Journal: Cancers

Article Title: JAK2 Inhibition Impairs Proliferation and Sensitises Cervical Cancer Cells to Cisplatin-Induced Cell Death

doi: 10.3390/cancers11121934

Figure Lengend Snippet: JAK2 is aberrantly phosphorylated in cervical disease and HPV+ cervical cancer cells. ( A ) Representative western blots from cytology samples of CIN lesions of increasing grade analysed for phosphorylated JAK2 and total JAK2 expression. GAPDH served as a loading control. ( B ) Scatter dot plot of densitometry analysis of a panel of cytology samples. Twenty samples from each clinical grade (neg, CIN I–III) were analysed by western blot and densitometry analysis was performed using ImageJ. ( C ) Representative western blot of from six cervical cancer cell lines—two HPV- (C33A and Dotc2 4510), two HPV16+ (SiHa and CaSKi) and HPV18+ (SW756 and HeLa)—for the expression of phosphorylated and total JAK2. GAPDH served as a loading control. Data are representative of at least three biological independent repeats. ( D ) Densitometry analysis from C. Error bars represent the mean ± standard deviation of a minimum of three biological repeats. ns- not significant, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: Total protein was resolved by SDS Polyacrylamide gel electrophoresis (SDS-PAGE) (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences, Little Chalfont, UK) and probed with antibodies specific for phospho-STAT3 (S727) (ab32143, Abcam, Cambridge, UK), phospho-STAT3 (Y705) (9131, Cell Signalling Technology, Danvers, MA, USA (CST)), STAT3 (124H6:9139, CST), phospho-STAT5 (Y694) (D47E7; 4322, CST), STAT5 (D3N2B; 25656, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), HPV18 E6 (G-7, Santa Cruz Biotechnology (SCBT)), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (ab70, abcam), HPV 16 E7 (ED17, SCBT), Cyclin D1 (ab134175, Abcam), p21 (2947, CST), GAPDH (G-9, SCBT), PARP-1 (9542, CST) and Bcl- xL (2764, CST).

Techniques: Western Blot, Expressing, Control, Standard Deviation

Journal: Cancers

Article Title: JAK2 Inhibition Impairs Proliferation and Sensitises Cervical Cancer Cells to Cisplatin-Induced Cell Death

doi: 10.3390/cancers11121934

Figure Lengend Snippet: STAT5 is a downstream mediator of JAK2 in HPV+ cervical cancer cells. ( A ) Representative western blots from cytology samples of CIN lesions of increasing grade analysed for phosphorylated STAT5 and total STAT5 expression. GAPDH served as a loading control. ( B ) Scatter dot plot of densitometry analysis of a panel of cytology samples. Twenty samples from each clinical grade (neg, CIN I-III) were analysed by western blot and densitometry analysis was performed using ImageJ. ( C ) Representative western blot of from six cervical cancer cell lines—two HPV- (C33A and Dotc2 4510), two HPV16+ (SiHa and CaSKi) and HPV18+ (SW756 and HeLa)—for the expression of phosphorylated and total STAT5. GAPDH served as a loading control. ( D ) Densitometry analysis from C. ( E ) Representative western blot of ruxolitinib dose response in HeLa and CaSKi cells after 48 h. GAPDH served as a loading control. Densitometry analysis is in . ( F ) Representative western blot of HeLa and CaSKi cells after transfection of a pool of four specific JAK2 siRNA for 72 h. GAPDH served as a loading control. Data are representative of at least three biological independent repeats. Densitometry analysis is in . Error bars represent the mean ± standard deviation of a minimum of three biological repeats. ns- not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (Student’s t -test).

Article Snippet: Total protein was resolved by SDS Polyacrylamide gel electrophoresis (SDS-PAGE) (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences, Little Chalfont, UK) and probed with antibodies specific for phospho-STAT3 (S727) (ab32143, Abcam, Cambridge, UK), phospho-STAT3 (Y705) (9131, Cell Signalling Technology, Danvers, MA, USA (CST)), STAT3 (124H6:9139, CST), phospho-STAT5 (Y694) (D47E7; 4322, CST), STAT5 (D3N2B; 25656, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), HPV18 E6 (G-7, Santa Cruz Biotechnology (SCBT)), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (ab70, abcam), HPV 16 E7 (ED17, SCBT), Cyclin D1 (ab134175, Abcam), p21 (2947, CST), GAPDH (G-9, SCBT), PARP-1 (9542, CST) and Bcl- xL (2764, CST).

Techniques: Western Blot, Expressing, Control, Transfection, Standard Deviation

Journal: Cancers

Article Title: JAK2 Inhibition Impairs Proliferation and Sensitises Cervical Cancer Cells to Cisplatin-Induced Cell Death

doi: 10.3390/cancers11121934

Figure Lengend Snippet: Phosphorylated JAK2 positively correlates with STAT3 and STAT5 phosphorylation in cervical disease and HPV+ cervical cancer cells. ( A ) Representative western blots from cytology samples of CIN lesions of increasing grade analysed for phosphorylated and total JAK2, STAT3 and STAT5 expression. GAPDH served as a loading control. ( B ) Scatter dot plot of densitometry analysis of a panel of cytology samples. Samples from CIN 3 were analysed by western blot and densitometry analysis was performed using ImageJ. Graphs represent the correlation between phosphorylated JAK2 and both phosphorylated STAT3 and STAT5 from matched cytology samples. ( C ) Representative western blot of from six cervical cancer cell lines—two HPV- (C33A and Dotc2 4510), two HPV16+ (SiHa and CaSKi) and HPV18+ (SW756 and HeLa)—for the expression of phosphorylated and total JAK2, STAT3 and STAT5. GAPDH served as a loading control. ( D ) Scatter Dot plot show correlation between phosphorylated JAK2 and both phosphorylated STAT3 and STAT5 in cervical cancer cell lines.

Article Snippet: Total protein was resolved by SDS Polyacrylamide gel electrophoresis (SDS-PAGE) (10–15% Tris-Glycine), transferred onto Hybond nitrocellulose membrane (Amersham biosciences, Little Chalfont, UK) and probed with antibodies specific for phospho-STAT3 (S727) (ab32143, Abcam, Cambridge, UK), phospho-STAT3 (Y705) (9131, Cell Signalling Technology, Danvers, MA, USA (CST)), STAT3 (124H6:9139, CST), phospho-STAT5 (Y694) (D47E7; 4322, CST), STAT5 (D3N2B; 25656, CST), Phospho-JAK2 (Y1007/1008) (3776, CST), Total JAK2 (3230, CST), HPV18 E6 (G-7, Santa Cruz Biotechnology (SCBT)), HPV18 E7 (8E2, Abcam (ab100953), HPV 16/18 E6 (ab70, abcam), HPV 16 E7 (ED17, SCBT), Cyclin D1 (ab134175, Abcam), p21 (2947, CST), GAPDH (G-9, SCBT), PARP-1 (9542, CST) and Bcl- xL (2764, CST).

Techniques: Phospho-proteomics, Western Blot, Expressing, Control

Journal: mBio

Article Title: Development of DNA Vaccine Targeting E6 and E7 Proteins of Human Papillomavirus 16 (HPV16) and HPV18 for Immunotherapy in Combination with Recombinant Vaccinia Boost and PD-1 Antibody

doi: 10.1128/mBio.03224-20

Figure Lengend Snippet: Characterization of HPV16 E7 and HPV18 E6 antigen presentation by cells transfected with the various DNA vaccines. 293-D b or 293 K b cells were transfected with either pBI-10.1, pBI-11, or pBI-12 DNA vaccine or mock transfected using Lipofectamine 2000. Twenty-four hours later, these cells were harvested and cocultured with either murine H-2D b -restricted HPV16 E7 (aa 49 to 57) peptide-specific CD8 + T cells or murine H-2K b -restricted HPV 18 E6 (aa 67 to 75) peptide-specific CD8 + T cells in the presence of GolgiPlug. The cells were then harvested, and IFN-γ intracellular staining was performed to determine the activation of HPV16 E7 or HPV18 E6 antigen-specific CD8 + T cells. (A) Bar graph to summarize flow cytometry data for activation of HPV16 E7-specific CD8 + T cells. (B) Bar graph to summarize the flow cytometry data for the activation of HPV18 E6-specific CD8 + T cells.

Article Snippet: PE-conjugated HPV16 E7 (aa 49 to 57) peptide-loaded H-2D b tetramers and PE-conjugated HPV18 E6 (aa 67 to 75) peptide-loaded H-2K b tetramers were purchased from MBL International (Japan).

Techniques: Transfection, Staining, Activation Assay, Flow Cytometry