Journal: Cell

Article Title: Ketogenic Diets Alter the Gut Microbiome Resulting in Decreased Intestinal Th17 Cells.

doi: 10.1016/j.cell.2020.04.027

Figure Lengend Snippet: Figure 2. Metagenomic Sequencing Reveals Decreased Abundance of Bifidobacterial Species on the Ketogenic Diet in Our Human Cohort (A) Heatmap showing fold change of the 50 most abundant species, comparing the ketogenic diet (KD) to baseline diet (BD) stages across all 17 subjects in our human study. Bacterial species are ordered by the magnitude and direction of the average fold change, from the most negative (top) to positive (bottom). (B) The ketogenic diet results in consistent reductions in the abundance of bifidobacterial species in our human cohort. Each line connects paired samples between the two diet arms from each study subject for bifidobacterial species detected in a study subject’s gut microbiota. See also Table S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Hemin Sigma Cat #51280 Resazurin Sigma Cat #199303 TOS-propionate agar Sigma Cat #43314 DTT Spectrum Cat #40400120-1 HBSS Life Technologies Cat #14025126 EDTA Invitrogen Cat #AM9260G Fetal bovine serum, heat inactivated Life Technologies Cat #10438026 Dispase II Roche Cat #501003345 DnaseI Sigma Cat #10104159001 Collagenase VIII Sigma Cat #C2139 Percoll VWR Cat #89428-524 PMA Fisher Scientific Cat #AAJ63916MCR Ionomycyin Sigma Cat #I0634 Cell stimulation cocktail Fisher Scientific Cat #501129036 Golgi plug Fisher Scientific Cat #BDB555029 Fixation/Permeabilization Solution Kit Fisher Scientific Cat #BDB555028 Sodium Pyruvate Life Technologies Cat #11360070 Penicillin-Streptomycin Thermo Fisher Cat #15070063 MEM Non-Essential Amino Acids Life Technologies Cat #11140050 GlutaMAX GIBCO Cat #35050061 RMPI 1640 Fisher Scientific Cat #11875119 HEPES Fisher Scientific Cat #NC0734307 Ham’s F-10 medium Fisher Scientific (Corning) Cat #MT10070CV Collagenase type I Worthington Cat #NC9633623 Bovine serum albumin Fisher Scientific Cat #BP1600-100 Critical Commercial Assays LiquiColor b-Hydroxybutyrate Colorimetric Assay VWR (Stanbio) Cat #10152-474 ZymoBIOMICS 96 MagBead DNA Kit Zymo Research Cat #D4302 Deposited Data 16S rRNA and shotgun metagenomic sequencing data NCBI SRA PRJNA529487 Experimental Models: Organisms/Strains Mouse: C57BL6/J, germ-free UCSF Gnotobiotics N/A Software and Algorithms GraphPad Prism 8 GraphPad Software https://www.graphpad.com/ FlowJo v10.6.1 TreeStar https://www.flowjo.com/ R v3.5.3 CRAN https://r-project.org DADA2 v1.8.0 Callahan et al., 2016 https://benjjneb.github.io/dada2/ PhILR v1.6.0 Silverman et al., 2017 https://github.com/jsilve24/philr Vegan v2.5-5 Dixon, 2003 https://cran.r-project.org/web/packages/ vegan/index.html Ape v5.3 Paradis et al., 2004 https://cran.r-project.org/web/packages/ ape/index.html DESeq2 v1.20.0 Love et al., 2014 https://bioconductor.org/packages/ release/bioc/html/DESeq2.html lmerTest v3.1-0 Kuznetsova et al., 2017 https://cran.r-project.org/web/packages/ lmerTest/index.html (Continued on next page) ll Cell 181, 1263–1275.e1–e8, June 11, 2020 e2 Article

Techniques: Sequencing

Journal: Cellular and Molecular Life Sciences

Article Title: JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells

doi: 10.1007/s00018-022-04140-5

Figure Lengend Snippet: JAM-A limits single cell migration. A Single cell migration of MDCKII cells with inducible expression of JAM-A shRNAs (pEmU6proT, −Dox: shRNA not induced, +Dox: shRNA induced). Control cells (−Dox) were stably transfected with LA-mCherry, JAM-A KD cells (+Dox) were stably transfected with LA-EGFP. Cells were cultured on collagen-I (Col-I), fibronectin (FN), or vitronectin (VN) as indicated. Left panels: representative images of a LA-mCherry-transfected control cell (Ctrl, −Dox) and a LA-GFP-transfected JAM-A knockdown cell (JAM-A KD, +Dox) grown on collagen-I. Black-and-white images show the motility paths (yellow lines); the blob diameters used to track the cell during migration are highlighted by purple circles. Quantifications of single cell migration speed are shown in the right panels. Number of cells analyzed: Col-I: −Dox: 45, +Dox: 38, FN: −Dox: 48, +Dox: 44; VN: −Dox: 52, +Dox: 52, five independent experiments. B Quantifications of single cell migration speed of JAM-A KO MDCKII cells reconstituted with an inducible expression vector for murine JAM-A (pInducer21, −Dox: not induced, +Dox: induced). As control cells, MDCKII cells with inducible JAM-A shRNA expression in the absence of Dox were used. Number of cells analyzed: Ctrl MDCKII: 63; JAM-A KO, −Dox: 64; JAM-A KO, +Dox: 63; three independent experiments. C Quantifications of single cell migration speed of JAM-A KO MDCKII cells reconstituted with an inducible expression vector encoding phospho-deficient mJAM-A mutants (mJAM-A Y281F or mJAM-A S285A in pInducer21, −Dox: not induced, +Dox: induced). Number of cells analyzed: Ctrl MDCKII: 70; JAM-A KO + mJAM-A Y281F: −Dox: 67; +Dox: 65; JAM-A KO + mJAM-A S285A: −Dox: 62; +Dox: 65; four independent experiments). All statistical analyses shown in this figure were performed using Mann–Whitney U test. Data are presented as mean values ± SD. NS not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following siRNAs and shRNAs directed against canine (c) genes were used: cJAM-A shRNA 5′-CCAGTAAGAAGGTGATTTA-3′ (in pLVTHM (Addgene #12247) and in pEmU6proT (provided by Dr. Karl Matter); cCD151 siRNA pool (NCBI Gene ID475992, siTOOLs Biotech, München), cCD9 siRNA (NCBI Gene ID611695, siTOOLs Biotech, München), cITGA3 (NCBI Gene ID491074, siTOOLs Biotech, München); negative control siRNA pool (neg. ctrl. siPOOL Neg D1, siTOOLs Biotech, München); negative control siRNA (Qiagen 1027280), OnTarget plus non-targeting pool (Dharmacon D-001810-10-05).

Techniques: Migration, Expressing, shRNA, Control, Stable Transfection, Transfection, Cell Culture, Knockdown, Plasmid Preparation, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells

doi: 10.1007/s00018-022-04140-5

Figure Lengend Snippet: JAM-A positively regulates collective cell migration. A Monolayer expansion assays of MDCKII cells with inducible expression of JAM-A shRNAs (described in the legend to Fig. ). Cells were seeded on Col-I, FN- or VN-coated microscope slides separated by a silicone stamp. Collective migration was triggered by stamp removal. The migration velocity of the cell collective was quantified by measuring the cell-free area directly after the removal of the stamp and 8 h later (see “ ” for details). Top panels: representative images of monolayer expansion immediately after stamp removal (0 h) and after 8 h (8 h). Bottom panels: quantifications of collective cell migration velocities on different ECM substrates. Each dot represents one biological replicate (one independent cell population). Number of independent cell populations analyzed: Col-I: n = 12 for each cell line (four independent experiments), FN: n = 10 for each cell line (three independent experiments), VN: n = 9 for each cell line (three independent experiments). B Quantifications of collective cell migration speed of JAM-A KO MDCKII cells reconstituted with an inducible expression vector for murine JAM-A (see legend to Fig. B). Number of independent cell populations analyzed: n = 12 for each condition, three independent experiments. C Quantifications of collective cell migration speed of JAM-A KO MDCKII cells reconstituted with an inducible expression vector encoding phospho-deficient mJAM-A mutants (see legend to Fig. C). Number of independent cell populations analyzed: n = 14 for ctrl MDCKII cells, n = 12 for JAM-A KO MDCKII cells and mJAM-A expressing JAM-A KO MDCKII cells; three independent experiments. D β1 integrin expression in JAM-A KO MDCKII cells. Top: western blot analysis of β1 integrin in MDCKII cells with doxycycline-regulated expression of JAM-A shRNAs (pEmU6proT, −Dox: shRNA not induced, +Dox: shRNA induced). Cells were grown either on plastic or on collagen-I. Bottom: quantitative analysis of β1 integrin protein levels. Data are presented as mean values ± SD ( n = 4 independent experiments). All statistical analyses shown in this figure were performed using two-tailed Student’s t test. Data are presented as mean values ± SD. NS not significant; ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The following siRNAs and shRNAs directed against canine (c) genes were used: cJAM-A shRNA 5′-CCAGTAAGAAGGTGATTTA-3′ (in pLVTHM (Addgene #12247) and in pEmU6proT (provided by Dr. Karl Matter); cCD151 siRNA pool (NCBI Gene ID475992, siTOOLs Biotech, München), cCD9 siRNA (NCBI Gene ID611695, siTOOLs Biotech, München), cITGA3 (NCBI Gene ID491074, siTOOLs Biotech, München); negative control siRNA pool (neg. ctrl. siPOOL Neg D1, siTOOLs Biotech, München); negative control siRNA (Qiagen 1027280), OnTarget plus non-targeting pool (Dharmacon D-001810-10-05).

Techniques: Migration, Expressing, Microscopy, Plasmid Preparation, Western Blot, shRNA, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells

doi: 10.1007/s00018-022-04140-5

Figure Lengend Snippet: JAM-A interacts with α3β1 integrin in MDCKII cells. A CoIP of α3β1 integrin with JAM-A. The interaction of JAM-A with α3β1 integrin is detectable in Brij98-based lysates but not in NP40-based lysates. Note that the weak JAM-A signal in Brij98 lysed cells is due to lower solubility of JAM-A in Brij98. The asterisks indicate IgG heavy chains. IP immunoprecipitation, Lys lysate. B CoIP of α3β1 integrin with JAM-A from MDCKII cells cultured on collagen-I, laminin or vitronectin. JAM-A immunoprecipitates were immunoblotted for α3 integrin and JAM-A as indicated. To increase the solubility of JAM-A, the direct IPs for JAM-A were performed with NP40-based lysates. C CoIPs of JAM-A phospho-deficient mutants (JAM-A/Y281F, JAM-A/S285A) with α3β1 integrin. The asterisk indicates IgG heavy chains. D CoIPs of JAM-A mutants with α3β1 integrin. JAM-A constructs with deletions of the entire cytoplasmic domain (Δ40), the membrane-distal Ig-like domain (ΔD1), the membrane-proximal Ig-like domain (ΔD2), the entire extracellular domain (MC), or with point mutations affecting the N-linked glycosylation site of JAM-A (N185Q), were transfected into JAM-A-deficient MDCKII cells and analyzed for interaction with α3β1 integrin by CoIP. f.l. full length, MC membrane and cytoplasmic. E Collective cell migration velocity of control MDCKII cells (Ctrl siRNA pool) and α3 integrin KD MDCKII cells (α3 ITGN siRNA pool) on collagen-I and laminin. Left: representative images of monolayer expansion immediately after stamp removal (0 h) and after 8 h (8 h). Right: statistical evaluation of monolayer expansion on collagen-I and laminin. Each dot represents one independent cell population. Number of independent cell populations analyzed: n = 12 for each condition. Data are derived from three independent experiments. Statistical analysis shown in this figure was performed using two-tailed Student’s t test. Data are presented as mean values ± SD. **** P < 0.0001

Article Snippet: The following siRNAs and shRNAs directed against canine (c) genes were used: cJAM-A shRNA 5′-CCAGTAAGAAGGTGATTTA-3′ (in pLVTHM (Addgene #12247) and in pEmU6proT (provided by Dr. Karl Matter); cCD151 siRNA pool (NCBI Gene ID475992, siTOOLs Biotech, München), cCD9 siRNA (NCBI Gene ID611695, siTOOLs Biotech, München), cITGA3 (NCBI Gene ID491074, siTOOLs Biotech, München); negative control siRNA pool (neg. ctrl. siPOOL Neg D1, siTOOLs Biotech, München); negative control siRNA (Qiagen 1027280), OnTarget plus non-targeting pool (Dharmacon D-001810-10-05).

Techniques: Solubility, Immunoprecipitation, Cell Culture, Construct, Membrane, Glycoproteomics, Transfection, Migration, Control, Derivative Assay, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells

doi: 10.1007/s00018-022-04140-5

Figure Lengend Snippet: JAM-A interacts with tetraspanin CD151. A CoIP of CD151 with JAM-A in HEK293T cells. GFP-CD151 immunoprecipitates were immunoblotted for JAM-A (top panels) and GFP (bottom panels). B CoIP of CD151 with JAM-A in Caco2 cells (left) and HK-2 cells (right). GFP-CD151 immunoprecipitates were immunoblotted for JAM-A and GFP as indicated. C CoIPs of JAM-A deletion mutants with CD151. JAM-A mutant constructs (described in Fig. ) were transfected into JAM-A-deficient MDCKII cells and analyzed for interaction with CD151 by CoIP. D IF analysis of MDCKII cells transfected with EGFP-tagged CD151 and immunostained for endogenous α3 integrin and JAM-A. Arrowheads indicate co-localization of CD151, α3 integrin and JAM-A at cell–cell contact sites. Scale bar: 10 µm. E Monolayer expansion assays of MDCKII cells after siRNA-mediated depletion of CD151. MDCKII cells were transiently transfected with siRNA pools directed against CD151 (CD151 KD cells). As control, MDCKII cells were transfected with a scrambled siRNA pool (Ctrl KD cells). Collective cell migration was analyzed as described in the legend to Fig. . Top: representative images of monolayer expansion immediately after stamp removal (0 h) and after 8 h (8 h). Collective cell migration was analyzed on collagen-I and laminin as indicated. Bottom: statistical evaluation of monolayer expansion on collagen-I and laminin. Each dot represents one independent cell population. Number of independent cell populations analyzed: n = 12 for each condition. Data are derived from three independent experiments. Statistical analysis was performed using two-tailed Student’s t test. Data are presented as mean values ± SD. *** P < 0.001, **** P < 0.0001

Article Snippet: The following siRNAs and shRNAs directed against canine (c) genes were used: cJAM-A shRNA 5′-CCAGTAAGAAGGTGATTTA-3′ (in pLVTHM (Addgene #12247) and in pEmU6proT (provided by Dr. Karl Matter); cCD151 siRNA pool (NCBI Gene ID475992, siTOOLs Biotech, München), cCD9 siRNA (NCBI Gene ID611695, siTOOLs Biotech, München), cITGA3 (NCBI Gene ID491074, siTOOLs Biotech, München); negative control siRNA pool (neg. ctrl. siPOOL Neg D1, siTOOLs Biotech, München); negative control siRNA (Qiagen 1027280), OnTarget plus non-targeting pool (Dharmacon D-001810-10-05).

Techniques: Mutagenesis, Construct, Transfection, Control, Migration, Derivative Assay, Two Tailed Test

Journal: Cellular and Molecular Life Sciences

Article Title: JAM-A interacts with α3β1 integrin and tetraspanins CD151 and CD9 to regulate collective cell migration of polarized epithelial cells

doi: 10.1007/s00018-022-04140-5

Figure Lengend Snippet: JAM-A interacts with tetraspanin CD9. A CoIP of α3β1 integrin with JAM-A in CD151-depleted MDCKII cells. Lanes with postnuclear supernatants (PNS) contain 5% of the input used for IPs. B CoIP of CD9 with JAM-A. JAM-A immunoprecipitates obtained from MDCKII cells were immunoblotted for CD9 (top panels) and JAM-A (bottom panels). C CoIPs of JAM-A deletion mutants with endogenous CD9. JAM-A deletion constructs lacking the PDZ domain-binding motif (Δ3) or the entire cytoplasmic domain (Δ40) were transfected into JAM-A-deficient MDCKII cells and analyzed for interaction with CD9 by CoIP. Note that the JAM-A–CD9 interaction is retained after deletion of the entire cytoplasmic domain. The asterisk indicates IgG heavy chains. ( D ) Monolayer expansion assays of MDCKII cells after siRNA-mediated depletion of CD9 and after combined depletion of CD151 and CD9. MDCKII cells were transiently transfected with siRNA pools directed against CD9, or both CD151 and CD9. As control, MDCKII cells were transfected with a scrambled siRNA pool (Ctrl KD cells). Collective cell migration was analyzed as described in the legend to Fig. . Top: representative images of monolayer expansion immediately after stamp removal (0 h) and after 8 h (8 h). Collective cell migration was analyzed on collagen-I and laminin as indicated. Bottom: statistical evaluation of monolayer expansion on collagen-I and laminin. Each dot represents one independent cell population. Number of independent cell populations analyzed: n = 12 for each condition. Data are derived from three independent experiments. Statistical analysis was performed using two-tailed Student’s t test. Data are presented as mean values ± SD. *** P < 0.001, **** P < 0.0001. The experiments were performed in parallel to the monolayer expansion assays shown in Fig. E. The data obtained for control cells are therefore identical to those shown in Fig. E. E Cartoon of JAM-A association with α3β1 integrin through tetraspanins CD151 and CD9 (summary of biochemical data). Note that the interaction of JAM-A with both tetraspanins is mediated by the extracellular domain of JAM-A (red arrows), and that the interaction of CD151 with α3β1 integrin is also mediated by the extracellular domains of the two proteins (green arrow) . Phosphorylations involved in the regulation of MDCK cell motility and collective cell migration are indicated. For simplicity, the two phosphorylations are depicted in the same JAM-A dimer. Western blot data shown in this figure are representatives of at least three independent experiments

Article Snippet: The following siRNAs and shRNAs directed against canine (c) genes were used: cJAM-A shRNA 5′-CCAGTAAGAAGGTGATTTA-3′ (in pLVTHM (Addgene #12247) and in pEmU6proT (provided by Dr. Karl Matter); cCD151 siRNA pool (NCBI Gene ID475992, siTOOLs Biotech, München), cCD9 siRNA (NCBI Gene ID611695, siTOOLs Biotech, München), cITGA3 (NCBI Gene ID491074, siTOOLs Biotech, München); negative control siRNA pool (neg. ctrl. siPOOL Neg D1, siTOOLs Biotech, München); negative control siRNA (Qiagen 1027280), OnTarget plus non-targeting pool (Dharmacon D-001810-10-05).

Techniques: Construct, Binding Assay, Transfection, Control, Migration, Derivative Assay, Two Tailed Test, Western Blot

Journal: Molecular Medicine Reports

Article Title: Eburicoic acid inhibits endothelial cell pyroptosis and retards the development of atherosclerosis through the Keap1/Nrf2/HO‑1/ROS pathway

doi: 10.3892/mmr.2025.13551

Figure Lengend Snippet: Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs via the HO-1/ROS pathway. HUVECs were pre-treated with 100 µg/ml ox-LDL for 24 h and further incubated with (A and B) 1, 5, 10 and 20 µg/ml eburicoic acid for 24 h or (C and D) incubated with 10 µg/ml eburicoic acid 6, 12, 24 and 48 h, respectively. The mRNA and protein levels of HO-1 were detected using RT-qPCR and western blotting, respectively. (E) Cells were transfected with HO-1 siRNA or scrambled control siRNA for 48 h. Western blotting was used to detect HO-1 protein level. Cells were pre-treated with 100 µg/ml ox-LDL for 24 h. Then, they were transfected with HO-1 siRNA for 48 h before treatment with 10 µg/ml eburicoic acid for 24 h. (F) RT-qPCR analyses of NLRP3, caspase-1, ASC and GSDMD mRNA levels. (G) Western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels. (H) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels. (I) Determination of LDH release using an LDH release assay kit. (J) Spectrophotometry analysis of caspase-1 activity. (K) Representative images of Hoechst/PI staining and ROS production. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; HO-1, heme oxygenase-1; ROS, reactive oxygen species; si, small interfering; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; IL, interleukin; LDH, lactate dehydrogenase; ROS, reactive oxygen species EA, eburicoic acid.

Article Snippet: siRNA targeting HO-1 (NCBI Gene ID: 3162), NF-E2-related factor 2 (Nrf2; NCBI Gene ID: 4780) and scrambled control siRNA were designed and synthesized by Shanghai GenePharma Co., Ltd. using GenePharma RNAi designer V2.0 software.

Techniques: Activation Assay, Incubation, Quantitative RT-PCR, Western Blot, Transfection, Control, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Spectrophotometry, Activity Assay, Staining

Journal: Molecular Medicine Reports

Article Title: Eburicoic acid inhibits endothelial cell pyroptosis and retards the development of atherosclerosis through the Keap1/Nrf2/HO‑1/ROS pathway

doi: 10.3892/mmr.2025.13551

Figure Lengend Snippet: Eburicoic acid inhibits NLRP3 inflammasome activation and pyroptosis in ox-LDL-treated HUVECs via the Nrf2/HO-1/ROS pathway. (A) Cells were transfected with Nrf2 siRNA or scrambled control siRNA for 48 h. western blotting was used to detect the Nrf2 protein level. (B-G) Cells were pre-treated with 100 µg/ml ox-LDL for 24 h. Then, they were transfected with Nrf2 siRNA for 48 h before treatment with 10 µg/ml eburicoic acid for 24 h. (B) RT-qPCR analyses of HO-1, NLRP3, caspase-1, ASC and GSDMD mRNA levels; (C) western blotting analyses of NLRP3, pro-caspase-1, cleaved caspase-1, ASC and GSDMD-N protein levels; (D) ELISA Analyses of IL-1β, IL-6 and IL-18 secretion levels; (E) Determination of LDH release using an LDH release assay kit; (F) Spectrophotometry analysis of caspase-1 activity; (G) Representative images of Hoechst/PI staining and ROS production. Data are represented as mean ± SD. ns, statistically insignificant, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Scale bar, 20 µm. n=3. NLRP3, NLR family pyrin domain-containing protein 3; ox-LDL, oxidized low-density lipoprotein; HUVECs, human umbilical vascular endothelial cells; Nrf2, NF-E2-related factor 2; HO-1, heme oxygenase-1; ROS, reactive oxygen species; si, small interfering; ASC, apoptosis-associated speck-like protein containing CARD; GSDMD-N, N-terminal gasdermin-D; LDH, lactate dehydrogenase; ROS, reactive oxygen species; RT-qPCR, reverse transcription-quantitative PCR EA, eburicoic acid.

Article Snippet: siRNA targeting HO-1 (NCBI Gene ID: 3162), NF-E2-related factor 2 (Nrf2; NCBI Gene ID: 4780) and scrambled control siRNA were designed and synthesized by Shanghai GenePharma Co., Ltd. using GenePharma RNAi designer V2.0 software.

Techniques: Activation Assay, Transfection, Control, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Spectrophotometry, Activity Assay, Staining, Reverse Transcription, Real-time Polymerase Chain Reaction