Journal: Scientific Reports

Article Title: Centrosomal P4.1-associated protein (CPAP) positively regulates endocytic vesicular transport and lysosome targeting of EGFR

doi: 10.1038/s41598-021-91818-8

Figure Lengend Snippet: EGFR is detected in CPAP overexpression associated vesicular structures and higher levels of CPAP diminish the cellular levels of EGFR. ( A ) HEK293T and HeLa cells expressing GFP-CPAP under doxy-inducible promoter were stained using anti-EGFR antibody, 24 h after induction of protein expression with doxy (1 μg/ml). Maximum Z-projection images (left panel) and single Z planes (right panel) of representative images from at least 3 independent experiments are shown. White arrows show examples of co-localization. ( B ) GFP-CPAP expressing HeLa cells were treated with Alexa fluor 555-conjugated EGF for different time-points and subjected to confocal microscopy to detect ligand-bound EGFR. Maximum Z-projection (left panel) and single Z-plane (right panel) of representative images from at least three independent experiments are shown. ( C ) HeLa cells stably expressing GFP-CPAP under doxy-inducible promoter were left untreated or treated with varying amounts of doxy for 24 h and subjected to WB to detect GFP-CPAP, EGFR and β-actin. Representative IB images of cell lysates (upper panel) and densitometry analysis (mean of three independent experiments) of EGFR levels at different time-points relative to 0 min time-points are shown (lower panel). P -value by paired t -test. ( D ) Untreated and Doxy treated cells were surface stained using fluorchrome linked anti-EGFR antibody and examined by FACS. Representative overlay graph with MFI values (upper panel) and mean MFI values of cells (three independent experiments) exposed to different doses of Doxy for 24 h (lower panel) are shown. Original scanned x-ray film images or ChemiDoc imager acquisition images of relevant IB panels are included as Supplemental Fig. . All images were processed using ImageJ software version 1.53 ( https://imagej.nih.gov/ij/ ). Flow cytometry data was processed using FlowJo software version 10.0 ( https://www.flowjo.com/solutions/flowjo ). All results are representative of at least three experiments that produced similar trend in outcomes or include cumulative values from three independent experiments. GraphPad Prism software version 9 ( https://www.graphpad.com/scientific-software/prism/ ) was used for determining p-values.

Article Snippet: RNAi resistant construct for expressing GFP-CPAP and mCherry-CPAP were kindly provided by Dr. Pierre Gonczy, Swiss Institute for Experimental Cancer Research, Switzerland and the CPAP siRNA targeting sequence has been reported earlier .Primary antibodies used in this study: anti-CPAP (Proteintech; cat#11517-1-AP),-GFP (Proteintech; cat#50430-2-AP) -actin (Proteintech; cat# HRP-600008), -EGFR (Santa Cruz Biotech;sc-373746), phospho EGFR (cell signal; cat# 2234; phospho Akt (Proteintech:, 66444-1-Ig); -CD63 (BD Biosciences; cat# 556019), -EEA1 (Bethyl labs; cat# A301-896A),EEA1 (BD biosciences; 610456)LAMP (DSHB; cat#H4B4).

Techniques: Over Expression, Expressing, Staining, Confocal Microscopy, Stable Transfection, Software, Flow Cytometry, Produced

Journal: Scientific Reports

Article Title: Centrosomal P4.1-associated protein (CPAP) positively regulates endocytic vesicular transport and lysosome targeting of EGFR

doi: 10.1038/s41598-021-91818-8

Figure Lengend Snippet: CPAP depletion increases surface and cellular levels of EGFR. HeLa cells stably expressing control-shRNA or CPAP-shRNA were examined for CPAP depletion and EGFR levels by WB ( A ) and surface EGFR levels by FACS ( B ) Panel ( A ) shows representative IB images (left panel) and mean densitometric values (relative to actin; right panel). Panel ( B ) shows representative overlay graph (left panel) and MFI values. ( C ) Cells were subjected to serum starvation overnight, treated with cycloheximide (chx) for 1 h, followed by EGF for 1 h on ice, washed and incubated at 37 °C for indicated durations, and subjected to FACS analysis to detect surface levels of EGFR after staining using anti-EGFR antibody. Representative overlay graphs for each time-point (upper panel), mean MFI values (lower left panel) and EGF treatment induced fold changes in EGFR specific MFI values, relative to 0 min time-point, (lower right panel) are shown. ( D ) Cells stimulated using EGF, as described in panel C, were subjected to IB to detect EGFR and β-actin. Representative IB images (upper panel) and the densitometric quantification of band intensities (lower left panel) and mean ± SD of values (lower right panel) are shown. ( E ) Cells were stimulated using EGF for different durations as described in panel C and subjected to IB to detect total EGFR, phospho-EGFR(Y1068), phospho-AKT(S473) and β-actin. Representative IB images (upper panel) and the densitometric quantification of band intensities (lower panels) are shown. ( F ) Cell were subjected to serum starvation and treated with chx as described for panel C, followed by addition of fresh warm medium containing EGF (no pre-binding of EGF before internalization), incubated for indicated durations at 37 °C, and subjected to IB to detect phospho-AKT (S473). IB images (upper panel) and densitometric values (lower panel) are shown. EGFR, phospho-EGFR and phospho-AKT dynamics under another experimental condition are shown in Supplemental Fig. . ( G ) HeLa cells were treated with control-siRNA, CPAP-siRNA1 or CPAP-siRNA2 for 72 h and subjected to IB to detect cellular CPAP, EGFR and β-actin. Representative IB images (upper panel) and mean densitometric values (lower panels) are shown. ( H ) siRNA treated cells were subjected to FACS to detect surface levels of EGFR and representative overlay graph with MFI values is shown. ( I ) siRNA treated cells that exogenously express siRNA-resistant GFP-CPAP under doxy-inducible promoter, were treated with EGF as described for panel D and subjected to IB analysis to detect GFP-CPAP, EGFR and β-actin. GFP-CPAP expressing cells were treated with doxy for 24 h before initiating this assay. Representative IB images (upper panel) and densitometric values (lower panel) are shown. Original scanned x-ray film images or ChemiDoc imager acquisition images of relevant IB panels are included as Supplemental Fig. . Flow cytometry data was processed using FlowJo software version 10.0 ( https://www.flowjo.com/solutions/flowjo ). All results are representative of at least three experiments that produced similar trend in outcomes or include cumulative values from three independent experiments. P -value by paired t -test. GraphPad Prism software version 9 ( https://www.graphpad.com/scientific-software/prism/ ) was used for determining p -values.

Article Snippet: RNAi resistant construct for expressing GFP-CPAP and mCherry-CPAP were kindly provided by Dr. Pierre Gonczy, Swiss Institute for Experimental Cancer Research, Switzerland and the CPAP siRNA targeting sequence has been reported earlier .Primary antibodies used in this study: anti-CPAP (Proteintech; cat#11517-1-AP),-GFP (Proteintech; cat#50430-2-AP) -actin (Proteintech; cat# HRP-600008), -EGFR (Santa Cruz Biotech;sc-373746), phospho EGFR (cell signal; cat# 2234; phospho Akt (Proteintech:, 66444-1-Ig); -CD63 (BD Biosciences; cat# 556019), -EEA1 (Bethyl labs; cat# A301-896A),EEA1 (BD biosciences; 610456)LAMP (DSHB; cat#H4B4).

Techniques: Stable Transfection, Expressing, shRNA, Incubation, Staining, Binding Assay, Flow Cytometry, Software, Produced

Journal: Molecular medicine reports

Article Title: Knockdown of long noncoding RNA DLEU1 suppresses the progression of renal cell carcinoma by downregulating the Akt pathway.

doi: 10.3892/mmr.2019.10705

Figure Lengend Snippet: Figure 4. DLEU1 knockdown inhibits the Akt pathway and disrupts EMT in renal cell carcinoma cells. (A) Following transfection for 48 h, western blot analysis was conducted to determine alterations in the expression of Akt pathway‑associated proteins. (B) Alterations in the expression of marker proteins (E‑cadherin, N‑cadherin and vimentin) in the EMT process were detected by western blotting. Data are presented as the means ± standard deviation (n=3). Results were obtained from three independent experiments. *P<0.05 vs. NC. Akt, protein kinase B; DLEU1, deleted in lymphocytic leukemia 1; EMT, epithelial‑mesenchymal transition; NC, negative control; p, phosphorylated; P70S6K, P70S6 kinase; si/siRNA, small interfering RNA.

Article Snippet: The primary antibodies used were as follows: antiBcl-2 (cat. no. 60178-1-ig; ProteinTech Group, inc.), Bax (cat. no. 50599-2-ig; ProteinTech Group, inc.), total caspase-3 (cat. no. 66470-2-ig; ProteinTech Group, inc.), cleaved caspase-3 (cat. no. 25546-1-aP; ProteinTech Group, inc.), total caspase-9 (cat. no.9502; cell Signaling Technology, inc.), cleaved caspase-9 (cat. no. 10380-1-aP; ProteinTech Group, inc.), akt (cat. no. 9272; cell Signaling Technology, inc.), phosphorylated (p)-akt (cat. no. 66444-1-ig; ProteinTech Group, inc.), cyclin d1 (cat. no. 60186-1-ig; ProteinTech Group, inc.), P70S6K (cat. no. 14485-1-aP; ProteinTech Group, inc.) and GaPdH (cat. no. cat. no. 60004-1-ig; ProteinTech Group, inc.).

Techniques: Knockdown, Transfection, Western Blot, Expressing, Marker, Standard Deviation, Negative Control, Small Interfering RNA