Journal: mBio

Article Title: An mRNA-encoded scFv antibody targeting the helix-α3 of HPV18 E7 oncoprotein as a novel antiviral strategy

doi: 10.1128/mbio.02627-25

Figure Lengend Snippet: Characterization of EC₅₀ and binding properties of six anti-HPV18E7 mAbs. ( A ) Antibody against HPV18E7 binding ability analysis by ELISA. Recombinant HPV18 E7 protein (1 μg/mL) was coated onto plates. Commercial antibody F-7 as a positive control. ( B ) Competition matrix of six anti-E7 mAbs. Yellow box and black lines indicate mAbs that compete with one another. The value contained in each box is 1 − a given mAb/control across duplicate; a value <0.5 was taken to be negative binding of the detection mAb. Non-competing pairs are shown in green. ( C ) Kinetic parameters for the binding of each antibody to HPV18E7, as determined by surface plasmon resonance analysis. ( D ) Six Anti-E7 mAbs could recognize HPV18 E7 protein in the HPV18-positive HeLa cells by immunofluorescence. Scale bar = 20 um. Commercial antibody F-7 as a positive control. No primary antibody group as a negative control. The rectangular artifacts on the right side of F-7, 14E5, and 17F2 are the result of a technical issue that occurred during file conversion. ( E ) Quantitative analysis of panel D . The signal targeting E7 (the green signal in panel D) was quantitatively analyzed using ImageJ and normalized. Y normalization = mean density (experimental group)/average of mean density (negative control). A pairwise difference analysis was performed between the experimental and control groups using t -test, n = 3, * P < 0.05, ** P < 0.01, and *** P < 0.001. ( F ) Western blot showed that Anti-E7 mAbs could recognize endogenously expressed HPV18 E7 protein in the HPV18-positive HeLa cells. β-actin as the control. This figure represents spliced gel segments combined for imaging.

Article Snippet: The primary antibodies were monoclonal antibodies (2E8, 5C5, 8A7, 9A5, 14E5, and 17F2) and anti-HPV18E7 (Santa Cruz, no. sc-365035; working concentration, 4 μg/mL), anti-HA-HRP (Abcam, no. ab128131, 1:1,000), anti-β-actin-HRP (ProteinTech, HRP-60008, 1:5,000), anti-Rb (CST, no.9309S, 1:100), anti- phospho-Rb (CST, no.8516S, 1:100).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Positive Control, Control, SPR Assay, Immunofluorescence, Negative Control, Western Blot, Imaging

Journal: International Journal of Cancer

Article Title: Th17 cells favor migration and invasiveness of cervical cancer cells under hypoxia in an IGF2BP2 ‐dependent manner

doi: 10.1002/ijc.70340

Figure Lengend Snippet: Th17 cells induce CAIX, hexokinase II and SLC2A1 mRNA expression in an oxygen‐dependent manner. SiHa (left), SW756 (middle) and Hela (right) were stimulated with medium (black bars), rhIL‐17 (100 ng/mL, red bars) and conditioned media of in vitro generated Th17 cells (CMTH17, 20%, blue bars) and incubated under normoxic (21% O 2 , striped bars) or hypoxic oxygen conditions (1% O 2 ). After 24 h, the cells were analyzed for (A) CAIX, (B) hexokinase II and (C) SLC2A1 expression by qRT‐PCR analysis and normalized to RPL13A housekeeping gene expression. The quotient of the gen of interest/RPL13A of medium‐ stimulated cells incubated by 21% oxygen was set at 1. Shown are the results (mean + SD) from six independent stimulations. Asterisks (* p < .05, ** p < .01, *** p < .001, **** p < .0001) represent statistical significances. The p ‐value according to the nonparametric Mann–Whitney U ‐test.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa) or DSMZ (HeLa).

Techniques: Expressing, In Vitro, Generated, Incubation, Quantitative RT-PCR, Gene Expression, MANN-WHITNEY

Journal: International Journal of Cancer

Article Title: Th17 cells favor migration and invasiveness of cervical cancer cells under hypoxia in an IGF2BP2 ‐dependent manner

doi: 10.1002/ijc.70340

Figure Lengend Snippet: Th17‐induced expression of CAIX, SLC2A1 and hexokinase II on protein level in 2D cultures and 3D spheroids. (A, B) 2D monolayers of SiHa, SW756 and HeLa were stimulated with medium or CMTH17 and incubated under hypoxic conditions. Twenty‐four hours later CAIX (A, blue bars) and SLC2A1 (B, purple bars) expression were investigated by IF. Bars represent quantification of relative fluorescence/cell of 20 independent pictures (scale bar: 20 μm) From n = 2 independent experiments. The values of medium‐stimulated cells by 21% oxygen was set at 1%. (C) SiHa (left), SW756 (middle) and HeLa cells (right) were stimulated with 100 ng/mL rhIL‐17 (red bars) or 20% CMTH17 (blue bars) for 24 h by hypoxic conditions. Whole cell extracts were analyzed for hexokinase II expression by Western blot analysis. The relative hexokinase II expression (hexokinase/β‐Actin) in cells incubated by 1% oxygen was set at 1. β‐Actin was used as a loading control. Shown are the results (mean + SD) from four independent stimulations. (D–F) 3D spheroids of SW756 and HeLa were generated over 11 days in the presence of medium, rhIL‐17 (red bars) or CMTH17 (blue bars). 5 μm sections of fixed paraffin‐embedded spheroids were validated by HE stainings and analyzed for (D) CAIX, (E) SLC2A1 and (F) hexokinase II expression by IF (scale bar: 200 μm). Sections from CMTH17‐stimulated SW756 spheroids were used for CAIX (D) and hexokinase II (F) stainings, sections from medium‐stimulated HeLa spheroids were used for CAIX (D) and SLC2A1 (E) stainings, sections from IL‐17‐stimulated HeLa spheroids were used for SLC2A1 (E) and hexokinase II (F) stainings and sections from CMTH17‐stimulated HeLa spheroids were used for SLC2A1 (E) and hexokinase II (F) stainings. Bars represent quantification of relative fluorescence/spheroid of n = 6 independent spheroids, respectively (mean + SD). Asterisks (* p < .05, ** p < .01, **** p < .0001) represent statistical significances. The p ‐value according to the nonparametric Mann–Whitney U ‐test.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa) or DSMZ (HeLa).

Techniques: Expressing, Incubation, Fluorescence, Western Blot, Control, Generated, MANN-WHITNEY

Journal: International Journal of Cancer

Article Title: Th17 cells favor migration and invasiveness of cervical cancer cells under hypoxia in an IGF2BP2 ‐dependent manner

doi: 10.1002/ijc.70340

Figure Lengend Snippet: Th17 cells mediate enhanced glucose uptake as well as proliferation and migration of cervical cancer cells under hypoxic conditions. (A) HeLa, SiHa and SW756 cells were stimulated with rhIL‐17 (100 ng) or CMTH17 and cultured in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. After 24 h, the cells were incubated with the fluorescent glucose analogon NBDG (10 mg/mL diluted in PBS) for 1 h. Glucose uptake was investigated by IF. Bars represent quantification of relative fluorescence/cell of 150 cells in total from five independent pictures (scale bar: 200 μm) of independent stimulation experiments, respectively. The values of medium‐stimulated cells incubated by 21% oxygen was set at 1. (B, C) Monolayers of SiHa cells stimulated with medium (black lines), rhIL‐17 (red lines), CMTH17 (blue lines) were scratched and (B) stimulated with 300 μM cobalt chloride every 24 h to create and retain hypoxic conditions or (C) incubated by hypoxic conditions (1% O 2 ). Pictures of the scratches were taken after 0, 24, 48, and 72 h (scale bar: 200 μm). The relative loss of area was calculated for (B) 3 days or (C) 2 days, respectively, indicated by lines (left picture), in relation to time point 0 h (middle, line graphics). The relative loss of the area after 72 or 48 h of the cells stimulated with medium was set at 1 (right, bar chart). The line graphics show one representative experiment performed in doubles (mean ± SD). Bars represent data (mean + SD) of n = 3 independent experiments performed in doubles. (D) SiHa cells were stimulated with medium (black bars), rhIL‐17 (red bars) or CMTH17 (blue bars) and cultured in normoxic (21% oxygen) or hypoxic (1% oxygen) conditions. After 24 h, cells were used in transwell migration assays. Transmigrated cells were calculated after 24 h. Representative pictures (left panel; scale bar: 100 μm); Quantification of n = 3 experiments with six independent pictures, respectively (mean ± SD), lower panel. The number of medium stimulated normoxic cells was set at 1. Asterisks (* p < .05, ** p < .01, **** p < .0001) represent statistical significances. The p ‐value according to the nonparametric Mann–Whitney U ‐test.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa) or DSMZ (HeLa).

Techniques: Migration, Cell Culture, Incubation, Fluorescence, MANN-WHITNEY

Journal: International Journal of Cancer

Article Title: Th17 cells favor migration and invasiveness of cervical cancer cells under hypoxia in an IGF2BP2 ‐dependent manner

doi: 10.1002/ijc.70340

Figure Lengend Snippet: Th17 cells increase IGF2BP2 expression on mRNA and protein expression levels in hypoxic cervical cancer cells. (A) SiHa, SW756 and HeLa cells were incubated by normoxic (21%) or hypoxic oxygen conditions (1% O 2 ). After 24 h, the cells were analyzed for IGF2BP2 expression by qRT‐PCR analysis and normalized to RPL13A housekeeping gene expression. The quotient of IFG2BP2/RPL13A of normoxic cells was set at 1. Shown are the results (mean + SD) from four independent experiments. (B, C) SiHa (left), SW756 (middle) and Hela (right) were stimulated with medium (black bars), rhIL‐17 (100 ng/mL, red bars) and conditioned media of in vitro generated Th17 cells (CMTH17, 20%, blue bars) and incubated by hypoxic oxygen conditions (1% O 2 ). (B) After 24 h, the cells were analyzed for IGF2BP2 expression by qRT‐PCR analysis and normalized to RPL13A housekeeping gene expression. The quotient of IFG2BP2/RPL13A of medium stimulated cells was set at 1. Shown are the results (mean + SD) from six independent stimulations. (C) In neutralization experiments, CM were prestimulated with neutralizing anti‐IL‐17 or respective isotype control antibodies for 2 h (light blue bars) and IGF2BP2 expression was analyzed in relation to RPL13A. (D) After 24 h, whole cell extracts were analyzed for IGF2BP2 expression by Western blot analysis. β‐Actin was used as a loading control. The relative IGF2BP2 expression (IGF2BP2/ β‐Actin) of medium stimulated cells was set at 1. Bars represent results (mean + SD) from four independent stimulations. (E) 3D spheroids of HeLa and SW756 were generated over 11 days in the presence of medium, rhIL‐17 (red bars) or CM of Th17 cells (blue bars). The 5‐μm sections of fixed paraffin‐embedded spheroids were validated by HE stainings and analyzed for IGF2BP2 expression by IF (scale bar: 200 μm). Sections from medium‐stimulated SW756 spheroids from Figure were used for IGF2BP2 stainings, sections from medium‐stimulated HeLa spheroids from Figure were used for IGF2BP2 stainings and sections from IL‐17‐stimulated HeLa spheroids from Figure were used for IGF2BP2 stainings. Bars represent quantification of relative fluorescence/spheroid of n = 6 independent spheroids, respectively (mean + SD). Asterisks (* p < .05, ** p < .01, *** p < .001) represent statistical significances. The p ‐value according to the nonparametric Mann–Whitney U ‐test.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa) or DSMZ (HeLa).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Gene Expression, In Vitro, Generated, Neutralization, Control, Western Blot, Fluorescence, MANN-WHITNEY

Journal: International Journal of Cancer

Article Title: Th17 cells favor migration and invasiveness of cervical cancer cells under hypoxia in an IGF2BP2 ‐dependent manner

doi: 10.1002/ijc.70340

Figure Lengend Snippet: Th17‐hypoxia‐induced enhanced migration and invasion is dependent on IGF2BP2. (A) SiHa cells were transfected with IGF2BP2‐specific siRNA (siIGF2BP2 #1, #2) or siControl and stimulated with 100 ng/mL rhIL‐17 or CMTH17 for 24 h under hypoxic conditions. Whole cell extracts were analyzed for IGF2BP2 expression by Western blot analysis. β‐Actin was used as a loading control. The relative IGF2BP2 expression (IGF2BP2/β‐Actin) of medium stimulated cells was set at 1. (B, C) SiIGF2BP2 (#1, #2) transfected SiHa cells were scratched and stimulated with medium, rhIL‐17 or CMTH17 24 h post‐transfection. (B) Pictures of the scratches were taken after 0, 24, 48, and 72 h (scale bar: 200 μm). The relative loss of area was calculated for 3 days, indicated by lines, in relation to time point 0 h. The relative loss of the area after 72 h of the cells stimulated with medium was set at 1. (C) Bars represent data (mean + SD) of n = 3 independent experiments performed in doubles. (D, E) SiHa cells were stimulated with IGF2BP2 inhibitors (IGF2BP2 inihibitors#1, #2, 50 μM) for 1 h. Cells were scratched followed by stimulation with medium, rhIL‐17 or CMTH17 in the presence of IGF2BP2 inhibitors (IGF2BP2 inihibitors#1, #2, 25 μM for 72 h). Pictures of the scratches were taken after 0, 24, and 48 h (scale bar: 200 μm). The relative loss of area was calculated for 2 days, indicated by lines, in relation to time point 0 h. The relative loss of the area after 48 h of the cells stimulated with medium was set at 1. (E) Bars represent data (mean + SD) of n = 3 independent experiments performed in doubles. (F–H) Spheroids of SW756 cells and (I–K) spheroids of HeLa cells were generated in the absence or presence of rhIL‐17, CMTH17 cells, medium or CM of naive CD4 + cells. On day 5, spheroids were incubated with inhibitors (#1/#2, 25 μM) or DMSO as a control for 24 h. On day 6, spheroids were embedded into Matrigel. Pictures were taken for 8 days and spheroid invasion was calculated. Shown are the pictures of one representative experiment which depicts the invasiveness of the spheroids over the time (F–J, scale bar: 200 μm). The relative invasiveness after 8 days was determined in relation to medium or CM of naive CD4 + T cells, respectively and DMSO stimulated cells which was set at 1 (H, K, gray stripped bars). Due to the invasiveness of spheroids from HeLa cells on day 8, pictures were merged with the Microsoft Image Composite Editor program. Shown are the results (mean + SD) from six independent spheroids. Asterisks (* p < .05, ** p < .01, *** p < .001, **** p < .0001, n.s. = not significant) represent statistical significances. The p ‐value according to the nonparametric Mann–Whitney U ‐test.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa) or DSMZ (HeLa).

Techniques: Migration, Transfection, Expressing, Western Blot, Control, Generated, Incubation, MANN-WHITNEY