Each color represents a distinct SC.

Article Snippet: Subsequent to surface staining and staining with live/dead fixable dye (Aqua, 1:1000, Thermo-Fisher), cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm and Perm/Wash Buffer; BD Biosciences), and stained for 30 min on ice for Granzyme A (GB9, 1:50, BioLegend), Granzyme B (GB11, 1:50, BioLegend), Perforin (B-D48, 1:50, BD Biosciences), and ISG15 (IC8044P, 1:50, R&D Systems).

Techniques: Flow Cytometry

Journal: Nature immunology

Article Title: Mapping Systemic Lupus Erythematosus heterogeneity at the single-cell level

doi: 10.1038/s41590-020-0743-0

Figure Lengend Snippet: (a). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on T cells SCs (T-SCs, n=6). Each color represents a distinct SC. (b). Percentage of GzB+, or Perforin+ CD8+ T cells within cHD and cSLE as categorized based on SLEDAI categories (cHD cells ‘none’ in gray, SLEDAI <=4 in yellow; n=9 and SLEDAI >4, in red; n=8). (c). Flow cytometry detection of GzB and perforin proteins in PBMCs, gated on CD8+ T cells, from cSLE (n=17) with different SLEDAI scores and cHD (n=14). T-test was used for statistical analysis. P-values are shown for the respective comparisons. (d). Individual UMAP plots from 11 cHD (left), 33 cSLE (right) based on NK SCs (NK-SCs, n=4). Each color represents a distinct SC. (e). Flow staining of ISG15 on PBMCs gated on CD57+ NK cells from six cSLE patients (in purple) with different SLEDAI scores and nine matched cHD (in green). MFI values are represented. (f). Boxplots representing the ESR (left panel), C4 (middle panel) and C3 (right panel) levels across the six cSLE subcluster groups (SCGs) depicted in . (g). Dotplot representing the correlation between hemoglobin (HGB) levels (g/dL) and proportion of cells from the Erythrocyte cluster across the cSLE samples.

Article Snippet: Subsequent to surface staining and staining with live/dead fixable dye (Aqua, 1:1000, Thermo-Fisher), cells were fixed and permeabilized according to the manufacturer’s instructions (Cytofix/Cytoperm and Perm/Wash Buffer; BD Biosciences), and stained for 30 min on ice for Granzyme A (GB9, 1:50, BioLegend), Granzyme B (GB11, 1:50, BioLegend), Perforin (B-D48, 1:50, BD Biosciences), and ISG15 (IC8044P, 1:50, R&D Systems).

Techniques: Flow Cytometry, Staining

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. The mRNA levels of ISG15 and ISG56 genes on lesion and adjacent non-lesion skin samples in vivo . Holstein cattle were intravenously injected with LSDV for 14 days, and then lesion and non-lesion skin samples from the same cattle were dissected for RT-qPCR analysis of mRNA levels of ISG15 and ISG56 genes. B. Effects of LSDV on IFN-β-induced transcription of ISGs. MDBK cells (5 × 10 5 ) were left un-infected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml, final concentration) for 6 hours followed by RT-qPCR analysis of mRNA levels of the indicated genes. C. Effects of LSDV on IFN-β-induced phosphorylation of STAT1 and STAT2. MDBK (5 × 10 5 ) cells were left uninfected or infected with wild-type LSDV (MOI = 1) for 12 hours and then treated with IFN-β (100 ng/ml) for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in A-B are mean ± SD (n = 11 in A and n = 3 in B) from one representative experiment. These experiments were repeated at least twice with similar results. ns nonsignificant, **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: In Vivo, Injection, Quantitative RT-PCR, Infection, Concentration Assay, Phospho-proteomics, Western Blot

Journal: PLOS Pathogens

Article Title: Lumpy skin disease virus protein LSDV122 impairs IFN-I receptor complex formation to evade host innate immunity

doi: 10.1371/journal.ppat.1013871

Figure Lengend Snippet: A. Effects of LSDV122-deficiency on LSDV-induced production of serum ISG15 and CXCL10. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). The orbital blood of viral infected mice was collected at 5 and 10 hours post infection, and the orbital blood of mock injected mice was collected at 5 hours post injection for ELISA analysis of CXCL10 and ISG15 levels. B-D. Effects of LSDV122-deficiency on LSDV-induced transcription of antiviral genes in different tissues of mice. Seven-week-old C57BL/6 mice were intravenously injected with medium (mock, n = 4), LSDV (2.6 × 10 5 pfu each mouse, n = 6) or LSDVΔ122 (2.6 × 10 5 pfu each mouse, n = 5). Spleen (B), liver (C) and lung (D) of mice were collected at 5 or 10 hours post viral infection or 5 hours post mock injection for RT-qPCR analysis of mRNA levels of the indicated genes. Data shown in A-D are mean ± SD. These experiments were repeated at least twice with similar results. *P < 0.05; **P < 0.01 (unpaired t-test).

Article Snippet: At 5 and 10 hours post-infection, orbital blood samples were collected, and the levels of Isg15 and Cxcl10 in the serum were measured using ELISA kits (CSB-EL011843MO, CUSABIO; EK0736, BOSTER).

Techniques: Injection, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Biomedical Science

Article Title: Localization, traffic and function of Rab34 in adipocyte lipid and endocrine functions

doi: 10.1186/s12929-023-00990-8

Figure Lengend Snippet: UBA1 conveys Rab34 action on FABP5 stability to regulate lipid metabolism. A Transfected 3T3-L1 cells with the indicated plasmids (GFP-Rab34 or FABP5-c-Myc) were treated with MG132 (10 μmol/L, 12 h) and lysed under denaturing conditions. c-Myc-tagged FABP5 was purified by anti-c-Myc immunoprecipitation and ubiquitinated FABP5 was detected by Western Blot. An expression vector coding for hemagglutinin (HA)-tagged ubiquitin (HA-Ubiquitin) was employed for cotransfection of cells expressing FABP5-c-Myc, alone or in combination with GFP-Rab34. The graph shows the ratio of Ubiquitinated-FABP5-c-Myc immunosignal to Ponceau S immunosignal. Data are referred to values in non-transfected cells (100%) and expressed as mean ± SEM (n = 3 biological replicates). **P < 0.01; ***P < 0.001. B Co-immunoprecipitation analysis in cells expressing GFP-Rab34 and vectors coding for LD-associated proteins related to ubiquitination/deubiquitination processes: UBA1-c-Myc (top panel), UCHL3-c-Myc (middle panel), or ISG15-c-Myc (bottom panel). In each experimental setting, proteins were purified by anti-c-Myc immunoprecipitation and detected by Western Blot using anti-c-Myc or anti-GFP antibodies. C Representative immunoblots and quantification of FABP5 levels in 3T3-L1 cells transfected with GFP-Rab34 (+ , 0.8 µg/µL), UBA1-c-Myc (+ , 0.8 µg/µL; + + , 1.6 µg/µL) or both expression vectors in the absence or presence of MG132 (10 µmol/L, 12 h). Data represent the ratio of FABP5 immunosignal to β-actin immunosignal and referred to values in non-transfected cells (100%). Data are expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001 vs. non-transfected cells. $$ P < 0.01; $$$ P < 0.001 vs. their respective condition treated with MG132. # P < 0.05; ## P < 0.01. D, E Rescue experiments of FABP5 in 3T3-L1 cells expressing GFP-Rab34 and UBA1 siRNA (siUBA1), alone or in combination. At the end of the experiments, cells were processed for immunoblotting studies ( D ) (see also Fig. S4) and for measurement of TGs (lipogenesis) and glycerol content (lipolysis) ( E ). Data are referred to values in control cells (100%; Scr), and expressed as mean ± SEM (n = 3 biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001

Article Snippet: Plasmids coding for Ubiquitin C-Terminal Hydrolase L3 (UCHL3) (pCMV6-UCHL3-c-Myc) and ISG15 Ubiquitin Like Modifier (ISG15) (pCMV6-ISG15-c-Myc) were purchased from OriGene (Rockville, USA).

Techniques: Transfection, Purification, Immunoprecipitation, Western Blot, Expressing, Plasmid Preparation, Ubiquitin Proteomics, Cotransfection, Control

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A) tSNE was used to visualize the single-cell global transcriptome data. Blue dots represent uninfected cells, derived from healthy donors. Beige dots represent bystander cells and red dots represent infected cells, both derived from patients infected with DV. (B) Differential expression of ISGs in PBMCs of patients infected with DV. The Gene Ontology term “type one interferon” was used to filter the results from the single-cell RNA sequencing. (C) Single cell ISG expression variability in uninfected, bystander and infected. ISGylation related genes are underlined. Blue bar represents uninfected cells, derived from healthy donors. Beige bar represents bystander cells and red bar represents infected cells, both derived from patients infected with DV. (D) Violin plot representing the expression of ISGylation family members in uninfected [U], bystander [B] and infected [I] PMBC. (E) Expression of ISG15 in DV infected PMBCs. Size is proportional to the percentage of infected cells in each cell type. Color intensity represents ISG15 average expression.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: Derivative Assay, Infection, Quantitative Proteomics, RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A) A549 WT and ISG15 KO were infected with 20 DV PFUs. At 36 hpi cells were fixed, permeabilized and stained for the flavivirus E protein using 4G2 antibody. Displayed images were acquired with a Leica DMI6000 B microscope. (B and C) DV relative plaque area (B) and the number of infected cells per plaque (C) quantified by ImageJ software and analyzed using Welch’s t test. Error bars represent mean ± SD. Results are representative of three independent experiments. (D, E and F) Multiple-step DV growth curve in A549 cells. Shown is the percentage of cells infected as measured by E protein staining (4G2+) (D), extracellular DV mRNA (PrM) relative expression by RT-qPCR (E) and titration by focus forming assay (FFA) (F). Statistical analyses were conducted using unpaired t tests. Error bars represent mean ± SD. Results are representative of three independent experiments. (G) Changes in genomic DV mRNA relative expression over time following binding of DV to A549 WT and ISG15 KO cells at 4°C and analyzed using unpaired t test. Error bars represent mean ± SD. Results are representative of two independent experiments. (H and I) Complementation of A549 ISG15 KO cells with ectopically expressed ISG15. Shown is the percentage of cells infected 36 hpi as measured by E protein staining (4G2+) (H) and DV mRNA relative expression by RT-qPCR (I). One-way ANOVA was used to analyze these experiments. EV: empty vector. Error bars represent mean ± SD. Results are representative of two independent experiments. (J and l) A549 WT and ISG15 KO were infected with 20 ZIKV PFUs. At 36 hpi cells were fixed, permeabilized and stained for the flavivirus E protein. ZIKV relative plaque area (J) and number of infected cells per plaque (I), quantified by ImageJ software and analyzed using unpaired t test with Welch’s correction. Images were acquired with an Olympus IX83 inverted microscope. Error bars represent mean ± SD. Results are representative of three independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). P values *<0.05; **<0.01; ***<0.001.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: Infection, Staining, Microscopy, Software, Expressing, Quantitative RT-PCR, Titration, Focus Forming Assay, Binding Assay, Plasmid Preparation, Inverted Microscopy

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A) ISGylation profile of A549 WT and HERC5 KO cells by Western blot. Cells were primed with IFNα2b (100 IU/ml) for 24 h and cell lysates were analyzed with an ISG15 antibody. (*) indicates antibody unspecific band. (B and C) A549 cells were infected with 20 DV PFUs. At 36 hpi cells were fixed, permeabilized and stained for the flavivirus E protein. DV relative plaque area (B) and the number of infected cells per plaque (C) quantified by ImageJ software and analyzed by one-way ANOVA. Images were acquired with an Olympus IX83 inverted microscope. Error bars represent mean ± SD. Results are representative of three or more independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). p values ****<0.0001.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: Western Blot, Infection, Staining, Software, Inverted Microscopy

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A-D) A549 cells were infected with 20 DV PFU. At 36 hpi, cells were harvested and the indicated mRNA transcripts were quantified by RT-qPCR (A, C and D). Cell lysates were analyzed by immunoblotting (B). Error bars represent mean ± SD. Results are representative of two independent experiments. Data was analyzed by unpaired t test. (E and F) A549 ISG15 KO cells were primed with IFNα2b (100 IU/ml) for 12 h, washed three times with DPBS and allowed to rest 12 h before infection with DV or ZIKV at an MOI of 0.1. Shown is the percentage of cells infected at 36 hpi, as measured by E protein staining (4G2+). Error bars represent mean ± SD. Results are representative of three independent experiments. Data was analyzed using unpaired t test. (G and H) A549 cells were infected with 20 DV PFUs. At 36 hpi cells were fixed, permeabilized and stained for the flavivirus E protein. DV relative plaque area (E) and number of infected cells per plaque (F) quantified by ImageJ software and analyzed by one-way ANOVA. Images were acquired with an Olympus IX83 inverted microscope. Error bars represent mean ± SD. Results are representative of three independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). p values *<0.05; ***<0.001.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: Infection, Quantitative RT-PCR, Western Blot, Staining, Software, Inverted Microscopy

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A) A549 WT and ISG15 KO were infected with 20 DV PFU. Cells were harvested at the indicated time points after infection (hpi) and cell lysates were analyzed by Western blot using STAT2 and Actin antibodies. (B, C and D) A549 WT and ISG15 KO immunofluorescence assay (IFA) 36 hpi for cellular IFIT3 and flavivirus E protein expression (D). Percentage of IFIT3 (B) and DV (C) positive cells per plaque were quantified by ImageJ software and analyzed using unpaired t test with Welch’s correction when appropriate. Displayed images were acquired with a Leica DMI6000 B microscope. (E) A549 cells were infected with DV at MOI 0.01. At 36 hpi, cells were fixed, permeabilized and stained for flavivirus E protein. Cell lysates were analyzed by Western blot with the indicated antibodies before (upper panel) and after (bottom panel) cells were sorted by fluorescence-activated cell sorting (FACS) based on E protein expression. U: uninfected. B: bystander. I: infected. Error bars represent mean ± SD. Results are representative of two independent experiments. Statistical analyses were performed using Prism 8 (GraphPad Software). p values ***<0.001.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: Infection, Western Blot, Immunofluorescence, Expressing, Software, Microscopy, Staining, Fluorescence, FACS

Journal: bioRxiv

Article Title: ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

doi: 10.1101/784678

Figure Lengend Snippet: (A) Flag-tag immunoprecipitation (IP) assay and Western blot analysis of HEK293 ISG15 KO cells transfected with ZIKV NS5-FLAG, human USP18 WT, human USP18 C64A mutant or the empty vector (pcDNA3.1) plasmids, followed by IFNα2b (100 IU/ml) priming for 18 h. WCL, whole cell lysate. Results are representative of three independent experiments. (B) STAT2 IP assay and Western blot analysis of HEK293 ISG15 KO cells transfected with the indicated plasmids, followed by IFNAα2b (100 IU/ml) priming for 18 h. Results are representative of three independent experiments. (C and D) Complementation with USP18 in A549 ISG15 KO cells. Cells were stably transfected with human USP18 or the empty vector and infected with DV at an MOI of 0.01. At 36 hpi, cells were harvested and cell lysates were analyzed by Western blot with the corresponding antibodies (C). Percentage of cells infected as measured by E protein staining (D). Error bars represent mean ± SD. Results are representative of two independent experiments. Statistical analyses were conducted using Mann-Whitney’s test in Prism 8 (GraphPad Software. p value **<0.01 (D). EV: empty vector.

Article Snippet: Human antibodies used for immunoblot were as follows: mouse mAb to β-actin (Abcam, ab6276, dilution 1:4000), rabbit mAb to IFIT1 (Abcam, ab137632, dilution 1:1000), mouse mAb to pSTAT1(Y701) [M135] (Abcam, ab29045, dilution 1:1000), mouse mAb to STAT1 (Abcam, ab3987, dilution 1:1000), rabbit pAb to IFIT3 (ProteinTech, 15201-1-AP, dilution 1:1000), rabbit mAb to USP18 [D4E7] (Cell Signaling Technologies, 4813, dilution 1:1000), rabbit mAb to STAT2 [D9J7L] (Cell Signaling Technologies, 72604, dilution 1:1000), mouse mAb to ISG15 (R&D System, MAB4845, dilution 1:1000), home-made mouse mAb anti-GFP ( , ) (dilution 1:1000) and mouse mAb to FLAG tag (Sigma, F3165, dilution 1:500).

Techniques: FLAG-tag, Immunoprecipitation, Western Blot, Transfection, Mutagenesis, Plasmid Preparation, Stable Transfection, Infection, Staining, Software