Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative Western blot for PI(4,5)P 2 in total cell extracts of RM+ N/TERTs and KGM N/TERTs. Equal protein loading was confirmed by immunoblotting for Tubulin. ( B ) Representative Western blot for PI(4,5)P 2 in total cell extracts of KGM N/TERT grown in low calcium and then switched to high calcium concentrations for up to 8 days. Calcium induced keratinocyte differentiation was confirmed by Western blotting for Loricrin. Equal protein loading was confirmed by immunoblotting for tubulin. ( C ) Representative Western blot for PI(4,5)P 2 in total cell extracts of empty vector positive KGM N/TERT-pLXSN or KGM N/TERT-8E6. Difference in intensity of PI(4,5)P 2 bands in KGM N/TERTs between Figure and is due to 80 μg total protein and long exposure of blot to film versus 40 μg total protein and short exposure. Equal protein loading was confirmed by immunoblotting for tubulin. ( D ) Representative Western blot for PI(4,5)P 2 in total cell extracts of KGM N/TERT-pLXSN and KGM N/TERT-8E6 treated with skimmed milk containing either PI(4,5)P 2 specific antibodies (top) or in milk containing100 mM Neomycin (bottom). ( E ) Total cell extracts of KGM N/TERT-8E6 were treated with 0, 5, 10 or 20 mU Phospholipase C (PLC). ( F ) Quantification of phosphoinositide levels in HPV8-E6 expressing keratinocytes. Relative amounts of PIP and PIP 2 levels in KGM N/TERT-pLXSN and KGM N/TERT-8E6 cells. Bars present phosphoinositide values normalized to phosphatidylcholine (PC) levels (PIP x /PC). Data are presented as mean ± SD ( n = 3). ( G – H ) Effect of E6 expression on the relative distribution of PIP ( G ) and PIP 2 ( H ) molecular species. Lipid species are annotated based on their fatty acyl composition x:y, with x = total number of C atoms in both fatty acyl chains, and y = total number of double bonds in both fatty acyl chains). Data are normalized to 100% within each lipid class and are displayed as mean ± SD ( n = 3).

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Western Blot, Plasmid Preparation, Expressing

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative immunocytochemical staining of PI(4,5)P 2 in KGM N/TERT-pLXSN and KGM N/TERT-8E6 showing an increase in nuclear PI(4,5)P 2 levels in HPV8-E6 positive cells (blue: DAPI; green: PI(4,5)P 2 ). ( B ) Representative immunofluorescence staining image of PI(4,5)P 2 on organotypic skin cultures based on de-epidermalized human dermis as matrix, which was repopulated with wt PHK or PHK coding for the empty retroviral vector pLXSN or pLXSN-8E6. The organotypic cultures were grown for 14 days at the air-liquid interphase, followed by fixing and embedding in paraffin.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Staining, Immunofluorescence, Plasmid Preparation

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Skin biopsies were taken from FVB/n-wt ( n = 4) and K14-HPV8-E6 ( n = 4) mice out of the UV irradiated skin area 13 days or 24 days following UV treatment. Additionally, skin biopsies were taken from non-irradiated ( n = 4) FVB/n-wt and K14-HPV8-E6 ( n = 4) mice, respectively. Paraffin sections were stained for PI(4,5)P 2 (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement membrane zone; d: dermis; e: epidermis). The magnified image of wt skin 24 days following UV treatment demonstrates unspecific staining in the cornified cell layer.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Irradiation, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Representative immunofluorescence staining for PI(4,5)P 2 in normal human skin, skin SCC of the normal population (SCC/NP) and HPV8 positive EV-SCC. Histology of the EV lesions is shown by HE staining (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement-membrane zone; d: dermis; e: epidermis). ( B ) Representative immunofluorescence staining for PI(4,5)P 2 in malignant melanoma (MM, n = 5), Merkel cell polyomavirus positive Merkel cell carcinoma (MCC, n = 5) and basal cell carcinoma (BCC, n = 5) (blue: DAPI; green: PI(4,5)P 2 ; dashed line: basement-membrane zone; d: dermis; e: epidermis).

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Representative immunofluorescence staining for PI(4,5)P 2 in normal cervix ( n = 3), and HPV16 positive CIN I ( n = 3), CIN II ( n = 5), CIN III ( n = 11) and cervical cancer (CC, n = 3) (blue: DAPI; green: PI(4,5)P 2 ). Histology of the tissue is shown in the HE staining images.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Immunofluorescence, Staining

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Extracts from C33a cells, transiently transfected with either empty vectors or plasmids coding for Flag-8E6-wt, -P31S, -P38S, -F41Y, -W63T, -L61A/W63A, -V68A, -E83A, -D96A, -D126A, -K136N, -K149A were incubated with M2-FLAG-agarose. Co-immunoprecipitated of MAML1 and 10% of the input extracts were subjected to Western blots with specific antibodies. Expression of HPV8-E6 was confirmed by a Western blot for the Flag-tagged HPV8-E6 protein. Equal protein loading was confirmed by immunoblotting for tubulin. ( B ) N/TERTs were transduced with retroviruses encoding for either the empty vector pLXSN-HA or HA-tagged HPV8-E6wt, -L61A, -W63T or L61A/W63A. PI(4,5)P 2 was detected with specific antibodies. Expression of HPV8-E6 was confirmed by Co-IP of the HA-tagged proteins. Equal protein loading was confirmed by immunoblotting for tubulin.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Transduction, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: ( A ) Quantification of PIP4KIIα, β, γ and PIP5KIα, β, γ mRNA expression by RT-qPCR in KGM N/TERTs and RM+ N/TERTs ( n = 3 independent experiments measured in duplicates). Data were normalized to the HPRT1 mRNA levels and are presented as mean ± SEM. The relative gene expression level of RM+ N/TERTs was set as 1 ( ** p < 0.01; *** p < 0.001; nq: not quantifiable; ns: not significant). ( B ) Quantification of PIP4KIIα, β, γ and PIP5KIα, β, γ mRNA expression by RT-qPCR in KGM N/TERT-pLXSN and KGM N/TERT-8E6 ( n = 3 independent experiments measured in duplicates). Data were normalized to HPRT1 mRNA levels and are presented as mean ± SEM. The relative gene expression level of KGM N/TERT-pLXSN was set as 1 ( * p < 0.05; ** p < 0.01; nq: not quantifiable). ( C ) Extracts from C33a cells, transiently transfected with expression vectors for empty vector or Flag-HPV8-E6wt were incubated with M2-FLAG-agarose. Co-immunoprecipitated OCRL-1 and 10% of the input extracts were detected by Western blot with specific antibodies ( n = 5). The expression of HPV8-E6 was confirmed by a Western blot against the Flag tag. Equal protein loading was confirmed by immunoblotting for tubulin. ( D ) Schematic diagram of the nitrocellulose membrane with immobilized phospholipids in 100 picomole spots (PIP-strip; left image); LPA: lysophosphatidic acid; LPC: lysophosphocholine; PI: phosphatidylinositol; SIP: sphingosine-1-phosphate; PA: phosphatidic acid. Representative images of PIP-strips incubated with total cell extracts of C33a cells transiently transfected with pXJ41-Flag (middle image) or pXJ41-8E6-Flag (right image). Membranes were washed and lipid-bound Flag-8E6 was detected with monoclonal anti-Flag antibody. ( E ) The left image shows the PI(4,5)P 2 pathway flow in a normal keratinocyte. The right image schematically summarizes HPV8-E6 targets involved in PI(4,5)P 2 metabolism. The figure also shows that the PIP5K pathway is the major and PIP4K the minor route of PI(4,5)P 2 synthesis.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Incubation, Immunoprecipitation, Western Blot, FLAG-tag, Stripping Membranes

Journal: Oncotarget

Article Title: Phospholipidation of nuclear proteins by the human papillomavirus E6 oncoprotein: implication in carcinogenesis

doi: 10.18632/oncotarget.26140

Figure Lengend Snippet: Extracts from KGM N/TERT-pLXSN and KGM N/TERT-8E6 were incubated with A/G-agarose coupled with either CAND1, SND1 or normal IgG-mouse antibodies. Co-immunopecipitated PI(4,5)P 2 and 10% of the input extracts were detected by Western blot with specific antibodies against PI(4,5)P 2 . Total levels of PI(4,5)P 2 , CAND1 and SND1 were detected using specific antibodies. Equal protein loading was confirmed by immunoblotting for tubulin.

Article Snippet: After the membrane had been blocked with 5 % skimmed milk in TBST (10 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.05 % Tween 20) for 1h, the blots were probed with antibodies to Loricrin (ab24722, Abcam, Cambridge, UK), PI(4,5)P 2 (clone 2C11, Santa Cruz, Heidelberg, Germany) [ ] and tubulin (clone YL1/2, Abcam), which was used as loading control.

Techniques: Incubation, Western Blot

Journal: Nature Communications

Article Title: An acquired phosphatidylinositol 4-phosphate transport initiates T-cell deterioration and leukemogenesis

doi: 10.1038/s41467-022-32104-7

Figure Lengend Snippet: a Heatmap of PM lipid changes in T-cells of ORP4L KI and littermate wild-type mice ( n = 3 mice). b , c PI(4)P ( b ) and PI(4,5)P 2 ( c ) contents in T-cells of ORP4L KI and littermate wild-type mice. Scale bar, 10 μm. The right panel indicates quantitation of relative fluorescence intensity. Black triangles present in dividual data points for n = 16 (panel b) and n = 20 (panel c ) cells from one mouse. The same experiment was repeated twice in T-cells from two mice. d Schematic representation of PIPs metabolism in the PM. e Time course of PI(4,5)P 2 probe GFP-PH PLCδ1 at the PM of ORPL4 KI T-cells subjected to PI4KIIIα or PIP5KB knockdown. The right panel indicates quantification of relative fluorescence intensity changes of GFP-PH PLCδ1 at the PM. Scale bar, 5 μm. f Time course of the PI(4)P probe GFP-P4M SidM at PM and Golgi complex of ORPL4 KI T-cells subjected to PI4KIIIα knockdown. The right panel indicates quantification of relative fluorescence intensity changes of GFP-P4M SidM at the PM (upper) and Golgi (lower). Scale bar, 5 μm. g Time course of the PI(4,5)P 2 probe GFP-PH PLCδ1 at the PM of ORPL4 KI T-cells subjected to PI4KIIα knockdown. The right panel indicates quantification of relative fluorescence intensity changes of GFP-PH PLCδ1 at the PM. Scale bar, 5 μm. In panel ( e – g ), the data are presented as Mean ± SD ( n = 10 cells from one mouse). The same experiments were repeated twice in T-cells from two mice. In each box plot of ( b , c ), the central mark indicates the median, the bottom and top edges of the box indicate the interquartile range, and the whiskers represent the maximum and minimum point. Two-tailed unpaired t -test with a confidence interval of 95% was used to compute statistics. P -values are indicated in the figures. Source data are provided as a Source Data file.

Article Snippet: Primary antibodies of PI(4)P (Enzo Life Sciences, Cat# Z-P004, 1:100), PI(4,5)P 2 (Enzo Life Sciences, Cat# ADI-915-062, clone: KT10, 1:100) and PI(3,4,5)P 3 (Enzo Life Sciences, Cat# Z-P345, 1:100) diluted in PBS containing 5% NGS were incubated at 4 °C overnight.

Techniques: Quantitation Assay, Fluorescence, Two Tailed Test