Journal: bioRxiv

Article Title: Addressing anemia severity in antimony-resistant Leishmania donovani infection at the nexus of oxidative outburst and iron transit

doi: 10.1101/2024.03.04.583250

Figure Lengend Snippet: (A.) Western blot of whole cell lysate of uninfected, LD-R and LD-S infected MФs at 24hrs pi showing the expression of precursor-ADAM10 (p-ADAM10, ~90 kDa) and mature-ADAM10 (m-ADAM10, ~64 kDa) keeping β-actin acts as a housekeeping control. (B.i.) Western blot of whole cell lysate showing furin expression of DFO treated MФs, LD-S, and LD-R infected MФs at 24hrs pi. (B.ii.) 40X Confocal images showing the localization of furin (red) in uninfected, LD-S, and LD-R infected MФs. (B.iii.) Western blot of furin expression in the supernatant fraction of same experiments sets as B.i. (C.) Confocal images showing Furin (red, 3 rd panel and edges,6 th panel), ADAM10 (purple), and CD11b (green) colocalization in LD-S and LD-R infected MФs at 24hrs pi. One representative small nucleus of LD has been marked in ( * ) to show the infected MФs. The scale bar indicates 20µm. (D.) Scheme showing Furin maturation starting from Endoplasmic reticulum (ER) where it remains inactivated (observed in LD-S infected MФs at 24hrs pi) to getting shed as an active enzyme (observed in LD-R infected MФs at 24hrs pi) to cleave prodomain of p-ADAM10 rendering it activated (m-ADAM10), which in turn cleaves extracellular domain of SIRPα in LD-R infected MФs at 24hrs pi.

Article Snippet: Antibody against ADAM10 was purchased from Abclonal (A10438) and Invitrogen (MA5-23867).

Techniques: Western Blot, Infection, Expressing

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 1. Histological analysis of primary RB tumors. ADAM17 (a) and ADAM10 (b) expression in exemplary paraffin sections of human RB patient tumors. Immunohistochemistry was detected by diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 600 µM and 200 µM (magnified insets).

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 2. Verification of ADAM10 and ADAM17 knockdown efficiency. Efficient stable, lentiviral ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) double knock- down was verified by quantitative real-time PCR (a,d) and western blot analyses in Y79 (b,c) and WERI-Rb1 cells (e,f). Indicated intensity ratios relative to ß-actin, used as a loading control, were cal- culated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control, Software

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 3. Effects of ADAM10/17 single and double knockdown on cell viability and proliferation of RB cells. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases cell viability and proliferation levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by WST-1 assays (a), and BrdU stains (b,c). Values are means of three independent experiments ± SEM. ns p > 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Control

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 4. Effects of ADAM10/17 single and double knockdown on cell growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown decreases growth of Y79 (a,c,e) and WERI-Rb1 cells (b,d,f) compared to control cells (ctr) as revealed by growth curve analysis. Values are means of three independent experiments ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Control

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 5. Effects of ADAM10/17 single and double knockdown on apoptosis and anchorage in- dependent growth of RB cell lines. Stable ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) knockdown increases caspase mediated apoptosis levels of Y79 and WERI-Rb1 cells compared to control cells (ctr) as revealed by DAPI cell counts (a) and caspase3/7 assays (b). Both RB cell lines show significantly reduced colony formation capacity (c) and colony sizes (d,e) after ADAM10 and ADAM 17 single or double knockdown as revealed by soft agarose assays. Values are means of three independent experiments ± SEM. ns > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Control

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 6. Effect of lentiviral ADAM10/17 single and double knockdown on tumor formation of RB cell lines in vivo as revealed by chick chorioallantoic membrane (CAM) assays. Photographs of CAM tumors in situ (upper row) and ruler measurements (in cm) of excised CAM tumors (lower row) 7 days after inoculating the Y79 (a) and WERI-Rb1 (b) RB cell onto the CAM. Quantification of weight (c) and size (d) of CAM tumors developing from ADAM10 (shADAM10), ADAM17 (shADAM17) and ADAM10/17 (shADAM10/17) depleted Y79 and WERI-Rb1 cell lines in comparison the control cells (ctr). Values are means of at least three independent experiments ± SEM. ns p > 0.05; * p < 0.05; *** p < 0.001; and **** p < 0.0001 statistical differences compared to the control group calculated by Student’s t-test. (e) GFP antibody staining of stably GFP expressing Y79 and WERI-Rb1 cells in CAM tumor paraffin sections. Immunohistochemistry was revealed using diaminobenzidine (brown signal) and hematoxylin counterstaining (blue nuclei staining). Scale bars, 2 mm and 300 µM (magnified insets). #: blood vessel in CAM mesoderm, *: GFP-positive (GFP+) RB tumor cells, arrowheads: solid GFP-positive RB tumor mass.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, In Vivo, Membrane, In Situ, Comparison, Control, Staining, Stable Transfection, Expressing, Immunohistochemistry

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 7. Effects of stable, lentiviral ADAM10/17 single or double knockdown on RB cell migration in vivo. (a) Desmin stains of blood vessels (red) in representative CAM whole mounts showing

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Migration, In Vivo

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 8. Analysis of L1CAM ectodomain shedding after ADAM10/17 single and double knockdown in Y79 and WERI-Rb1 cell lines. Western blot analysis of ADAM mediated L1CAM ectodomain (L1– 200) shedding in cell culture supernatant of Y79 and WERI-Rb1 cells 72 h after ADAM10 (shADAM10), ADAM17 (shADAM17) single and ADAM10/17 (shADAM10/17) double knockdown in comparison to control cells (ctr). Representative western blots of L1 shedding upon ADAM knockdown (a) and quantification of the L1CAM ectodomain expression reveals a significant reduction of L1-200 shed- ding after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1 (b). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. * p < 0.05 and ** p < 0.01 statistical differences compared to the control (ctr) group calculated by Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Knockdown, Western Blot, Cell Culture, Comparison, Control, Expressing, Software

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 9. AKT and phospho-AKT (pAKT) expression levels after shRNA-mediated ADAM10/17 single (shADAM10 and shADAM17) or double knockdown (shADAM10/17) in the RB cell lines Y79 and WERI-Rb1 as revealed by western blot analysis. Representative western blots showing AKT and p-AKT expression levels (a,e) and quantification of the p-AKT/AKT ratio (b–d,f–h) after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1. Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. ns p > 0.05 and * p < 0.05 statistical differences compared to the control group calculated by paired Student’s t-test.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Expressing, shRNA, Knockdown, Western Blot, Control, Software

Journal: International journal of molecular sciences

Article Title: ADAM10 and ADAM17-Novel Players in Retinoblastoma Carcinogenesis.

doi: 10.3390/ijms232012621

Figure Lengend Snippet: Figure 11. Correlation of miRNA-145, miRNA-152 and miRNA-365 expression with ADAM10 (a–c) and ADAM17 (d–f) expression levels in 15 individual RB patients as revealed by real-time PCR.

Article Snippet: Immunostaining was performed using a rabbit monoclonal antibody against ADAM10 (#14194; Cell Signaling Technology, Danvers, USA), ADAM17 (PA5-17079; Invitrogen, Darmstadt, Germany), and GFP (2955S; Cell Signaling Technology, Danvers, USA) at a dilution of 1:100 (ADAM10), 1:100 (ADAM17) and 1:200 (GFP) in antibody dilution buffer provided in the Vectastain kit (Biozol, Eching, Germany) at 4 ◦C overnight in a humidified chamber.

Techniques: Expressing, Real-time Polymerase Chain Reaction