Journal: bioRxiv

Article Title: Matrix tropism influences endometriotic cell attachment patterns

doi: 10.1101/2025.02.22.639314

Figure Lengend Snippet: Methodology for preparing, seeding, and analyzing microarrays. a) 1 kPa and 6kPa PA solutions are prepared as described in 2.2. b) Slides are washed, etched, and silanized before prepolymer solution is deposited in the printing region of interest and cured and dehydrated. c) ECM combinations are diluted in printing buffer to a final concentration of 250 μg/mL and arrayed onto the slide(s) with a liquid handler. d) 200,000 cells per microarray were seeded in monoculture or coculture and incubated for 30 minutes. e) Microarrays were washed with PBS and then incubated for 24 hours at which point f) microarrays were fixed in 4% PFA and stained with phalloidin and DAPI with fluoromount. g) Fixed and mounted slides were imaged on the Zeiss AxioScan.Z1. h) Images were exported and cropped into identified islands using MATLAB. CellProfiler was used to identify fluorescent regions of the cell (nuclei, actin) and partition information including cell count, cell area, etc. i) The outputs of cell profiler are then analyzed in R Studio.

Article Snippet: After the washes, the PBS was removed from the slides, 100 μL of DAPI Fluoromount-G (SouthernBiotech, 0100-20) solution was added on top of the microarrays and sandwiched with a coverslip (Electron Microscopy Sciences, 63765-01), sealed with clear nail polish, and stored protected from light at 4°C until imaging.

Techniques: Concentration Assay, Microarray, Incubation, Staining, Cell Counting

Journal: Cell

Article Title: FBXO44 promotes DNA replication-coupled repetitive element silencing in cancer cells

doi: 10.1016/j.cell.2020.11.042

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After 3 × 10 min washes with KCMT buffer, cells were stained with DAPI (Thermo Fisher Scientific no. D1306), coverslips mounted with anti-fade mounting medium (Vector Laboratories no. H-1000), and slides examined by fluorescence microscopy (Nikon Inverted TE300) or confocal microscopy (Zeiss LSM 710 NLO).

Techniques: Marker, Microarray, Recombinant, Plasmid Preparation, Imaging, Staining, Immunoprecipitation, Cell Isolation, Software

Journal: Oncogene

Article Title: Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0567-7

Figure Lengend Snippet: ( a ) Heatmap illustrating correlation between basal gene expression and 72 h single-agent EC 50 s of obatoclax from MTT assays in 47 diagnostic infant ALL samples. Note two major probeset clusters partitioning cases into resistant, sensitive and mixed groups in which EC 50 s were, in general, high (>176 nM; i.e. C max achieved at recommended adult phase II dose) (left), low (<176 nM) (right), or mix of high and low (middle). Functional annotation of genes in clusters is summarized (far right). Asterisks indicate cases with elevated HOXA gene expression pattern. ( b ) Correlations determined by IPA between obatoclax EC 50 and canonical signaling pathways in all 47 (left) and 25 KMT2A-AFF1 cases (right), respectively. ( c ) Independent QPCR confirmation of lack of difference in EIF4E transcript levels between three sample groups with differing obatoclax sensitivities in 42 available cases from microarray (left), and in 23 of these 42 cases that were KMT2A-AFF1 (right). Horizontal lines represent median; bars, range.

Article Snippet: Cells (6–12×10 4 /sample) were smeared onto slides, dried at 37°C, fixed and permeabilized with 100% methanol at −20°C ×20 min, washed twice with PBS and blocked with 10% FBS/0.2% Tween-20/PBS at 37°C ×1 h. After labeling with 1:50 anti-EIF4E-FITC (BD Transduction) in 2% FBS/0.2% Tween-20/PBS at 37°C ×2 h, slides were mounted using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Expressing, Diagnostic Assay, Functional Assay, Microarray

Journal: Oncogene

Article Title: Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0567-7

Figure Lengend Snippet: ( a ) Western blot analysis of basal EIF4E expression in available diagnostic infant ALL samples from microarray in and healthy adult PBMC controls. Microarray clusters by obatoclax response correspond to . Note elevated EIF4E protein (normalized to GAPDH) in 18 of 20 infant ALL samples compared to PBMC controls. Also note that proportions of cases with EIF4E elevation does not differ between three groups of samples with differing obatoclax sensitivities. EIF4E levels normalized to GAPDH are shown at top of blot. RS4:11 cells with elevated EIF4E are shown as a reference. Experiment was repeated twice on the same samples. ( b ) QPCR analysis of EIF4E mRNA expression in infant ALL genetic subsets (42 COG P9407 samples from microarray, 5 samples from CHOP). Plots show normalized expression relative to 6 healthy adult PMBC controls expressed as 2-ΔΔC T for all samples by genetic subset. p-values indicate significance; ns, not significant. Horizontal lines represent median 2-ΔΔC T ; bars, range. ( c ) Western blot analysis of EIF4E in diagnostic samples from 12 childhood cases of pre-B ALL. Note elevated EIF4E in 9 cases (asterisks). Cytogenetic abnormalities are indicated for each sample. Complex karyotype included structural 1q, 3q, 4q and 7p abnormalities; high resolution SNP array showed 9p13.2 deletion including part of PAX3 locus, homozygous 7q34 and 14q11.2 TCR loci deletions, and 16q22.1 deletion including part of CTCF locus. EIF4E levels normalized to GAPDH are shown at top of blot. Due to sample limitations experiment was done once.

Article Snippet: Cells (6–12×10 4 /sample) were smeared onto slides, dried at 37°C, fixed and permeabilized with 100% methanol at −20°C ×20 min, washed twice with PBS and blocked with 10% FBS/0.2% Tween-20/PBS at 37°C ×1 h. After labeling with 1:50 anti-EIF4E-FITC (BD Transduction) in 2% FBS/0.2% Tween-20/PBS at 37°C ×2 h, slides were mounted using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Western Blot, Expressing, Diagnostic Assay, Microarray

Journal: Oncogene

Article Title: Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0567-7

Figure Lengend Snippet: ( a ) Proliferating fractions plotted as inhibitory sigmoid E max models to estimate ribavirin EC 50 s in 8 of 9 cases with sufficient material for ribavirin treatment at multiple concentrations ( c.f. , all cases except PAPAWG). An EC 50 cut-off of <28 μM, based on clinical C max in adult AML trial, was used to define sensitivity. ( b ) Western blot analysis of primary infant ALL samples from cases in showing correlation between ribavirin response and basal eIF4E expression (left). Ribavirin EC 50 s are above the lanes for all samples where determined. N/A indicates EC 50 not determined because material was not available (Case PAPAWG). EIF4E levels normalized to GAPDH are shown at top of blot. Note greater ribavirin sensitivity (lower EC 50 s) in samples with higher EIF4E expression. Red box indicates resistant cases. Experiment was performed once due to sample limitations. Normalized EIF4E levels in sensitive vs. resistant cases in the Western blot are compared in bar graph (right). Bars represent standard error. Note significantly higher EIF4E levels in ribavirin-sensitive than ribavirin-resistant cases. ( c ) Western blot analysis showing abrogation of expression of MYC, MCL1 and/or NBN target proteins in EIF4E regulon by ribavirin treatment of infant ALL cells at clinically relevant concentrations. Note the greater protein reductions in the sensitive cases, which also show more abundant basal EIF4E expression. Band intensities normalized to ATCB and then made relative to vehicle for each protein are shown at top of blots. Blue boxes show decreased proteins after ribavirin treatment in the sensitive cases. Also note decreases in EIF4E in sensitive cases indicated by blue boxes. Red box indicates lack of consistent protein down-regulation by ribavirin treatment in resistant cases. Experiment was performed once due to sample limitations. ( d ) Re-localization of nuclear EIF4E to the cytoplasm in ribavirin-treated infant ALL cells. The cells are splenocytes from secondary xenografts expanded in NSG mice created from Case PAPAWG. The primary cells responded to single-agent ribavirin on BMSCs ( , top). The cells were treated for 72 h with vehicle or ribavirin at 20 μM. DAPI-labeled DNA in nucleus is blue; EIF4E labeled with anti-EIF4E-FITC is green. Image shows change in EIF4E nuclear localization to the cytoplasm in most cells in the field after ribavirin treatment compared to the nuclear localization of EIF4E in vehicle-treated cells. Experiment was performed once, but same result was seen in RS4:11 cell line ( c.f. ).

Article Snippet: Cells (6–12×10 4 /sample) were smeared onto slides, dried at 37°C, fixed and permeabilized with 100% methanol at −20°C ×20 min, washed twice with PBS and blocked with 10% FBS/0.2% Tween-20/PBS at 37°C ×1 h. After labeling with 1:50 anti-EIF4E-FITC (BD Transduction) in 2% FBS/0.2% Tween-20/PBS at 37°C ×2 h, slides were mounted using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Western Blot, Expressing, Labeling

Journal: Oncogene

Article Title: Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0567-7

Figure Lengend Snippet: ( a ) MTT assay data represented as surviving fraction plots after treatment of KMT2A- R cell lines × 72 h with increasing ribavirin concentrations. Plots are mean values from 10 experiments for RS4:11, 8 experiments for SEM-K2 and 3 experiments for KOPN-8 cells. Each experiment included 3–6 replicates/condition. Error bars show standard error. Note lower ribavirin EC 50 within clinically relevant concentrations for RS4:11 cells. ( b ) Western blot showing correlation between ribavirin sensitivity (lower EC 50 ) and high-level basal EIF4E expression. EIF4E levels normalized to ACTB and then made relative to RS4:11 are shown at top of blot. The blot is representative of data reproduced in 3 separate experiments. Note that the most ribavirin-sensitive cell line RS4:11 expresses the most EIF4E and KOPN-8 with highest EC 50 has the least EIF4E expression. ( c ) Bar graph of relative 3H-ribavirin uptake after 8 h treatment of KMT2A -R cell lines with 3H-ribavirin (RS4:11 cell line set to 1). Note that KOPN-8 with highest EC 50 has the lowest uptake, and RS4:11 with lowest EC 50 has the highest uptake ( c.f. ). Experiment performed twice with 3 intra-experimental replicates per experiment. Black bars indicate standard deviation. ( d ) Western blot analysis of basal expression of ADK, SLC29A1 and UGT1A proteins. Band intensities for each protein normalized to ACTB and then made relative to RS4:11 cells are at top of blots. Blots are representative of two independent experiments. Note similar expression of ADK, SLC29A1 and UGT1A in RS4:11 cells compared to SEM-K2 cells, and lower expression of all three proteins in KOPN-8 cells. ( e ) Analysis of cell cycle in ribavirin-treated RS4:11 cells. Representative flow cytometric analysis of propidium iodide labeling showing modeled cell cycle distribution after treatment × 24 h with vehicle or ribavirin at indicated concentrations (top). The histogram is representative of 3 separate experiments. Bar graphs (bottom) show percentages of cells in G0/G1, S, and G2/M in the 3 experiments. Note dose-dependent increase in cells in S phase indicative of cell cycle arrest with ribavirin treatment. ( f ) Western blot analysis of EIF4E targets in ribavirin-treated RS4:11 cells. Cells were treated for 72 h with vehicle or ribavirin at 5 μM and 20 μM. Vehicle-treated cells are in center lanes. Band intensities normalized to ACTB and then made relative to vehicle for each protein are at top of blots. Experiments were done 3–5 times except BCL2 and BIRC5 done twice for 5 μM ribavirin. The blots show down-regulation of MYC, MCL1, NBN, BCL2 and BIRC5. ( g ) Change in EIF4E subcellular distribution in ribavirin-treated RS4:11 cells. Confocal images show nuclear to cytoplasmic shift in most cells in population after 72 h ribavirin treatment compared to the nuclear EIF4E location in most cells following vehicle treatment. DAPI labeled DNA in the nucleus is blue and EIF4E labeled with anti-eIF4E-FITC is green. The images are representative of 3 separate experiments.

Article Snippet: Cells (6–12×10 4 /sample) were smeared onto slides, dried at 37°C, fixed and permeabilized with 100% methanol at −20°C ×20 min, washed twice with PBS and blocked with 10% FBS/0.2% Tween-20/PBS at 37°C ×1 h. After labeling with 1:50 anti-EIF4E-FITC (BD Transduction) in 2% FBS/0.2% Tween-20/PBS at 37°C ×2 h, slides were mounted using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: MTT Assay, Western Blot, Expressing, Standard Deviation, Labeling

Journal: Oncogene

Article Title: Targeting EIF4E signaling with ribavirin in infant acute lymphoblastic leukemia

doi: 10.1038/s41388-018-0567-7

Figure Lengend Snippet: (a) Analysis of cytoplasmic:nuclear ratios of EIF4E target mRNAs. ( a, left ) Verification of cytoplasmic and nuclear mRNA fractionation by semi-quantitative RT-PCR analysis of tRNA lys (cytoplasmic marker) and U6 snRNA (nuclear marker). Ethidium bromide stained agarose gel shows tRNA lys product in cytoplasmic but not nuclear fraction, and product for U6 snRNA in nuclear but not cytoplasmic fraction. ( a, middle ) Cytoplasmic:nuclear mRNA ratios of EIF4E target transcripts in RS4:11 cells determined by QPCR after treatment with vehicle or ribavirin (20 μM) for 72h. The data show decreased export of MYC , MCL1 , NBN and BCL2 but not BIRC5 mRNAs. ( a, right ) Total mRNA levels for transcripts analyzed in the same experiment. Data were normalized to ACTB and shown relative to vehicle (set to 1). Experiment was carried out three independent times. (b) Polysomal profiling of RS4:11 cells after treatment with vehicle or ribavirin (20 μM) for 72h. ( b, top ) Unchanged total polysomal profiles with ribavirin treatment. (b, middle) Polysomal profiling of known EIF4E translation targets. The data show shifts of MYC, MCL1, BCL2 and BIRC5 mRNAs and also NBN mRNA from higher to lower polysomal fractions (right side in plots) with ribavirin treatment. ACTB mRNA was analyzed as negative control. Fractions are shown relative to monosome fraction of vehicle control (Fraction 1). (b, bottom) Input levels of analyzed transcripts normalized to UBC and shown relative to vehicle (set to 1). Experiments were carried out two independent times. (c) Western blot analysis of PI3K/AKT1/mTOR and eIF4/p70S6K signaling in ribavirin-treated RS4:11 cells. Cells were treated for 72 h with vehicle or ribavirin at 5 μM and 20 μM. Vehicle-treated cells are in center lanes. Band intensities normalized to ACTB and then made relative to vehicle for each protein are at top of blots. The blots show decreased phospho-AKT1Thr308 but not AKT1 total protein; decreased phospho-EIF4EBP1Thr37/46 with only modestly decreased EIF4EBP1 total protein; >90% decrease in phospho-RPS6Ser235/236 with only modestly decreased RPS6 total protein, and decreased phospho–EIF4ESer209 but not EIF4E total protein. Experiments were done at least 3 independent times. The same blot was used for BCL2 and EIF4E, and the same ACTB panel is shown in both figures.

Article Snippet: Cells (6–12×10 4 /sample) were smeared onto slides, dried at 37°C, fixed and permeabilized with 100% methanol at −20°C ×20 min, washed twice with PBS and blocked with 10% FBS/0.2% Tween-20/PBS at 37°C ×1 h. After labeling with 1:50 anti-EIF4E-FITC (BD Transduction) in 2% FBS/0.2% Tween-20/PBS at 37°C ×2 h, slides were mounted using Fluoro-Gel II with DAPI (Electron Microscopy Sciences, Hatfield, PA, USA).

Techniques: Fractionation, Quantitative RT-PCR, Marker, Staining, Agarose Gel Electrophoresis, Negative Control, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway

doi: 10.1186/s13046-022-02348-8

Figure Lengend Snippet: IL-32 expression positively correlates with the infiltration of M2 macrophage in ESCC. A Correlation coefficient between IL-32 and infiltrative monocytes, M1 and M2 macrophages in ESCC tumor tissues, and the subsets of infiltrative cells were performed by the bioinformatic tool xCELL. Each symbol represents an individual patient ( n = 84). Pearson’s rank correlation test, *P < 0.05. B Infiltrative M2 macrophages in ESCC tumor and paired peritumor tissues were analyzed based on RNA microarray ( n = 84). Paired one-tailed Student’s t -test, ***P < 0.001. C IHC tissue microarray was used to analyze CD206 expression in ESCC tumor and paired peritumor tissues ( n = 16). Paired one-tailed Student’s t -test, *P < 0.05. D Representative IHC images of IL-32 and CD206 in the paired ESCC tumor tissues. Scale bar, 200 μm. E Correlation coefficient between IL-32 and CD206 according to IHC scores in ESCC ( n = 16), Pearson’s rank correlation test, *P < 0.05. F Immunofluorescence images of the indicated markers (CD206 and IL-32) in ESCC tumor tissue. Scale bar, 50 μm. G Schematic diagram of the EV cocultured with induced MDMs. H Confocal fluorescence microscopy was used to detect the process that MDMs (Red) internalized EV (Green) derived from EC109. Scale bar, 15 μm. The data are representative of at least three independent experiments

Article Snippet: For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions.

Techniques: Expressing, Microarray, One-tailed Test, Immunofluorescence, Fluorescence, Microscopy, Derivative Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway

doi: 10.1186/s13046-022-02348-8

Figure Lengend Snippet: EV-IL-32 promotes macrophage M2 polarization. A Schematic chart showed MDMs cocultured with EV treated with or without GW4869. B MDMs cocultured with EV (50 µg/mL) isolated from EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β for 72 h. The percentage of CD14 + CD206 + macrophages were tested by flow cytometry. C The EV were derived from EC109 shNC cells which were treated with or without GW4869 at 10 µM. Western blot analyzed the expression of TSG101 in EV. D EV-IL-32 in lysate was detected by ELISA. E MDMs cocultured with EV (Derived from 1 × 10 7 EC109 shNC cells treated with or without GW4869) for 72 h. Flow cytometry analysis of the CD14 + CD206 + macrophages. F The EV derived from KYSE150 IL-32β cells treated with or without GW4869 at 10 µM. Western blot analysis of the expression of TSG101 in EV. G EV-IL-32 in lysate was detected by ELISA. H MDMs cocultured with EV (Derived from 1 × 10 7 KYSE150 IL-32β cells treated with or without GW4869) for 72 h. Flow cytometry analysis of the CD14 + CD206 + macrophages. I MDMs cocultured with EV (50 µg/mL) derived from EC109 shNC, EC109 shIL-32, KYSE150 vector and KYSE150 IL-32β for 48 h. The mRNA expression of M1 ( IL-1β and iNOS ) and M2 ( IL-10 and Arg1 ) macrophage associated genes were detected by qRT-PCR. All data are representative of three independent experiments and represented as means ± SEM. Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions.

Techniques: Isolation, Plasmid Preparation, Flow Cytometry, Derivative Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway

doi: 10.1186/s13046-022-02348-8

Figure Lengend Snippet: IL-32 promotes M2 macrophage polarization in the primary tumor microenvironment. A , B Unpolarized MDMs (1 × 10 6 /mouse) and tumor cells (1 × 10 6 /mouse) were subcutaneously injected into nude mice on the right back for 3 days. Phenotypic analysis of M2 macrophages (Anti-human CD45 + CD14 + CD206 + ) was performed by flow cytometry. ( n = 3). C , D Tumor growth curves and tumor weight showed that IL-32 promoted tumor growth in subcutaneous tumor-bearing mouse model with KYSE150 vector and KYSE150 IL-32β cells ( n = 5). E Intratumoral macrophages were defined as CD45 + F4/80 + CD11b + and the proportion was showed ( n = 5). F The percentage of CD206 + macrophages was significantly higher in KYSE150 IL-32β group, which was defined as CD45 + F4/80 + CD11b + CD206 + ( n = 5). G , H Tumor growth curves and tumor weight showed that knockdown IL-32 inhibited tumor growth in subcutaneous tumor-bearing mouse model with EC109 shNC and EC109 shIL-32 cells ( n = 4). I Intratumoral macrophages were defined as CD45 + F4/80 + CD11b + and the proportion was showed ( n = 4). J The percentage of CD206 + macrophages was significantly higher in EC109 shNC group, which was defined as CD45 + F4/80 + CD11b + CD206 + ( n = 4). Data are represented as means ± SEM. Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions.

Techniques: Injection, Flow Cytometry, Plasmid Preparation, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Extracellular vesicle IL-32 promotes the M2 macrophage polarization and metastasis of esophageal squamous cell carcinoma via FAK/STAT3 pathway

doi: 10.1186/s13046-022-02348-8

Figure Lengend Snippet: IL-32 promotes lung metastasis in the EC109 and KYSE150 mouse models in vivo. A EC109 shIL-32 and EC109 shNC (2 × 10 6 cells/mouse) were intravenously injected into nude mice. Representative images of excised lungs. Graph showed the number of metastatic foci in the lungs ( n = 4). B Lung metastasis was confirmed by H&E. Scale bars, 400 or 200 μm. C KYSE150 IL-32β and KYSE150 vector (1 × 10 6 cells/mouse) were intravenously injected into nude mice. Representative images of excised lungs were shown. D Lung metastasis was confirmed by H&E. Graph showed the number of metastatic foci in the lungs ( n = 4). Scale bars, 400 or 200 μm. E , F Immunofluorescence of IL-32 and CD206 in the lungs from mice inoculated with EC109 shIL-32, EC109 shNC, KYSE150 vector and KYSE150 IL-32β cells. Scale bar, 50 μm. G BALB/c nude mice were intravenously injected with 100 µL clodronate liposome or control liposome every 4 days after tail vein injection of 2 × 10 6 EC109 shNC and EC109 shIL-32 cells until 7 weeks. Representative images of excised lungs. Graph showed the number of metastatic foci in the lungs ( n = 5). Data are represented as means ± SEM. Student’s t -test, * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: For CD206/IL-32/DAPI triple staining, slides were stained with rabbit anti-human CD206 (Servicebio, GB11062, China) and mouse anti-human IL-32 (BioLegend, 513501, USA) according to the manufacturer’s instructions.

Techniques: In Vivo, Injection, Plasmid Preparation, Immunofluorescence, Control