Journal: Molecular Oncology

Article Title: Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

doi: 10.1002/1878-0261.13546

Figure Lengend Snippet: Th17 Cells induced the expression of EMT markers in cervical cancer cells and enhanced their migration and invasiveness. (A, B) SiHa and SW756 cells were stimulated with rhIL‐17 or conditioned media (CM) of Th17 cells for 24 h. (A) Transcriptomic profile of Epithelial‐to‐Mesenchymal Transition (EMT) markers. Fold changes of stimulated cells to unstimulated cells were illustrated by color code (reduction in blue, induction in red). Shown are the results from an array analysis performed in quadruplicates. (B) Expression profile of EMT markers was validated by qRT‐PCR (B upper panel; rhIL17 stimulation: Light red and light blue bars, CM Th17 stimulation: dark red and dark blue bars) and western blot analysis after 72 h (lower panel). β‐Actin was used as a loading control. Bars represent quantification of n = 3 independent experiments (mean ± SD; E‐cadherin: Blue bars; vimentin: Red bars). (C) Monolayers of SiHa and HeLa cells stimulated with medium, rhIL‐17 or CM of Th17 cells were scratched. Pictures were taken after 0, 24, 48 and 72 h. The area of the gap, indicated by lines (left panel), was determined in relation to time point 0 h (right panel). Scale bar: 100 μm. Bars represents results (mean ± SD) of n = 3 experiments. The area of 0 h was set at 100%, respectively. (D) SiHa, SW756 and HeLa cells were stimulated with medium, rhIL‐17 or CM of Th17 cells and used in transwell migration assays. Transmigrated cells were calculated after 24 h. Representative pictures (upper panel; scale bar: 100 μm); Quantification of n = 3 experiments with five independent pictures, respectively (mean ± SD), lower panel. The number of medium stimulated cells was set at 1. (E) Spheroids of SW756 cells were generated in the absence or presence of rhIL‐17 or CM of Th17 cells. Spheroids were embedded into matrigel, pictures were taken for 5 days and spheroid invasion was calculated. Shown are the results mean ± SD from four independent experiments performed in doubles. P ‐value according to the nonparametric Kruskal–Wallis test (B–D) or Mann–Whitney U ‐test (E). Asterisks represent statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL_0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa, Manassas, VA, USA) or DSMZ (HeLa) and authenticated by a multiplex human cell line authentication test (Multiplexion; DSMZ, Braunschweig, Germany) in August 2020.

Techniques: Expressing, Migration, Quantitative RT-PCR, Western Blot, Control, Generated, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

doi: 10.1002/1878-0261.13546

Figure Lengend Snippet: Th17 cells induced the expression of miR‐142‐5p in cervical cancer cells that mediated enhanced migration and invasion. (A) SiHa, SW756 and HeLa cells were stimulated with rhIL‐17 (orange), conditioned media (CM) of Th17 cells (blue) or medium (black bars) as a control for 24 h and miR‐142‐5p expression was analyzed in relation to RNU48. (B) In neutralization experiments, CM were prestimulated with neutralizing anti‐IL‐17 or respective isotype control antibodies for 2 h (light blue bars) and miR‐142‐5p expression was analyzed in relation to RNU48. (C) SiHa, SW756 and HeLa cells were transfected with miR‐142‐5p expressing plasmid (green bars) or pSG5 (black bars) as empty vector control. MiR‐142‐5p expression was analyzed in relation to RNU48 48 h later. Shown are the results mean ± SD from three independent experiments performed in triplicates in 2A‐2C. (D) MiR‐142‐5p transfected SiHa and HeLa cells were scratched 24 h post‐transfection. Pictures were taken after 0, 24, 48 and 72 h. The area of the gap, indicated by lines (left panel), was determined in relation to time point 0 h (right panel). Scale bar: 100 μm. Bars represents results (mean ± SD) of n = 3 experiments. (E) MiR‐142‐5p transfected SiHa and HeLa cells were used in transwell migration assays 48 h post‐transfection. Transmigrated cells were evaluated 24 h later. Numbers of pSG5 transfected cells were set at 1. Representative pictures (upper panel); quantification of n = 3 experiments with five independent pictures, respectively (mean ± SD) lower panel. Scale bar: 100 μm. (F) Spheroids of mir‐142‐5p transfected SW756 cells were generated. Spheroids were embedded into matrigel, pictures were taken for 5 days and spheroid invasion was calculated. Shown are the results mean ± SD from three independent experiments performed in duplicates. Scale bar: 200 μm. P ‐value according to the nonparametric Kruskal–Wallis test (A, D) or Mann–Whitney U ‐test (B, C, E, F). Asterisks represent statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL_0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa, Manassas, VA, USA) or DSMZ (HeLa) and authenticated by a multiplex human cell line authentication test (Multiplexion; DSMZ, Braunschweig, Germany) in August 2020.

Techniques: Expressing, Migration, Control, Neutralization, Transfection, Plasmid Preparation, Generated, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

doi: 10.1002/1878-0261.13546

Figure Lengend Snippet: Identification and validation of SDHC and SDHD as new targets of miR‐142‐5p. (A) Schematic diagram of reporter gene plasmids. The position of the predicted miR‐142‐5p binding sites in the respective 3′UTR reporter plasmids and their corresponding sequences as well as the sequences of the mutated binding sites (underlined) are shown. SDHC‐3′UTR reporter construct (upper panel), SDHD‐3′UTR reporter construct (lower panel). (B) HEK 293T cells were transfected with the wild type reporter plasmids of the respective target genes SDHC (light blue bar) or SDHD (dark blue bar) or mutated reporter plasmids (mut) of the target genes (striped bars). The luciferase activities were normalized with respect to the luciferase activity measured with empty reporter construct (gray bar). The results represent the mean of three independent experiments carried out in duplicates. (C) SiHa, SW756 and HeLa cells were transfected with miR‐142‐5p expressing plasmid or pSG5 as empty vector control. Whole cell extracts were analyzed 72 h later for SDHC (light blue bars) or SDHD (dark blue bars) expression by western blot analysis. β‐Actin was used as a loading control. Bars represent quantification of n = 3 independent experiments (mean ± SD). P ‐value according to the nonparametric Mann–Whitney U ‐test. Asterisks represent statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL_0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa, Manassas, VA, USA) or DSMZ (HeLa) and authenticated by a multiplex human cell line authentication test (Multiplexion; DSMZ, Braunschweig, Germany) in August 2020.

Techniques: Biomarker Discovery, Binding Assay, Construct, Transfection, Luciferase, Activity Assay, Expressing, Plasmid Preparation, Control, Western Blot, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

doi: 10.1002/1878-0261.13546

Figure Lengend Snippet: Th17 cells reduced the expression of SDHC and SDHD in cervical cancer cells. (A) SiHa, HeLa and SW756 cells were stimulated with conditioned media (CM) of Th17 cells or medium for 48 h. Expression of SDHC and SDHD was evaluated by qRT‐PCR. Bars represent quantification of n = 3 independent experiments (mean ± SD) performed in triplicates. (B) SW756 cells were analyzed for SDHC, SDHD and TOM22 expression by IF. Shown are representative pictures from n = 3 independent experiments. Scale bar: 20 μm. (C) 2D monolayers of SiHa cells were stimulated with rhIL‐17, CM of Th17 cells or medium for 72 h and SDHC and SDHD expression was investigated by IF. Bars represent quantification of relative fluorescence/cell of 20 independent pictures (mean ± SD; magnification 400×) from n = 2 independent experiments performed in duplicates. Scale bar: 20 μm. (D) 3D spheroids of HeLa cells were generated over 10 days in the presence of medium, rhIL‐17 or CM of Th17 cells. 5 μm sections of fixed paraffin‐embedded spheroids were validated by HE stainings and analyzed for SDHC and SDHD expression by IF. Bars represent quantification of relative fluorescence/spheroid of n = 6 independent spheroids (mean ± SD), respectively. Scale bar: 100 μm. (E, F) SiHa, SW756 and HeLa cells were stimulated with medium, rhIL‐17 or CM of Th17 cells for 72 h. (E) Whole cell extracts were analyzed for SDHC and SDHD expression by western blot analysis. β‐Actin was used as a loading control. Bars represent quantification of n = 3 independent experiments (mean ± SD; SDHC: Light blue bars; SDHD: Dark blue bars). (F) SDH activity of SiHa, SW756 and HeLa cells stimulated with medium (black bars), rhIL‐17 (orange bars) or CM of Th17 cells (blue bars) was determined after 72 h. Shown are the results from n = 3 independent experiments (mean ± SD) performed in triplicates. P ‐value according to the nonparametric Kruskal–Wallis test. (A, C–F) Asterisks represent statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (G) Serum samples of 59 cervical cancer patients (purple dots) and 38 healthy female controls were analyzed for succinate. (H) Serum levels of succinate were evaluated in patients with ( n = 34; blue dots) and without ( n = 25; gray dots) lymph node metastases. Black line: Median value of the respective groups. P ‐value according to the nonparametric Mann–Whitney U ‐test. Asterisks represent statistical significance: * P < 0.05; ** P < 0.01.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL_0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa, Manassas, VA, USA) or DSMZ (HeLa) and authenticated by a multiplex human cell line authentication test (Multiplexion; DSMZ, Braunschweig, Germany) in August 2020.

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Generated, Western Blot, Control, Activity Assay, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Th17 cells target the metabolic miR ‐142‐5p–succinate dehydrogenase subunit C/D ( SDHC / SDHD ) axis, promoting invasiveness and progression of cervical cancers

doi: 10.1002/1878-0261.13546

Figure Lengend Snippet: Th17‐induced increased migration of cervical cancer cells is dependent on miR‐142‐5p‐mediated reduced SDHC and SDHD expression. SiHa, SW756 and HeLa cells were transfected with two specific siRNAs for SDHC (dotted and stripped light blue bars) or SDHD (dotted and stripped dark blue bars), respectively, or mock siRNA (black bars) as a control. (A) After 48 h, SDHC and SDHD expression was calculated by qRT‐PCR (left panel) and whole cell extracts were analyzed for SDHC and SDHD expression by western blot analysis (right panel). β‐Actin was used as a loading control. Bars represent quantification of n = 3 independent experiments performed in duplicates. (mean ± SD). (B) 24 h post‐transfection, SiHa cells were scratched. Pictures were taken after 0, 24, 48 and 72 h. The area of the gap, indicated by lines (upper panel), was determined in relation to time point 0 h (lower panel). Scale bar: 100 μm. Bars represent results (mean ± SD) of n = 3 experiments. Additionally, SiHa cells were transfected with specific siRNAs for SDHC and SDHD (purple bars). 24 h post‐transfection, SiHa cell were scratched. Pictures were taken after 0, 24, 48 and 72 h. The area of the gap, indicated by lines (upper panel), was determined in relation to time point 0 h (lower panel). Scale bar: 100 μm. Bars represents results (mean ± SD) of n = 3 experiments. (C) Transfected SiHa and HeLa cells were used in transwell migration assays 48 h post‐transfection. Representative pictures (left panel); quantification of transmigrated cells of n = 3 experiments with five independent pictures, respectively (mean ± SD, right panel). Scale bar: 100 μm. (D–G) SiHa cells were transfected with inhibitor of miR‐142‐5p or inhibitor control. After 24 h, cells were stimulated with medium, rhIL‐17 or CM of Th17 cells. (D) Whole cell extracts were analyzed for SDHC and SDHD expression by western blot analysis. β‐Actin was used as a loading control. Fold expression of SDHC and SDHD per β‐Actin expression was calculated. Medium stimulated cells transfected with inhibitor control was set at 1. Shown are the results from n = 2 independent experiments. (E) Transfected cells were stimulated with medium, rhIL‐17 (orange bars) or CM of Th17 cells (light blue bars). After 24 h cells were scratched. Pictures were taken after 0, 24, 48 and 72 h. The area of the gap, indicated by lines (left panel), was determined after 72 h in relation to time point 0 h (right panel). Scale bar: 100 μm. Bars represent results (mean ± SD) after 72 h of n = 3 experiments. The area of medium stimulated cells transfected with inhibitor control was set at 100%. (F) 24 h post‐transfection, transfected cells were stimulated with medium, rhIL‐17 (orange bars) or CM of Th17 cells (light blue bars). After 24 h, cells were used in transwell migration assays. Representative pictures (left panel) after 24 h; quantification of transmigrated cells of n = 3 experiments with five independent pictures, respectively (mean ± SD; right panel). The number of medium stimulated cells transfected with inhibitor control was set at 1. Scale bar: 100 μm. (G) 24 h post‐transfection, spheroids were generated out of transfected cells for 4 days in the presence of medium, rhIL‐17 (orange bars) or CM of Th17 cells (light blue bars). Spheroids were embedded into matrigel, pictures were taken for 5 days and spheroid invasion was calculated. Shown are the results mean ± SD from four independent spheroids. Scale bar: 200 μm. P ‐value according to the nonparametric Kruskal–Wallis test. Asterisks represent statistical significance: * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: HPV18‐positive cervical carcinoma cell lines SW756 (RRID:CVCL_1727), HeLa (RRID:CVCL_0030) and HPV16‐positive SiHa cells (RRID:CVCL_0032) were received from ATCC (SW756, SiHa, Manassas, VA, USA) or DSMZ (HeLa) and authenticated by a multiplex human cell line authentication test (Multiplexion; DSMZ, Braunschweig, Germany) in August 2020.

Techniques: Migration, Expressing, Transfection, Control, Quantitative RT-PCR, Western Blot, Generated

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Indirect immunofluorescence assay (IFA) of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. With 2.5 × 10 5 IFU/well inoculum (MOI = 1), Chlamydia -infected HeLa cells were photographed using optical microscopy at 6, 12, 18, and 24 h p.i. Chlamydial inclusion bodies (green) are visible in both TC0668 wt - and TC0668 mut -infected cells, whereas the TC0668 protein (red) is only visible in TC0668 wt -infected HeLa cells. Magnification, ×200. TC0668 wt -infected HeLa cells were photographed by using phase contrast microscopy (A) and IFA (B) at 6, 12, 18, and 24 h p.i. TC0668 mut -infected HeLa cells were photographed by using phase contrast microscopy (C) and IFA (D) at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Immunofluorescence, Infection, Microscopy

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: qRT-PCR analysis of tc0668 gene copy number in C. muridarum TC0668 wt - and TC0668 mut -infected HeLa cells. With 1 × 10 6 IFU/well inoculum (MOI = 1), the copy number of the C. muridarum gene tc0668 was determined using qRT-PCR, and the C. muridarum plasmid gene pgp8 was used as the control. 16S rRNA was used to normalize tc0668 and pgp8 signals. Three biological replicates of each time point were performed, and points represent mean and standard errors. Copy number differences of tc0668 between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Quantitative RT-PCR, Infection, Plasmid Preparation, Control

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Quantitative proteomic analysis of HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains. (A) Basic statistics of proteome results from iTRAQ. Spectra, secondary mass spectra after quality control; Unique peptides, identified peptides that belong to only a group of proteins; and protein, identified proteins using Mascot 2.3.02 software. (B) Trends of differentially expressed proteins in TC0668 wt -infected cells at 6, 12, 18, and 24 h p.i. (C) Trends of differentially expressed proteins in TC0668 mut -infected cells at 6, 12, 18, and 24 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Control, Software

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Four-way Venn diagram of the total number of proteins significantly differentially expressed ( P < 0.05) between HeLa cells infected with C. muridarum TC0668 wt or TC0668 mut strains at 6, 12, 18, and 24 h p.i. Numbers of shared or unique proteins are indicated at the intersections of the circles in the Venn diagram.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Validation of profiling data with qRT-PCR. mRNA expression levels of seven up-regulated (encoded by SRPRB, JAK1, PMM1, HLA-DQB1, BCAP31, ITPR1, and THBS1) and three down-regulated (encoded by MAPKAPK2, TRAFD1, and IFI16) proteins in C. muridarum TC0668 mut -infected HeLa cells were determined using qRT-PCR at 18 h p.i, and compared with those of C. muridarum TC0668 wt -infected group. mRNA levels from three replicates for each group are expressed as mean and the standard errors. *represents that all copy number differences between TC0668 wt and TC0668 mut were statistically significant ( t test, P < 0.05).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Biomarker Discovery, Quantitative RT-PCR, Expressing, Infection

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Activation of NF-κB and PI3K/Akt signal pathways as determined using western blotting and IFA. With 1 × 10 6 IFUs/well inoculum (six-well plate) or 2.5 × 10 5 IFUs/well inoculum (24-well plate), expression of PI3K, p-Akt, p53, and NF-κB (p65) representing activation of PI3K/Akt and NF-κB signal pathways were determined using western blotting or IFA, respectively. Gel quantification software was used to calculate the relative intensity of the corresponding signals. The relative value (target protein/GAPDH) differences of PI3K, p53, and NF-κB (p65) molecules and the relative expression level of p-Akt (p-Akt/Akt) between TC0668 wt - and TC0668 mut -infected cells were statistically significant (one-way ANOVA, P < 0.05). (A) Expression of PI3K molecules in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (B) Expression of p-Akt and total Akt in TC0668 mut - and TC0668 wt -infected cells were determined using western blotting at 6, 12, 18, and 24 h post-infection. (C) Expression of p53 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (D) Expression of p65 molecules in the TC0668 mut - and TC0668 wt -infected cells at 6, 12, 18, and 24 h post-infection. (E) NF-κB molecules in the cytoplasm and nuclei of TC0668 mut - and TC0668 wt -infected HeLa cells at 6, 12, 18, and 24 h post-infection. DAPI dye core (blue), NF-κB fluorescence secondary antibody is 488 dye (green).

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Activation Assay, Western Blot, Expressing, Software, Infection, Fluorescence

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with inflammation in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics, Histone Deacetylase Assay, Membrane, Variant Assay

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Differentially expressed proteins related with fibrosis in TC0668 mut - vs. TC0668 wt -infected HeLa cells at 18 h p.i.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Infection, Multiplex sample analysis, Ubiquitin Proteomics

Journal: Frontiers in Microbiology

Article Title: iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and Wild-Type Strains

doi: 10.3389/fmicb.2019.02553

Figure Lengend Snippet: Protein-protein interaction network of statistically differentially expressed proteins ( P < 0.05) associated with inflammation and fibrosis at 18 h p.i. The proteins interconnectivity of two categories (inflammation and fibrosis) are shown. The yellow circles marked with symbols combining numbers and letters, represent the protein ID of differentially expressed proteins screened by comparison of TC0668 mut -infected and TC0668 wt -infected HeLa cells. The corresponding information of proteins can be analyzed via UniProt ( https://www.uniprot.org/ ), each protein ID corresponds to a protein. For example, the protein ID “P01023” corresponds to the protein “pha-2-macroglobulin”.

Article Snippet: HeLa cells (human cervical carcinoma epithelial cells, CCL-2; American Type Culture Collection) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (DMEM-10) at 37°C in 5% CO 2 .

Techniques: Comparison, Infection

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Overexpression of progerin to induce premature aging in the immortalized microglial cell line HMC3. (A) Schematic overview of lentiviral transduction strategy using pLenti‐CMV‐TRE3G‐Neo‐GFP‐Progerin and pLenti‐CMV‐rtTA3‐Hygro constructs, resulting in a doxycycline inducible microglial cell line. (B) QPCR analysis revealed a significant upregulation of progerin mRNA in HMC3‐Progerin cells after 72 h of doxycycline treatment (+DOX) compared to non‐induced controls (−DOX) [ n = 8, mean ± SD, unpaired students t ‐test]. (C, D) Western Blot analysis confirmed a corresponding increase in progerin protein levels in doxycycline‐treated cells (+DOX) relative to untreated controls (−DOX) after 72 h [ n = 3, mean ± SD, unpaired students t ‐test]. (E) Representative pictures of GFP‐progerin expression in induced HMC3‐Progerin cells. Images show robust GFP‐progerin expression in induced cells. Quantification indicated a transduction efficiency of 89.42% after 72 h of doxycycline induction and selection [scale bar = 20 μm; n = 6 (each n tested > 20 cells), mean ± SD, unpaired student's t ‐test].

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Over Expression, Transduction, Construct, Western Blot, Expressing, Selection

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Determination of the AgeScore in prematurely aged HMC3‐Progerin cells. (A) Schematic overview of the AgeScore that includes: Age markers reflecting the primary (DNA damage, histone modification, telomere attrition) and antagonistic (cell cycle arrest, senescence‐associated β‐Galactosidase (SA‐βGAL), senescence‐associated secretory phenotype (SASP), Lamin B1 expression, morphology) hallmarks of aging that include typical markers of cellular senescence (green bar). (B) Representative images and quantification of γH2A.X foci after 3, 7 and 14 days of doxycycline treatment show increased DNA damage in induced (+DOX) HMC3‐Progerin cells compared to non‐induced (−DOX) controls, with progressive accumulation over time. (C) H3K9 trimethylation (H3K9Me3) levels were significantly reduced in induced (+DOX) HMC3‐Progerin cells at all time points, as determined by corrected total cell fluorescence (CTCF) analysis. (D) Telomere length, assessed by monochrome multiplex quantitative PCR (MM‐qPCR), remained unchanged relative to a standard derived from healthy mixed‐aged individuals. (E) mRNA expression of cell cycle regulators CDKN2A (p16) and CDKN1A (p21) showed no significant induction following progerin expression, as measured by qRT‐PCR. (F) SA‐βGal activity, assessed by Spider βGal staining, was significantly increased in induced (+DOX) HMC3‐Progerin cells at all time points. For each n we tested > 100 cells. G: Representative images and CTCF quantification revealed a marked reduction in Lamin B1 expression after 3, 7 and 14 days of progerin induction. (H) Quantification of nuclear area (in pixel units) showed a significant increase only after 14 days of doxycycline treatment. (I) Enzyme‐linked immunosorbent assay (ELISA) of conditioned medium indicated elevated secretion of interleukin 6 (IL6) and interleukin 8 (IL8) in induced (+DOX) HMC3‐Progerin cells. (J) Calculation of AgeScore demonstrate an increase from 0 to 6 (after 3 and 7 days) and an increase from 0 to 7 after 14 days of doxycycline treatment in progerin expressing HMC3 microglia. *All data are presented as mean ± SD; statistical significance was determined by two‐way ANOVA followed by Sidak's post hoc test (* p < 0.05, ** p < 0.001, ** p < 0.0001); scale bar = 20 μm.

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Modification, Expressing, Fluorescence, Multiplex Assay, Real-time Polymerase Chain Reaction, Derivative Assay, Quantitative RT-PCR, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Functional decline of prematurely aged HMC3‐Progerin cells. (A) RNA expression analysis of pro‐ and anti‐inflammatory genes by qRT‐PCR after 72 h of doxycycline‐induced aging and 24 h of stimulation with 10 μg/mL lipopolysaccharide (LPS). Prematurely aged HMC3‐Progerin cells (+DOX) showed increased expression of pro‐inflammatory markers compared to non‐induced controls (−DOX). (B) Schematic overview of the migration assay using 2‐well culture inserts to generate a defined cell‐free gap. (C) Representative images and quantification of microglial migration 24 h after insert removal revealed significantly reduced migration capacity in doxycycline‐induced (+DOX) HMC3‐Progerin compared to non‐induced (−DOX) control cells. Cells were pretreated with 7.5 μg/mL mitomycin C to inhibit proliferation (see Figure ). (D) Schematic overview of phagocytosis assay using pHrodo Red Zymosan BioParticles, which fluoresce under acidic conditions in phagosomes. (E) Representative images and quantification demonstrated significantly reduced phagocytic activity in doxycycline‐induced (+DOX) HMC3‐Progerin cells compared to non‐induced (−DOX) controls. *All data are shown as mean ± SD; statistical significance was determined using unpaired student's t ‐test (C, E) or one‐way ANOVA with Tukey's post hoc test (A); * p < 0.05, ** p < 0.001, ** p < 0.0001; scale bars = 50 μm.

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Functional Assay, RNA Expression, Quantitative RT-PCR, Expressing, Migration, Control, Phagocytosis Assay, Activity Assay

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Transcriptomic profiling reveals aging‐associated signatures in HMC3‐Progerin microglia after 72 h of doxycycline induction. (A) Principal component analysis (PCA) of normalized RNA‐seq data demonstrates distinct clustering of +DOX and −DOX conditions. PC1 and PC2 account for 81% and 7% of the total variance, respectively, with each dot representing one biological replicate. (B) Log 2 fold change of selected microglial marker genes, including homeostatic and disease‐associated microglia (DAM stage 1 and 2) markers, reveals a transcriptional shift towards a DAM‐like phenotype upon progerin induction. (C) Analysis with the Microglia Annotation Tool revealed that a subset of the DEGs identified by RNA sequencing (–Dox vs. +Dox) partially overlaps with established reference signatures defined by specific phenotype marker genes: 31.0% homeostatic (HM), 37.4% disease‐associated (DAM), 40.7% antigen‐presenting (HLA), 44.6% inflammatory (CRM), and 62.3% interferon‐associated (IRM) microglia. (D) Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) highlights biological processes altered in induced (+DOX) HMC3‐Progerin cells. (E) Venn diagram shows DEG overlap with published human microglia datasets. A total of 2 genes ( CEACAM1 , PDPN ) were shared across all datasets; 8 overlapped with Galatro et al. , and 38 with Olah et al. . (F–H) RT‐qPCR validation of selected DEGs confirms upregulation of CEACAM1 and PDPN (F), AGTR1 and COL1A (G), and RAGE (H) in induced (+DOX) HMC3‐Progerin relative to non‐induced (−DOX) controls. (I) Transcriptomic age estimation using the Multi‐Timer algorithm revealed a significant increase in the aging score in induced (+DOX) HMC3‐Progerin cells, indicating an accelerated transcriptomic aging profile. *All data are shown as mean ± SD; * p < 0.05, ** p < 0.001, ** p < 0.0001; unpaired student's t ‐test.

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: RNA Sequencing, Marker, Quantitative RT-PCR, Biomarker Discovery

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Senolytic treatment partially reverses aging‐associated phenotypes in HMC3‐Progerin microglia. HMC3‐Progerin cells were treated with 500 nM rapamycin (RAPA) or a combination of 200 nM dasatinib and 10 μM quercetin (DQ) for 3, 7, or 14 days. (A) γH2A.X foci, indicative of DNA damage, were significantly reduced after 3 and 7 days of senolytic treatment, but not at day 14. (B) H3K9Me3 expression remained reduced in all treatment conditions, indicating that this histone modification was not restored. (C) Telomere length, measured by MM‐qPCR, remained unchanged across all time points and treatments. (D) Expression levels of cell cycle regulators CDKN2A (p16) and CDKN1A (p21) measured via qRT‐PCR were unaffected by senolytic treatment. (E) SA‐βGal activity, assessed by Spider βGal staining (each n tested > 100 cells), revealed a reduction in senescent cells following DQ treatment at 3 and 7 days, but not at day 14. (F) Representative images and quantification of CTCF of Lamin B1 expression remained diminished across all treatment conditions and time points. (G) Nuclear area showed partial restoration only after 14 days of treatment. (H) Enzyme‐linked immunosorbent assay (ELISA) of conditioned medium of interleukin 6 (IL6) and interleukin 8 (IL8) secretion showed no significant decrease following RAPA or DQ treatment. (I) AgeScore analysis revealed a partial reduction at 3 and 14 days with DQ and at 7 days with both RAPA and DQ. *All data are presented as mean ± SD; statistical significance was determined by two‐way ANOVA with Sidak's post hoc test; * p < 0.05, ** p < 0.001, **p < 0.0001; scale bar = 20 μm.

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Expressing, Modification, Quantitative RT-PCR, Activity Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Aging Cell

Article Title: Prematurely Aged Human Microglia Exhibit Impaired Stress Response and Defective Nucleocytoplasmic Shuttling of ALS Associated FUS

doi: 10.1111/acel.70232

Figure Lengend Snippet: Nucleocytoplasmic transport (NCT) defect in HMC3‐Progerin cells leads to FUS mislocalization and altered stress response. (A) Immunofluorescence staining of RAN protein after 3, 7, and 14 days of doxycycline treatment, with or without senolytics (500 nM rapamycin [RAPA] or 200 nM dasatinib +10 μM quercetin [DQ]). In induced (+DOX) HMC3‐Progerin cells, RAN redistributed from the nucleus to the cytoplasm at days 7 and 14, as shown by the nucleus‐to‐cytoplasm intensity ratio (N/C). This effect was partially reversed after 7 days of senolytic treatment. (B) Representative images of the shuttling assay using a dtTomato reporter containing classical nuclear localization (NLS) and nuclear export (NES) sequences. Doxycycline‐induced (+DOX) HMC3‐Progerin cells showed impaired nuclear import and increased cytoplasmic accumulation compared to non‐induced (−DOX) controls, quantified by mean intensity ratios. (C) Immunofluorescence analysis of FUS protein revealed nuclear‐to‐cytoplasmic mislocalization in induced (+DOX) HMC3‐Progerin cells, as measured by intensity ratios between nucleus and cytoplasm. (D) Stress granule (SG) formation following exposure to 200 μM sodium arsenite (SA) for 1 h. Quantification showed a significant increase in SGs in both groups, with a more pronounced response in non‐induced (−DOX) controls. *All data are shown as mean ± SD; statistical analysis by two‐way ANOVA with Sidak's post hoc test (A) or one‐way ANOVA with Tukey's post hoc test/unpaired student's t ‐test (B–D); * p < 0.05, ** p < 0.001, *** p < 0.0001; scale bar = 50 μm.

Article Snippet: Human HMC3 cells (ATCC, CRL‐3304) were cultured in MEM α medium (Gibco, #12571063) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin.

Techniques: Immunofluorescence, Staining

Journal: Nature cell biology

Article Title: Regulation of Mitochondrial Biogenesis in Erythropoiesis by mTORC1-Mediated Protein Translation

doi: 10.1038/ncb3527

Figure Lengend Snippet: ( a ) Heatmap is shown for transcription factors significantly upregulated at the protein but not mRNA levels. The genes are ranked based on the significance of fold changes in protein expression between HSPCs (A0) and ProEs (A5). n.d. not detected. ( b ) qRT-PCR analysis of TFAM and PHB2 in HSPCs and differentiating erythroid cells. ( c ) Western blot of TFAM, PHB2, GATA1 and GATA2 in HSPCs and erythroid cells. ( d ) Quantification of Western blot analysis. ( e ) The protein half-lives of TFAM and PHB2 were determined by CHX chase experiments. ( f ) Quantification of TFAM and PHB2 half-lives in HSPCs and ProEs. The dashed lines indicate the calculated half-lives for both proteins. Results are mean ± s.e.m. of n =3 independent experiments ( b,d,f ). Differences relative to HSPCs (day0) were assessed using a repeated-measures one-way ANOVA followed by Dunnett's test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001 ( b,d,f ). ( g ) Schematic of lentiviral shRNA-mediated depletion of TFAM or PHB2 in HSPCs, followed by erythroid differentiation ( top ). Validation of TFAM or PHB2 depletion by Western blot ( bottom ). Cells transduced with non-targeting shRNA (shNT) were analyzed as controls. The quantified protein expression of TFAM or PHB2 from three independent experiments is shown as mean ± s.e.m. on the bottom. ( h ) Depletion of TFAM significantly decreased mtDNA ( top ). TFAM and PHB2 depletion significantly decreased mitochondrial mass ( bottom ). ( i-n ) Depletion of TFAM or PHB2 significantly decreased MMP ( i ), intracellular ATP ( j ), protein synthesis in CD71 + CD235a - and CD71 + CD235a + erythroid progenitors ( k ), erythroid differentiation measured by the expression of CD34, CD71 and CD235a ( l ), proliferation ( m ), and increased apoptosis by AnnexinV and 7-AAD staining ( n ). Results are mean ± s.e.m. of n =3 independent experiments ( h,i,k-n ) or mean ± s.d. of n =8 independent measurements from three experiments ( j ). Differences relative to shNT were assessed using a repeated-measures one-way ANOVA followed by Dunnett's test for multiple comparisons; * P < 0.05, ** P < 0.01, *** P < 0.001 ( h-n ). ( o ) GSEA analysis of mitochondrial genes, ProE or HSPC-specific genes using the RNA-seq of shTFAM or shPHB2 relative to shNT, respectively. See Statistics Source Data in .

Article Snippet: Western blot was performed as described using the following antibodies: TFAM (7495S, Cell Signaling Technology; sc-23588, Santa Cruz Technology), PHB2 (14085S, Cell Signaling Technology), ATP5A1 (sc-136178, Santa Cruz Technology), ATP5B (sc-16690, Santa Cruz Technology), ATP5D (ab97491, Abcam), ATP5O (ab110276, Abcam, clone 4C11C10D12), 4EBP1 (9644S, Cell Signaling Technology), phos-4EBP1 (Thr37/46) (2855S, Cell Signaling Technology), p70S6K (2708S, Cell Signaling Technology), phos-S6K (Ser235/236) (2211S, Cell Signaling Technology), GATA1 (ab47490, Abcam), GATA2 (ab22849, Abcam), TSC1 (6935, Cell Signaling Technology), TSC2 (4308, Cell Signaling Technology), H3K9ac (9649, Cell Signaling Technology), H3K27ac (8173, Cell Signaling Technology), H3K56ac (4243, Cell Signaling Technology), H4K5ac (ab51997, Abcam), H3K4me3 (9571, Cell Signaling Technology), H3K9me3 (13969, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), H3K36me3 (4909, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), ACTB (MAB1501, Millipore, clone C4), and GAPDH (sc-26778, Santa Cruz Biotechnology).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, shRNA, Biomarker Discovery, Transduction, Staining, RNA Sequencing

Journal: Nature cell biology

Article Title: Regulation of Mitochondrial Biogenesis in Erythropoiesis by mTORC1-Mediated Protein Translation

doi: 10.1038/ncb3527

Figure Lengend Snippet: ( a ) Schematic of ex vivo differentiation of human primary HSPCs into ProEs or granulocytes. ( b ) A representative profile is shown for polysome profiling of human erythroid cells treated with control (DMSO) or PP242 (2.5μM) for 12h. ( c ) qRT-PCR analysis of indicated transcripts in cells treated with DMSO or PP242. Results are mean ± s.d. of n=4 independent measurements and shown as the percentage (%) of total mRNA of all fractions combined. ( d ) The number of downregulated proteins (fold change ≥ 1.3, P ≤ 0.01), Protein-only, or mitochondrial proteins were determined by iTRAQ-based proteomics in ProEs or granulocytes treated with PP242 (2.5μM) or control (DMSO) for 12h. P -values were calculated by hypergeometric distribution using All ( n =935 and 529 in ProEs and granulocytes, respectively), Protein-only ( n =205 and 50) and Mitochondria ( n =245 and 33) genes. n.s. not significant. ( e ) The changes in the levels of indicated proteins were determined by iTRAQ-based proteomics in ProEs or granulocytes treated with PP242. Results are shown for one representative proteomic experiment. ( f ) Top enriched GO terms of proteins downregulated in PP242-treated ProEs or granulocytes. ( g ) The most frequent TSS of the indicated transcripts were annotated using dbTSS, RefSeq and Ensembl databases. The sequences of TOP, TOP-like or TISU motifs were shown as the pink color. ( h ) The frequency of TOP-like motifs in Protein-only, RNA-only or all genes. P -values were calculated by hypergeometric distribution using All ( n =4,228 out of 6,624 total detected proteins), Protein-only ( n =418 out of 594 total detected proteins) and RNA-only ( n =363 out of 612 total detected proteins) genes. ( i ) Mutation of TOP-like motifs at the 5′UTRs of TFAM , PHB2 and ATP5D impaired GFP reporter expression in human primary erythroid cells but not granulocytes. Cells treated control (DMSO) or PP242 (2.5μM) for 12h were analyzed. Results are mean ± s.e.m. of n =3 independent experiments and analyzed by a two-tailed t -test. * P < 0.05, ** P < 0.01, n.s. not significant. See Statistics Source Data in . ( j ) Model of mTORC1-mediated post-transcriptional control of mitochondrial biogenesis during erythropoiesis.

Article Snippet: Western blot was performed as described using the following antibodies: TFAM (7495S, Cell Signaling Technology; sc-23588, Santa Cruz Technology), PHB2 (14085S, Cell Signaling Technology), ATP5A1 (sc-136178, Santa Cruz Technology), ATP5B (sc-16690, Santa Cruz Technology), ATP5D (ab97491, Abcam), ATP5O (ab110276, Abcam, clone 4C11C10D12), 4EBP1 (9644S, Cell Signaling Technology), phos-4EBP1 (Thr37/46) (2855S, Cell Signaling Technology), p70S6K (2708S, Cell Signaling Technology), phos-S6K (Ser235/236) (2211S, Cell Signaling Technology), GATA1 (ab47490, Abcam), GATA2 (ab22849, Abcam), TSC1 (6935, Cell Signaling Technology), TSC2 (4308, Cell Signaling Technology), H3K9ac (9649, Cell Signaling Technology), H3K27ac (8173, Cell Signaling Technology), H3K56ac (4243, Cell Signaling Technology), H4K5ac (ab51997, Abcam), H3K4me3 (9571, Cell Signaling Technology), H3K9me3 (13969, Cell Signaling Technology), H3K27me3 (9733, Cell Signaling Technology), H3K36me3 (4909, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), ACTB (MAB1501, Millipore, clone C4), and GAPDH (sc-26778, Santa Cruz Biotechnology).

Techniques: Ex Vivo, Control, Quantitative RT-PCR, Multiplex sample analysis, Mutagenesis, Expressing, Two Tailed Test

Journal: Molecules

Article Title: A Subcellular Quantitative Proteomic Analysis of Herpes Simplex Virus Type 1-Infected HEK 293T Cells

doi: 10.3390/molecules24234215

Figure Lengend Snippet: Subcellular quantitative proteomic analysis of herpes simplex virus type 1 (HSV-1)-infected HEK 293T cells. ( A ) Intracellular levels of HSV-1 genome DNA and IFNB1 and ISG56 mRNAs in HSV-1 infected HEK 293T cells. Mock- or HSV-1 (MOI = 5)-infected HEK 293T cells were harvested at 4 h p.i. and 20 h p.i. Total RNA was extracted and reverse transcribed into the cDNA to quantify the intracellular mRNA level of IFNB1 and ISG56 using quantitative RT-PCR. The total DNA was extracted, and the intracellular DNA level of the HSV-1 genome was measured with quantitative RT-PCR. ( B ) The MS analysis workflow of the stable isotope-labeled amino acid culture (SILAC). ( C ) Confirming the subcellular fractionation efficiency by Western blot. Cytoplasmic and nuclear fractions from the three biological replicates (R1, R2, and R3) were subjected to Western blot analyses. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lamin B1 were used as markers of cytoplasmic and nuclear proteins, respectively. H: hours.

Article Snippet: A mouse monoclonal antibody against GAPDH (ABclonal, Wuhan, China), a rabbit monoclonal antibody against IFITM3 (CST, Danvers, MA, USA), and a rabbit polyclonal antibody against IRF3 (Proteintech) and Lamin B1 (Proteintech) were purchased from indicated companies.

Techniques: Virus, Infection, Reverse Transcription, Quantitative RT-PCR, Labeling, Multiplex sample analysis, Fractionation, Western Blot

Journal: Molecules

Article Title: A Subcellular Quantitative Proteomic Analysis of Herpes Simplex Virus Type 1-Infected HEK 293T Cells

doi: 10.3390/molecules24234215

Figure Lengend Snippet: Validation of the protein regulation data by quantitative RT-PCR and Western blots. ( A ) Western blot analysis of the host proteins. Proteins from the cytoplasmic and nuclear fractions of the mock- or HSV-1-infected (MOI = 5) HEK 293T cells were extracted for Western blot analysis. Lamin B1 and GAPDH were used as internal controls for nuclear (nucleo) and cytoplasmic (cyto) proteins, respectively. ( B ) Quantitative RT-PCR analysis of the intracellular mRNA level of selected proteins. Mock- or HSV-1-infected (MOI = 5) HEK 293T cells were harvested, and the total mRNA was extracted and reverse transcribed into cDNA for quantitative RT-PCR analysis. The values are presented as the mean ± SD of three replicates. ( C ) SILAC-MS data for selected proteins. ND: not detected; H: hours.

Article Snippet: A mouse monoclonal antibody against GAPDH (ABclonal, Wuhan, China), a rabbit monoclonal antibody against IFITM3 (CST, Danvers, MA, USA), and a rabbit polyclonal antibody against IRF3 (Proteintech) and Lamin B1 (Proteintech) were purchased from indicated companies.

Techniques: Biomarker Discovery, Quantitative RT-PCR, Western Blot, Infection, Reverse Transcription, Multiplex sample analysis

Journal: Nature Communications

Article Title: RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia

doi: 10.1038/s41467-024-55643-7

Figure Lengend Snippet: a Schematic of clinical information and sample collection of patient 551599 through disease transformation. b Cell population proportion of 551599 PBMC samples by CyTOF. c tSNE plots showing surface CD14 + protein and key cytokine expression of 551599 samples. d Percent positive cytokine expression in 551599 CD14 + monocytes across disease stages and healthy peripheral blood donor (NPB) control by CyTOF. e Fold change of cytokine expression in 551599 CD14 + monocytes following 4h ex vivo treatment with 5 μM ruxolitinib by CyTOF. f Expression of signaling proteins in 551599 CD34 + HSCs and CD14 + monocytes at MF pPV compared to Early PV by CyTOF. Values represent the median arcsinh ratio. g UMAPs of 551599 from scRNA-seq by disease state and cell identity. h Expression dot plot of key genes from 551599 across disease states by scRNA-seq. i Trajectory branching of HSPC and CD14 + monocyte populations. j Pseudotime trajectory analysis of TNF , CCL3 , and RPS6KA1 expression across disease progression.

Article Snippet: Ruxolitinib was purchased from Selleck Chemicals (Houston, TX).

Techniques: Transformation Assay, Expressing, Control, Ex Vivo, Biomarker Discovery

Journal: Nature Communications

Article Title: RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia

doi: 10.1038/s41467-024-55643-7

Figure Lengend Snippet: a CyTOF analysis of CD14 + monocytes from MF #5 under treatment conditions. Cells were treated with 5 μM ruxolitinib, 5 μM PMD-026, 10 ng/mL TPO, or combination for 4 h. b Changes to cytokine expression from ( a ) and in four additional MF patients. Comparisons were performed between TPO alone versus TPO + inhibitor. c Immunoblot of CD14 + monocytes from three unique MF patients treated with indicated PMD-026 doses and time points. d Immunoblot of THP-1 cells stimulated with 20 ng/mL TNF and treated with 10 μM PMD-026 alone or in combination for 30 min. Immunoblot is representative of two independent experiments. e Left: Venn diagram showing overlapping DEGs between WashU Cohort MF CD14 + monocytes compared to those from NBM and CMML CD14 + monocytes compared to old healthy controls from (GSE135902). Middle: Key shared candidates from 279 total shared DEGs between MF and CMML monocytes. Right: Top altered Hallmark pathways by enrichment analysis from 279 shared DEGs. Gene count represents the number of genes enriched across each pertinent gene set. Gene ratio represents the number of genes enriched/total number of genes in each gene set. f Immunoblot of CD14 + monocytes from two unique CMML patients treated with indicated PMD-026 doses for 4 h. g qRT-PCR of key effectors from CD14 + monocytes from three CMML patients after 6 h 5 μM PMD- 026 treatment relative to control.

Article Snippet: Ruxolitinib was purchased from Selleck Chemicals (Houston, TX).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia

doi: 10.1038/s41467-024-55643-7

Figure Lengend Snippet: a White blood cell count following drug treatment in the JAK2 V617F mouse model. Mice were treated with control ( n = 7), 50 mg/kg PMD-026 ( n = 5), or 100 mg/kg PMD-026 ( n = 5). Statistics were assessed by two-way ANOVA between treatment groups versus control. Data are presented as mean values +/− SD. b Altered hematopoietic and disease parameters following drug treatment in the MPL W515L mouse model. Mice were treated with control ( n = 8), 90 mg/kg ruxolitinib ( n = 8), 100 mg/kg PMD-026 ( n = 8) or combination ( n = 8). White blood cells, monocytes, hematocrit, and platelets were assessed by Hemavet. Statistics were assessed by two-way ANOVA between treatment groups as indicated. Data are presented as mean values +/− SD. Spleen and liver weights were normalized to mouse weights at the endpoint. Statistics were assessed by one-way ANOVA with Dunnett’s multiple comparisons test. c Kaplan-Meier survival analysis. Statistics were assessed by log-rank test between treatment groups as indicated. d Histology of femur bone marrow at endpoint. Images show representative H&E and reticulin staining from 3 mice per group. Scale bar = 100 μM. e Luminex multiplex assays detecting plasma cytokines of treated MPL W515L mice. Vehicle ( n = 7), ruxolitinib ( n = 5), PMD-026 ( n = 7), combo ( n = 7). Statistics assessed by two-tailed Student’s t test. Boxplot showing median, upper, and lower quartiles with whiskers representing highest and lowest values excluding outliers, and potential outliers.

Article Snippet: Ruxolitinib was purchased from Selleck Chemicals (Houston, TX).

Techniques: Cell Counting, Control, Staining, Luminex, Multiplex Assay, Clinical Proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: RSK1 is an exploitable dependency in myeloproliferative neoplasms and secondary acute myeloid leukemia

doi: 10.1038/s41467-024-55643-7

Figure Lengend Snippet: a Schematic of the PDX model setup and mutation burden of five patient samples utilized to establish PDX models. b Efficacy in the 201943 MF model. Plots show the percentage of hCD45 + cells in the PB and BM of transplanted mice treated with vehicle, 60 mg/kg ruxolitinib, 100 mg/kg PMD-026, or combination ( n = 5 per group), spleen, liver weights of mice at endpoint normalized by mouse weight, and survival data. %hCD45 + in PB statistics comparing treatment group to control assessed by one-way ANOVA with Dunnett’s multiple comparisons; PMD-026 to combo by two-way Student’s t test. %hCD45 + in BM, and normalized spleen and liver weights statistics were assessed by one-way ANOVA with Dunnett’s multiple comparisons. Data are presented as mean values +/− SD. Kaplan-Meier survival analysis. Statistics were assessed by log-rank test between treatment groups as indicated. c Efficacy in the 290375 sAML model. Data are presented as mean values +/− SD. %hCD45 + in PB ( n = 5 in vehicle; n = 6 in PMD-026) statistics assessed by two-way ANOVA. %hCD45 + in BM ( n = 5 in vehicle; n = 6 in PMD-026) statistics assessed by two-tailed Student’s t test. d Efficacy in the 310914 sAML model. Data are presented as mean values +/− SD. %hCD45 + in PB ( n = 5 per group) statistics assessed by two-way ANOVA. %hCD45 + in BM ( n = 4 in vehicle; n = 5 in PMD-026) statistics assessed by two-tailed Student’s t test. e Efficacy in the 728508 sAML model. Data are presented as mean values +/− SD. %hCD45 + in PB ( n = 5 per group) statistics assessed by two-way ANOVA. %hCD45 + in BM ( n = 5 per group) statistics assessed by two-tailed Student’s t test. f Efficacy in the 676151 CMML model. Data are presented as mean values +/− SD. %hCD45 + in PB ( n = 5 per group) statistics assessed by two-way ANOVA. %hCD45 + in BM assessed by two-tailed Student’s t test.

Article Snippet: Ruxolitinib was purchased from Selleck Chemicals (Houston, TX).

Techniques: Mutagenesis, Control, Two Tailed Test

Journal: eLife

Article Title: An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

doi: 10.7554/eLife.103328

Figure Lengend Snippet: ( A ) Schematic diagram depicting the human chromosome 22q11.2 region. Bright grey and red horizontal bars indicate the two most common hemizygous genomic deletions found in the 22q11.2 Deletion Syndrome. The location of the coding genes and non-coding RNAs (miRNAs, underlined) are shown for chromosome 22q11.2. The microprocessor DGCR8 (DiGeorge Syndrome Critical Region Gene 8) and MIR185 are shown in bold. ( B ) Cortical marker TBR1 and pan-neuronal marker TUJ1 expression in cortical neurons as detected by immunocytochemistry at day 13 of differentiation. TBR1 (red), TUJ1 (green) and DAPI (blue) expression are shown. Scale bar: 100 μm. ( C ) Volcano plot showing differentially expressed human mature miRNAs (DEmiRs) in cortical neurons at day 8 of differentiation. Significant DEmiRs (p-value <5%) are shown above red line; Q5 (Ctrl) n=3, Q6 (22q11.2) n=3. 153/133 miRNAs were significantly up- and downregulated in Q6 (22q11.2) hiPSC-derived cortical neurons, respectively. 22q11.2 deletion region residing miRNAs miR-185, miR-1286 and miR-1306 are highlighted. ( D–F ) Consistent upregulation of EMC10 mRNA in Q6 (22q11.2) line derived cortical neurons as assayed by qRT-PCR at ( D ) day 8 (p=0.031; Q5: n=3, Q6: n=3), ( E ) day 20 (p=0.0478; Q5: n=4, Q6: n=4) and ( F ) day 34 (p=0.0358; Q5: n=3, Q6: n=3) of differentiation. ( G ) Western blot analysis showing upregulated EMC10 protein levels in Q6 (22q11.2) line derived cortical neurons at day 8 of differentiation. Tubulin was probed as a loading control. ( H ) Immunofluorescence images of NGN2 generated cells. Representative images of NGN2-iNs at DIV21 from Q5 (Ctrl) and Q6 (22q11.2) hiPSC lines identified via EGFP fluorescence and immunostained for neuronal dendrite marker MAP2 and the nuclear marker DAPI. Scale bar = 100 µm. ( I–K ) qRT-PCR assay of EMC10 mRNA expression level in NGN2-iNs at DIV21. ( I ) Upregulation of EMC10 mRNA in Q6 (22q11.2) line derived neurons compared to the healthy control line Q5 (p=0.0222). Q5 (Ctrl) n=4, Q6 (22q11.2) n=4. ( J ) Upregulation of EMC10 mRNA in Q1 (22q11.2) patient line compared to healthy control line Q2 (p0.0441). Q2 (Ctrl) n=5 and Q1 (22q11.2) n=7. ( K ) Upregulation of EMC10 mRNA in QR27 (22q11.2) patient line compared to healthy control line QR20 (p=0.0414). QR20 (Ctrl) n=5 and QR27 (22q11.2) n=5. Data are presented as mean ± SEM, unpaired two-tailed t-test, *p<0.05, **p<0.01. Figure 1—source data 1. PDF file containing original western blots for , indicating the relevant bands. Figure 1—source data 2. Original files for western blot analysis shown in .

Article Snippet: Sequence-based reagent , TaqMan Hs00232429 ( TBR1 ) , Thermo Fisher , cat#4331182 , Human TBR1 qRT-PCR.

Techniques: Marker, Expressing, Immunocytochemistry, Derivative Assay, Quantitative RT-PCR, Western Blot, Control, Immunofluorescence, Generated, Fluorescence, Two Tailed Test

Journal: eLife

Article Title: An antisense oligonucleotide-based strategy to ameliorate cognitive dysfunction in the 22q11.2 Deletion Syndrome

doi: 10.7554/eLife.103328

Figure Lengend Snippet: ( A ) Characterization of Q5 (Ctrl) and Q6 (22q11.2) hiPS cells demonstrate normal karyotype distribution in both lines. ( B–D ) Characterization of the Q5 and Q6 hiPSC lines. Multiplex Ligation-dependent Probe Amplification (MLPA) assay of gene copies within and around 22q11.2 locus for Q5 (Ctrl) ( B ) and Q6 (22q11.2) ( C ) line shows that copy number of genes in the 22q11.2 locus (highlighted in light blue) are reduced by half in the Q6 (22q11.2) hiPSC line. ( D ) qRT-PCR assays of embryonic stem cell marker OCT4/POU5F1 and NANOG shows that they are highly expressed in both hiPSC lines (Q5 Ctrl and Q6 22q11.2). ( E ) TBR1 and TUJ1 expression of cortical neurons by immunocytochemistry at day 20 of differentiation indicates the efficiency of hiPSC differentiation into cortical neurons. TBR1 (red), TUJ1 (green) and DAPI (blue). Scale bar: 100 μm. ( F–G ) Time-course qRT-PCR analysis at day 0 and day 20 of differentiation of human pluripotency marker ( F ) OCT4/POU5F1 (p=0.0012) and ( G ) TBR1 (p=0.0273), a preplate, subplate and cortical Layer VI neuron marker in Q5 (Ctrl) line. Day 0: n=4, Day 20 n=4. Data are presented as mean ± SEM, unpaired two-tailed t-test, *p<0.05, **p<0.01. Figure 1—figure supplement 1—source data 1. PDF file containing original gel for . Figure 1—figure supplement 1—source data 2. Original file for hiPSC validation shown in .

Article Snippet: Sequence-based reagent , TaqMan Hs00232429 ( TBR1 ) , Thermo Fisher , cat#4331182 , Human TBR1 qRT-PCR.

Techniques: Multiplex Assay, Ligation, Amplification, Quantitative RT-PCR, Marker, Expressing, Immunocytochemistry, Two Tailed Test, Biomarker Discovery

Journal: Nature

Article Title: H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

doi: 10.1038/s41586-023-05780-8

Figure Lengend Snippet: ( a ) Genomic confirmation of the APEX-modified Rpb1 loci in DPY30–mAID and RBBP5–FKBP degron cells. ( b ) Sanger sequencing of the wild type and Flag-APEX2-RPB1 knock-ins. ( c ) Western blot showing the expression of APEX-modified RPB1 in DPY30–mAID and RBBP5–FKBP degron cells. ( d ) Representative brightfield images of mES cell colonies. ( e ) RT-qPCR analysis of the expression of pluripotency and differentiation genes in the knock-in cells. Data are from three biological replicates (n = 3) and are analysed using Two-way ANOVA and represented as mean ± s.d. ( f ) Titration of biotin phenol (BP). Cells stably expressing Flag-APEX2-RPB1 were pre-incubated for 30 min with the indicated concentrations of BP, followed by the addition of 1 mM H2O2 for 1 min. Cell lysates were probed with Streptavidin-HRP. Proximity biotinylation is optimal at a BP concentration of 4 mM. ( g ) Confirmation of the APEX2 functionality by protein biotinylation in the APEX2-engineered DPY30–mAID and RBBP5–FKBP degron cells. The discrete bands, denoted with asterisks, show APEX2-independent biotinylation by native enzymes. The Connexin-APEX2 overexpressed cell was severed as positive control for the APEX2 system. ( h ) SILAC-based chromatin proteomic strategy for mapping the neighbourhood interaction networks of APEX2-tagged RNAPII. ( i ) Principal component analysis (PCA) of SILAC signal in the RNAPII-APEX2 cells with or without agonist. ( j ) Distribution of SILAC ratio of RNAPII interactions quantified in the chromatin proteomic analyses. Mean log2 SILAC ratio is shown. In total, 1,901 proteins were identified in this experiment. The RNAPII-APEX2-bait (BP+H2O2) population has a right-shifted distribution compared with the no agonist negative control population, which indicates that the log2(SILAC) ratio allows us to distinguish bona fide RNAPII interactions from non-RNAPII interactions. ( k ) GO network showing significantly (q value < 0.001) enriched terms for positive RNAPII interaction neighbourhoods from RNAPII-APEX2 experiment. The most prominent pathways are indicated. Connecting lines show interaction of protein nodes. ( l ) KEGG enrichment and GO network showing significantly ( q value < 0.001) enriched terms for positive RNAPII interaction neighbourhoods from RNAPII-APEX2 experiment. ( m ) Principal component analysis (PCA) of SILAC signal in the RNAPII-APEX2 DPY30–mAID cells with or without Auxin treatment. Time trajectory is shown by the dashed arrow. ( n ) Venn diagram indicating overlap between up or downregulated targets in the indicated samples. ( o ) Heatmap representing relative protein abundance of DPY30 and selected targets in Auxin treated DPY30–mAID cells. n = 3 independently samples. ( p ) Scatterplot analysis of proteins identified by SILAC in RNAPII-APEX2 DPY30–mAID cells following Auxin treatment for 2 and 8 h. ( q ) Gene ontology-based functional classification of 228 downregulated proteins in RNAPII-APEX2 DPY30–mAID cells following Auxin treatment for 2 and 8 h. The dot size is proportional to the number of members in an enrichment set, and colour intensity reflects the p value. Significance based on clusterProfiler analysis with Benjamini-Hochberg-adjusted P values.

Article Snippet: Approximately 4 mg lysates from SILAC heavy or light cells were mixed 1:1 and incubated with 50 μl Streptavidin magnetic beads (Pierce, 88817) at 4 °C on a rotating wheel overnight.

Techniques: Modification, Sequencing, Western Blot, Expressing, Quantitative RT-PCR, Knock-In, Titration, Stable Transfection, Incubation, Concentration Assay, Positive Control, Multiplex sample analysis, Negative Control, Quantitative Proteomics, Functional Assay

Journal: Nature

Article Title: H3K4me3 regulates RNA polymerase II promoter-proximal pause-release

doi: 10.1038/s41586-023-05780-8

Figure Lengend Snippet: ( a ) Venn diagram indicating overlap of H3K4me3 interactors and RNAPII-APEX2 dependent interactors from ChIP-MS (chromatin proteomic profiling) data. ( b ) Relative enrichments of selected targets in various ChIP preparations based on ChIP-MS. ( c ) Validation of INTS11 interaction with H3K4me3 in RBBP5–FKBP degron cells at different times after dTAG-13 addition. Biotinylated proteins within lysates were enriched using Streptavidin-coated magnetic beads and analysed by Western blot. In parallel, sample in which H 2 O 2 was omitted was prepared as negative control. ( d ) Schematic representation of the dTAG INTS11 targeting strategy for the INTS11–FKBP degron mES cells. ( e ) Western blot showing the expression of INTS11 and INTS11–FKBP–HA, using antibodies recognizing INTS11 or the HA in parental and knock-in degron cells. The arrow indicates the specific HA-tagged INTS11–FKBP–HA protein. ( f ) RT-qPCR analysis showing the expression of selected pluripotency and differentiation genes in the parental and INTS11–FKBP knock-in cells. Data are from three biological replicates (n = 3) and are analysed using Two-way ANOVA and represented as mean ± s.d. ( g ) Growth curve analysis of parental and INTS11–FKBP E14 cells treated with or without dTAG-13. ( h ) INTS11 enrichment profiles and heat maps as determined by using the HA-tag in control (0 h) and Auxin-treated (2 h) DPY30–mAID; INTS11–FKBP degron cells. Genome-wide binding averages showed enrichments at the TSS regions (TSS ± 2 kb) of protein coding genes. TSS, transcription start site. Rows were sorted by decreasing ChIP–seq occupancy in the control (0 h) cells. ( i ) Correlations between TT chem -seq replicate experiments in INTS11–FKBP degron cells treated with or without dTAG-13 for the indicated times. ( j ) Average profiles for TT chem -seq for the upstream anti-sense RNAs of each annotated protein-coding gene in INTS11 degron cells. TSS, transcription start site. ( k ) RNAPII profiles of various subclasses of annotations in INTS11–FKBP degron cells with or without dTAG-13 treatment. TSS, transcription start site. mRNA, messenger RNA. snRNA, small nuclear RNA. ncRNA, non-coding RNA. eRNA, enhancer RNA.

Article Snippet: Approximately 4 mg lysates from SILAC heavy or light cells were mixed 1:1 and incubated with 50 μl Streptavidin magnetic beads (Pierce, 88817) at 4 °C on a rotating wheel overnight.

Techniques: Biomarker Discovery, Magnetic Beads, Western Blot, Negative Control, Expressing, Knock-In, Quantitative RT-PCR, Control, Genome Wide, Binding Assay, ChIP-sequencing

Journal: Frontiers in Oncology

Article Title: An overview of glioblastoma multiforme and temozolomide resistance: can LC-MS-based proteomics reveal the fundamental mechanism of temozolomide resistance?

doi: 10.3389/fonc.2023.1166207

Figure Lengend Snippet: List of selected studies conducted (from year 2000 to date) to understand the TMZ resistance in human GBM and other types of cancers (TMZ treatment) using LC-MS-based proteomics approach.

Article Snippet: 2022 , SRPX Emerges as a Potential Tumor Marker in the Extracellular Vesicles of Glioblastoma ( ) , 1. Human GBM cell lines: IN IN-GB-11, IN-GB-28, IN-GB-29, and IN-GB-9 prepared from fresh tumor biopsies Human GBM U87-MG, 2. U251-MG and HEK293T purchased from ATCC 3. Human primary astrocytes (HPAs) purchased from Applied Biological Materials Inc. , EVs , 1. Cell line establishment 2. EVs isolation 3. Nanoparticle tracking analysis (NTA) to determine the average size of particles 4. nLC-MS/MS 5. Immunohistochemistry 6. RNA isolation and RT-qPCR 7. Generation of TMZ resistance cell line 8. Transfection with SRPX siRNAs 9. Cell viability, clonogenic survival and TMZ sensitivity assay 10. Statistical analysis , MASCOT 2.4.1 and Scaffold 5.0.1 , 1.Only one protein SRPX was detected in GBM derived EVs, which was absent in HPA derived EVs 2. Highest level of SRPX expression was observed for TMZ resistant GBM 3. Endogenous transcript level of ARPX was assessed by RT-qPCR and the U251-MG-R (TMZ-resistant cells) showed higher level of SRPX expression compared with U251-MG-P (parental cells) 4. TMZ treatment for 72 h resulted in an increased level of SRPX mRNA in both cell lines U251-MG-R and U251-MG-P 5. Cell viability tests in various experiments showed that depletion of SRPX inhibits tumor growth 6. Silencing SRPX sensitizes GBM cells to TMZ treatment , SRPX is highly enriched in EVs and plays a role in GBM tumorigenesis.

Techniques: Derivative Assay, Protein-Protein interactions, Control, Clone Assay, Migration, Cell Adhesion Assay, Nucleic Acid Electrophoresis, Quantitative Proteomics, Protein Extraction, Sample Prep, Ubiquitin Proteomics, Expressing, Activity Assay, Labeling, Quantitation Assay, Cell Cycle Assay, Transfection, Knockdown, Immunofluorescence, Biomarker Discovery, Extraction, Protein Quantitation, Western Blot, Xenograft Assay, Software, Viability Assay, WST-1 Assay, Gene Expression, shRNA, Sequencing, Proliferation Assay, Clinical Proteomics, Membrane, Multiplex sample analysis, Immunohistochemistry, Staining, Isolation, Knock-Out, Marker, Sensitive Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Trp53 p.R245Q promotes prostate tumor growth and remodels the tumor microenvironment. a Schematic of CRISPR/Cas9-mediated knock-in of Trp53 p.R245Q (arginine→glutamine) in prostate cancer cells and the workflow for in vivo validation and single-cell RNA sequencing. b-d Subcutaneous tumor growth of Myc-CaP cells in FVB/NJ mice (1 × 10 6 cells, WTp53 n = 5; Mutp53 n = 5): representative images of tumors at the experimental endpoint ( b ), tumor growth curves ( c ), and endpoint tumor weights ( d ). Tumors derived from Mutp53-expressing Myc-CaP cells exhibited significantly accelerated growth compared with those derived from control cells ( p < 0.05). e-g Subcutaneous tumor growth of RM-1 cells (1 × 10 6 cells in a Matrigel/PBS mixture) in C57BL/6 mice: representative endpoint tumor images ( e ), longitudinal tumor volume curves ( f ), and final tumor weights at sacrifice ( g ). Compared with those derived from WTp53 control cells, the tumors derived from Mutp53-expressing RM-1 cells markedly accelerated progression (WTp53 n = 5; Mutp53 n = 5; p < 0.05). h Kaplan-Meier survival analysis of subcutaneous RM-1 tumor-bearing mice injected with 5 × 10 5 cells: Compared with WTp53 controls, Mutp53-bearing mice presented significantly shorter overall survival (WTp53 n = 8; Mutp53 n = 10; log-rank test, p = 0.0156). i Dot plot visualization of canonical marker gene expression across major cell types in the single-cell transcriptome dataset derived from orthotopic RM-1 prostate tumors (5 × 10 5 cells implanted), including epithelial cells, endothelial cells, monocytes/macrophages, T/NK cells, fibroblasts, and pericytes, used for cell type annotation. j UMAP visualization of single-cell transcriptomes showing the distribution of major annotated cell types, along with representative top differentially expressed gene modules. k Fractional abundance of major cell populations in the tumor microenvironment. Box plots showing the relative fractions of epithelial cells, endothelial cells, pericytes, monocytes/macrophages, fibroblasts, and T/NK cells in tumors from the WTp53 and Mutp53 groups derived from single-cell transcriptome analysis. Among these populations, fibroblasts were obviously reduced in Mutp53 tumors, whereas changes in other cell types did not reach statistical significance. l Heatmap of observed-to-expected ratios for major cell populations. The Ro/e ratio was calculated for fibroblasts, pericytes, epithelial cells, monocytes/macrophages, endothelial cells, and T/NK cells in WTp53 and Mutp53 tumors. Compared with WTp53 tumors, Mutp53 tumors presented reduced fibroblast and pericyte enrichment, whereas monocytes/macrophages were relatively enriched. m Multiplex immunofluorescence staining of tumor sections for Pan-CK, CD4, FoxP3, CD8, CD68, CD163, and CTLA-4; Mutp53 tumors exhibit increased infiltration of CD4 + FoxP3+ regulatory T cells, CD8 + cytotoxic T cells, and CD68+/CD163 + macrophages, alongside elevated CTLA-4 expression. Scale bars: 100 μm. Note: Unless otherwise specified, p < 0.05 was considered statistically significant. Significance levels are indicated as follows: p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***). Abbreviations: WTp53: Wild-type p53; Mutp53: Mutant p53 ( Trp53 p.R245Q, arginine to glutamine substitution); CRISPR: Clustered Regularly Interspaced Short Palindromic Repeats; Cas9: CRISPR-associated protein 9; scRNA-seq: Single-cell RNA sequencing; UMAP: Uniform Manifold Approximation and Projection; Ro/e: Ratio of observed-to-expected frequency; Mon/Macro: Monocytes/Macrophages; T/NK cells: T lymphocytes/Natural Killer cells; Fibro: Fibroblasts; Peri: Pericytes; Endo: Endothelial cells; Epi: Epithelial cells

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: CRISPR, Knock-In, In Vivo, Biomarker Discovery, Single Cell, RNA Sequencing, Derivative Assay, Expressing, Control, Injection, Marker, Gene Expression, Multiplex Assay, Immunofluorescence, Staining, Mutagenesis

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutant p53 tumors exhibit enhanced sensitivity to PD-1 blockade through augmented CD8 + cytotoxic programs. a Schematic of the experimental design for the subcutaneous and orthotopic RM-1 tumor models treated with an anti-PD-1 antibody. b-d Subcutaneous tumor growth of WTp53 and Mutp53 RM-1 tumors established by injection of 1 × 10 6 cells, with or without anti-PD-1 therapy: representative images ( b ), tumor volume curves ( c ), and endpoint tumor weights ( d ) ( n = 5 per group, one-way ANOVA with Tukey’s test). e-g Orthotopic tumor growth of WTp53 and Mutp53 RM-1 tumors established by prostate implantation of 5 × 10 5 cells following PD-1 blockade: representative images ( e ), tumor volumes ( f ), and endpoint tumor weights ( g ) ( n = 4–5 per group, one-way ANOVA with Tukey’s test). h-i Flow cytometric analysis of intratumoral CD8 + GZMB + T cells at baseline and after PD-1 blockade; quantification is shown in (I) (one-way ANOVA with Tukey’s test). j-k Single-cell RNA-seq clustering of CD8 + T-cell subsets, highlighting enrichment of CD8+, CD8 + CD69+, CD8 + GZMA+GZMB+, CD8 + NKG7+, and CD4 + CD8+ T cells in Mutp53 tumors relative to WTp53 tumors. l-s Functional module scoring of CD8 + T cells, showing comparable costimulatory ( l-m ), exhausted ( n-o ), and resident ( r-s ) signatures between groups but markedly increased cytotoxic signatures in Mutp53 tumors ( p-q ). t Expression of representative effector genes in intratumoral CD8 + T cells, showing upregulation of Nkg7 , Gzmb , and Slamf7 in Mutp53 tumors, whereas Ifng expression remained unchanged (see also Fig. S4a). u Multiplex immunofluorescence staining for CD8 and GZMB, showing increased infiltration of CD8 + GZMB + T cells in Mutp53 tumors at baseline and after PD-1 blockade, alongside reduced IFN-γ expression, compared with WTp53 (see also Fig. S4b-c). Note: Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; ns, not significant. Abbreviations: Mutp53, mutant p53; WTp53, wild-type p53; PD-1, programmed cell death protein-1; scRNA-seq, single-cell RNA sequencing; GZMA, granzyme A; GZMB, granzyme B; IFN-γ, interferon-γ; UMAP, uniform manifold approximation and projection

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Mutagenesis, Injection, Single Cell, RNA Sequencing, Functional Assay, Expressing, Multiplex Assay, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutp53 promotes immunotherapy responsiveness through CXCL10-CXCR3-mediated recruitment and activation of CD8 + T cells. a-b Heatmap ( a ) and qRT-PCR analysis ( b ) of immune-related chemokine expression in subcutaneous WTp53 and Mutp53 tumors, showing significant upregulation of Cxcl10 together with Cxcl1 , Cxcl2 , Cxcl12 , Ccl2 , and Cxcl9 in Mutp53 tumors compared with WTp53 tumors. c Correlation analysis of CXCL10 expression with immune-related signaling pathways in the IMvigor210 urothelial carcinoma cohort treated with immune checkpoint blockade, revealing strong positive associations with IFN-γ-related signatures and the antigen presentation machinery (APM). d Representative immunohistochemical (IHC) staining of CXCL10 in WTp53 and Mutp53 tumors. e-f Immunoblot analysis ( e ) and quantitative densitometry ( f ) of CXCL10 protein expression in WTp53 and Mutp53 tumor cell lines and corresponding tumor tissues. g Transwell coculture assays demonstrating enhanced chemotaxis of CD8 + T cells toward Mutp53-derived tumor cells compared with WTp53-derived tumor cells ( n = 3). h Clinical association between intratumoral CXCL10 expression and immune checkpoint blockade (ICB) benefit in the IMvigor210 cohort, with higher CXCL10 levels predicting improved therapeutic response. i Schematic illustration of the in vivo experimental design for pharmacological disruption of the CXCL10-CXCR3 axis using the CXCR3 antagonist AMG-487 during anti-PD-1 treatment, with RM-1 tumors established by subcutaneous injection of 1 × 10⁶ cells. j-l Representative tumor images ( j ), tumor growth curves ( k ), and endpoint tumor weights ( l ) of WTp53- and Mutp53-bearing mice treated with anti-PD-1 therapy in the presence or absence of AMG-487 ( n = 5 per group). CXCR3 blockade markedly attenuated the therapeutic efficacy of PD-1 blockade, with a substantially stronger reversal observed in Mutp53 tumors. m Serum biochemical analyses evaluating liver and kidney function, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (BUN), and creatinine (CREA), showing no significant differences among treatment groups, indicating minimal systemic toxicity of AMG-487. n Multiplex immunofluorescence staining of tumor sections for CD8 (green), granzyme B (GZMB; red), and nuclei (DAPI; blue), revealing that anti-PD-1 therapy markedly increased infiltration of GZMB+CD8 + T cells in Mutp53 tumors, whereas CXCR3 inhibition significantly impaired this recruitment. o Quantification of GZMB+CD8 + T cells across treatment groups, confirming that the enrichment induced by PD-1 blockade in Mutp53 tumors was largely abolished by AMG-487 treatment. Note: Data are presented as the mean ± SD. Statistical significance was assessed using one-way or two-way ANOVA with Tukey’s multiple-comparison test, as appropriate. p < 0.05, p < 0.01, * p < 0.001; ns, not significant

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Protein-Protein interactions, Immunopeptidomics, Immunohistochemical staining, Immunohistochemistry, Western Blot, Chemotaxis Assay, Derivative Assay, Clinical Proteomics, In Vivo, Disruption, Injection, Drug discovery, Multiplex Assay, Immunofluorescence, Staining, Inhibition, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mutant p53 epigenetically rewires CXCL10 to promote CD8⁺ T-cell infiltration and enhance the anti-PD-1 response in advanced prostate cancer

doi: 10.1186/s13046-026-03672-z

Figure Lengend Snippet: Mutant p53 occupies the Cxcl10 promoter and remodels promoter-proximal chromatin to enable Cxcl10 transcription. a Immunoblot of RM-1 tumors showing that siRNA-mediated Trp53 silencing reduces the CXCL10 protein. p53 and GAPDH were used as target and loading controls, respectively. b JASPAR motif analysis identifying putative p53-binding sequences within the Cxcl10 promoter. The schematic shows the positions of Primer 1 and Primer 2 relative to the transcription start site and Exon 1. c-d ChIP-qPCR analysis of p53 occupancy at the Cxcl10 promoter. Mutp53 was more strongly enriched than WTp53 at Primer 2, whereas enrichment at Primer 1 was modest. The signals are normalized to the input signal and expressed relative to the IgG signal. e-f Histone-mark ChIP-qPCR analysis of the Cxcl10 promoter. At Primer 1 ( e ), H3K4me3 is minimally enriched in WTp53 tumors but is increased in Mutp53 tumors. At Primer 2 ( f ), Mutp53 tumors display increased H3K4me3 together with reduced H3K27me3 and H3K36me3, which is consistent with a promoter environment permissive for transcription. IgG, negative control. g Agarose-gel electrophoresis of representative ChIP amplicons validating the expected products for the indicated antibodies in WTp53 and Mutp53 tumors; left, bp ladder. h Working model: Mutp53 preferentially occupies the Cxcl10 promoter (primer 2), recruits the activating machinery, increases H3K4me3, and relieves repressive marks (H3K27me3, H3K36me3) relative to WTp53, thereby facilitating RNA polymerase II-driven Cxcl10 transcription. Note: Statistics. The dots represent biologically independent samples (typically n = 3 per group); the bars represent the means ± SEMs. Unless otherwise indicated, ChIP-qPCR datasets in c-f were analyzed via two-way ANOVA followed by multiple comparisons tests. Significance thresholds: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. Abbreviations: WTp53, wild-type p53; Mutp53, mutant p53; siRNA, small interfering RNA; ChIP, chromatin immunoprecipitation; qPCR, quantitative PCR; IgG, immunoglobulin G control IP; Pol II, RNA polymerase II; TSS, transcription start site; SEM, standard error of the mean; bp, base pairs; kDa, kilodaltons

Article Snippet: The mouse prostate cancer cell lines RM-1 (CRL-3310TM) and MyC-CaP (CRL-3255TM) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), which provides authenticated lines originally sourced from the American Type Culture Collection (ATCC; Manassas, VA, USA).

Techniques: Mutagenesis, Western Blot, Binding Assay, ChIP-qPCR, Negative Control, Agarose Gel Electrophoresis, Small Interfering RNA, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Control

Journal: EMBO Molecular Medicine

Article Title: Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis

doi: 10.15252/emmm.201708699

Figure Lengend Snippet: A SILAC‐based proteomics data identify known (red) and novel (green) hypoxia‐induced proteins in SW620 cells. One‐sample t ‐test was performed. B Western blotting confirmed GPRC5A as a hypoxia‐induced protein in SILAC lysates. C Validation of GPRC5A Western blot data using siRNA. *Non‐specific band of ˜60 kDa not depleted by GPRC5A siRNA. D Confocal microscopy showing plasma membrane GPRC5A expression in hypoxic SW620 cells (scale bars: 75 μm). E Western blotting showing GPRC5A upregulation by hypoxia in a panel of colorectal tumour cell lines. F Basal & hypoxia‐induced GPRC5A protein expression was decreased by HIF‐1/2α depletion. G Depletion of HIF‐1β decreased GPRC5A protein upregulation in hypoxia. H Hypoxia mimetic DMOG induced HIF‐1/2α, CA9 and GPRC5A protein expression. Dual HIF‐1/2α depletion reduced GPRC5A induction by DMOG. I qRT–PCR demonstrating that GPRC5A mRNA was upregulated by hypoxia ( n = 3). GPRC5A was normalised to HPRT (error bars ± SD). J qRT–PCR demonstrating that HIF‐1/2α depletion decreased GPRC5A induction during hypoxia ( n = 3). GPRC5A was normalised to HPRT (error bars ± SD). K ChIP‐PCR analyses identify HIF‐1α binding to the GPRC5A promoter region containing a putative optimal HRE (error bars ± SD, n = 3). Data information: Asterisks (*) indicate non‐specific band. Level adjustments were made to images in Adobe Photoshop post‐acquisition for clarity (equal changes applied to the entire image). Representative examples of n = 3 independent experiments are shown. Source data are available online for this figure.

Article Snippet: The human colorectal tumour cell lines Caco2, DLD1, HT29, HCT15, HCT116, LOVO, LS174T, RKO, SW480 and SW620 were obtained from the American Type Culture Collection (ATCC; Rockville, USA).

Techniques: Multiplex sample analysis, Western Blot, Biomarker Discovery, Confocal Microscopy, Clinical Proteomics, Membrane, Expressing, Quantitative RT-PCR, Binding Assay

Journal: EMBO Molecular Medicine

Article Title: Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis

doi: 10.15252/emmm.201708699

Figure Lengend Snippet: A, B Expression of CA9 and GPRC5A in formalin‐fixed paraffin‐embedded hypoxic SW620 cells by IHC. Reduced CA9 and GPRC5A expressions with siRNA confirm antibody specificity (scale bars: 200 μm). C IHC analysis of serial sections from human colorectal tissue from patients with mesenteric ischaemia (strangulated colon). GPRC5A is co‐expressed with CA9 in the colonic epithelial cells (scale bars: 50 μm). D Quantitative RT–PCR analysis of mouse intestinal tissue. Gene expression was normalised to housekeeping gene Tbp . Raw data from three independent experiments ( n = 3 mice) are shown (error bars ± SEM). E Tg[ fli1 :eGFP; vhl −/− ] and Tg[ fli1 :eGFP] zebrafish embryos (5 days post‐fertilisation) demonstrate excessive angiogenesis and increased expression of HIF target genes (scale bars: 100 μm). F gprc5ba was induced in vhl mutant zebrafish embryos and fli1 :eGFP zebrafish embryos exposed to 5% O 2 (vs. normoxia) for 24 h (RT–PCR). G, H Bioinformatic analysis of transcriptomics dataset GSE24551. Gene set analyses reveal GPRC5A mRNA strongly correlated with HIF/hypoxia gene signatures. GSEA datasets used were Semenza_HIF1_Targets (M12299) Broad_Hallmark_Hypoxia (M5891). Analysis was performed using R2 ( http://r2.amc.nl ). I Kaplan–Meier curve following analysis of transcriptomics dataset GSE24551. Event‐free survival is significantly reduced in patients with tumours expressing high levels of GPRC5A mRNA. Analysis was performed using R2 ( http://r2.amc.nl ). Data information: Level adjustments were made to images in Adobe Photoshop post‐acquisition for clarity (equal changes applied to the entire image). Representative examples of n = 3 independent experiments are shown. Source data are available online for this figure.

Article Snippet: The human colorectal tumour cell lines Caco2, DLD1, HT29, HCT15, HCT116, LOVO, LS174T, RKO, SW480 and SW620 were obtained from the American Type Culture Collection (ATCC; Rockville, USA).

Techniques: Expressing, Formalin-fixed Paraffin-Embedded, Quantitative RT-PCR, Gene Expression, Mutagenesis, Reverse Transcription Polymerase Chain Reaction

Journal: EMBO Molecular Medicine

Article Title: Cancer cell adaptation to hypoxia involves a HIF‐GPRC5A‐YAP axis

doi: 10.15252/emmm.201708699

Figure Lengend Snippet: A Lentivirally transduced and puromycin‐selected SW620:GPRC5A si1R cells were doxycycline‐induced (2.5 μg/ml) for 48 h and flow‐sorted (BD Influx) based on medium/high TurboGFP expression (note that pCW57‐GFP‐2A‐GPRC5A si1R uses the P2A self‐cleaving peptide to produce separate TurboGFP and GPRC5A si1R cDNA). Pre‐ and post‐flow‐sorted profiles are shown, confirming near 100% expression. Related to Figs D, and H, I, and L. B Confirmation of TurboGFP expression in doxycycline (2.5 μg/ml)‐treated SW620:GPRC5A si1R cells. Phase contrast and green (TurboGFP) images were obtained using the IncuCyte ZOOM live cell imaging system (scale bars: 300 μm). Level adjustments were made to images in Adobe Photoshop post‐acquisition for clarity (equal changes applied to the entire image). Related to Figs D, and H, I, and L. C Western blotting confirms GPRC5A overexpression in doxycycline‐treated SW620:GPRC5A si1R cells. Cells were treated for 72 h with doxycycline (2.5 μg/ml) in normoxia prior to harvest. Related to Figs D, and H, I, and L. Asterisk (*) indicates non‐specific band. D Optimisation of the violet ratiometric membrane asymmetry apoptosis assay for flow cytometry. SW620 cells were treated for 24 h with the pan‐BCL2 family inhibitor ABT‐737 (10 μM). Live, dead and apoptotic cells were gated using FlowJo (v10). A representative example is shown. Related to Fig E. Source data are available online for this figure.

Article Snippet: The human colorectal tumour cell lines Caco2, DLD1, HT29, HCT15, HCT116, LOVO, LS174T, RKO, SW480 and SW620 were obtained from the American Type Culture Collection (ATCC; Rockville, USA).

Techniques: Expressing, Live Cell Imaging, Western Blot, Over Expression, Membrane, Apoptosis Assay, Flow Cytometry