Journal: American Journal of Translational Research

Article Title: A novel NSUN5/ENO3 pathway promotes the Warburg effect and cell growth in clear cell renal cell carcinoma by 5-methylcytosine-stabilized ENO3 mRNA

doi:

Figure Lengend Snippet: NSUN5 (NOP2/Sun RNA methyltransferase 5)-mediated m5C (5-methylcytosine) modification of mRNA upregulates ENO3 (enolase 3) expression. A. Total RNA from normal and RCC (renal cell carcinoma) tissues were extracted, and MeRIP-qPCR (m5C RNA immunoprecipitation-quantitative polymerase chain reaction) was used to detect ENO3 (enolase 3) mRNA enrichment using the m5C (5-methylcytosine) antibody. B. MeRIP-qPCR (m5C RNA immunoprecipitation-quantitative polymerase chain reaction) was used to determine m5C (5-methylcytosine) modification levels in ENO3 (enolase 3) mRNA. C. RT-qPCR (real-time quantitative polymerase chain reaction) was performed to detect the candidate genes of m5C (5-methylcytosine) methyltransferase in 293A, 786-0, or Caki-1 cells. D. Genes were knocked down in 786-0 cells, and RT-qPCR (real-time quantitative polymerase chain reaction) was used to determine ENO3 (enolase 3) mRNA expression. E. 786-0 cells were transfected with si-NSUN5 (small interfering-NOP2/Sun RNA methyltransferase 5) or control siRNA (small interfering RNA), and then MeRIP-qPCR (m5C RNA immunoprecipitation-quantitative polymerase chain reaction) was used to detect ENO3 (enolase 3) mRNA enrichment using the m5C (5-methylcytosine) antibody. F and G. NSUN5 (NOP2/Sun RNA methyltransferase 5) mRNA or protein levels in RCC (renal cell carcinoma) or normal kidney tissues were measured using RT-qPCR (real-time quantitative polymerase chain reaction) and Western blotting. H. Kaplan-Meier analysis was used to analyze the survival of patients with ccRCC (clear cell renal cell carcinoma) using The Cancer Genome Atlas data of patients with low (n = 130) and high (n = 130) NSUN5 (NOP2/Sun RNA methyltransferase 5) levels (http://www.oncolnc.org/). I. Correlation analysis (Gene Expression Profiling Interactive Analysis; http://gepia.cancer-pku.cn/) revealed a positive correlation between ENO3 (enolase 3) and NSUN5 (NOP2/Sun RNA methyltransferase 5) in ccRCC tissues. J and K. Caki-1 cells were infected with oeNSUN5 (overexpression NOP2/Sun RNA methyltransferase 5) or pWPI vector, and then double immunofluorescence was used to detect ENO3 (enolase 3) and NSUN5 (NOP2/Sun RNA methyltransferase 5) co-expression. Scale bar, 20 μm. Data are expressed as mean ± standard deviation. *P < 0.05, **P < 0.01 vs. corresponding controls.

Article Snippet: RNA isolation and real-time quantitative polymerase chain reaction (RT-qPCR) Total RNA from tumor tissues or cultured cells was separated and extracted using Total RNA Kit I (Omega Bio-Tek) according to the manufacturer’s instructions.

Techniques: Modification, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Small Interfering RNA, Western Blot, Infection, Over Expression, Plasmid Preparation, Immunofluorescence, Standard Deviation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Dendritic cells in the bladder microenvironment of mice responding to CD40 agonism have higher expression of IL-15Rα. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 24 post-tumor implantation (n = 3-6 mice per group; bars represent SD). Responders (gray circles) and non-responders (blue circles) to anti-CD40 antibody therapy are indicated. ( C ) IL-15Rα mean fluorescence intensity across mice on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 24 post-tumor implantation (n = 3-6 mice per group). *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Luciferase, Imaging, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Endogenous IL-15 participates in the therapeutic activity of CD40 agonism. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody and/or anti-IL-15 blocking antibody or isotype-matched control antibody. ( B ) Representative intravital luciferase imaging (left) and quantification of luminescence (center) and bladder weights (right) across mice at day 12 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 5 mice per group) of CD44 hi CD122 + CD8 T cells (top) and CXCR6 + CD44 hi CD122 + CD8 T cells (bottom) in the bladders of mice treated as outlined in A. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Blocking Assay, Control, Luciferase, Imaging, Tumor Implantation, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: DCs in human non-muscle invasive bladder cancer express high levels of IL-15. Eight primary tumor samples from either untreated NMIBC patients (n=3) or from NMIBC patients that had progressed from NMIBC to MIBC following BCG therapy (n=5) were included in this single-cell RNA sequencing analysis. A) Phenograph clustering was performed and 29 clusters were identified. Canonical lineage markers for cell populations were utilized to assign cell identities to each cluster. B) tSNE plots were created for four representative markers: CD40, CD40L, IL15, and IL15RA. C) A matrix plot with column scaled expression of the same four markers of interest is shown to highlight the distribution and intensity of expression for each gene across each subset.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: RNA Sequencing, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15/IL-15Rα upregulation on dendritic cells in the bladder tumor microenvironment. Mice bearing orthotopic MB49 bladder tumors were treated intravesically with anti-CD40 antibody, BCG, or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). ( C ) Representative flow cytometry plots and ( D ) quantification across mice of IL-15 and IL-15Rα surface expression on cDC1s in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). Left graph is gated on cDC1s and depicts proportions of cDC1s that are double-positive for surface IL-15 and IL-15Rα. Right graph is gated on IL-15Rα-expressing cDC1s and depicts proportions of cDC1s expressing IL-15Rα that is occupied by IL-15. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: CD40 agonism induces IL-15Rα upregulation on dendritic cells in the UPPL1541 bladder tumor microenvironment. Mice bearing orthotopic UPPL1541 bladder tumors were treated intravesically with anti-CD40 antibody or isotype-matched control antibody on days 6 and 9 post-tumor implantation. ( A ) Representative histogram and ( B ) quantification across mice of IL-15Rα mean fluorescence intensity on type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 5 mice per group). **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances primary anti-tumor activity. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic MB49 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative intravital luciferase imaging (left) and luminescence quantification (right) across mice at day 14 post-tumor implantation (n = 5 mice per group; bars represent SD). ( C ) Survival (top) and representative intravital luciferase imaging (bottom) of surviving mice at day 85 post-tumor implantation treated as outlined in A. ( D ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across mice treated with anti-CD40 antibody 2141-V11 and IL-15 or control in the absence or presence of additional anti-CD8 T cell depleting antibody (n = 5 mice per group; bars represent SD). ( E ) Representative intravital luciferase imaging (left) and quantification of luminescence (right, top) and bladder weights (right, bottom) at day 14 post-tumor implantation across WT or Batf3 -/- mice treated with anti-CD40 antibody 1C10 and IL-15 or control (n = 5 mice per group; bars represent SD). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Luciferase, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination of Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor activity against UPPL1541 tumors. ( A ) Schematic of the treatment of humanized hCD40/hFcyR mice bearing orthotopic UPPL1541 bladder tumors with anti-CD40 antibody 2141-V11 and/or IL-15 or control (isotype-matched control antibody and/or vehicle). ( B ) Representative bladder ultrasound imaging (left) at day 45 post-tumor implantation and disease-free survival (right) of mice treated as outlined in A. *p < 0.05, **p < 0.01.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Activity Assay, Control, Imaging, Tumor Implantation

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: IL-15 therapy does not modulate myeloid CD40 expression. ( A ) Schematic of the treatment of mice bearing orthotopic MB49 bladder tumors with IL-15 or vehicle control. ( B ) Bladder weights across mice at day 12 post-tumor implantation (n = 4-5 mice per group; bars represent SD). ( C ) CD40 mean fluorescence intensity across mice on total dendritic cells (DC; defined as F4/80 − Ly-6G − CD11c + MHCII + ), type-1 conventional DCs (cDC1; defined as F4/80 − Ly-6G − CD11c + MHCII + XCR1 + ), type-2 conventional DCs (cDC2; defined as F4/80 − Ly-6G − CD11c + MHCII + SIRPα + ), macrophages (defined as CD11b + F4/80 + Ly-6G − ), monocytes (defined as CD11b hi Ly6G − ), and neutrophils (defined as CD11b + Ly-6G + ) in the bladder microenvironment as assessed by flow cytometry at day 12 post-tumor implantation (n = 4-5 mice per group). ns = not significant.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Expressing, Control, Tumor Implantation, Fluorescence, Flow Cytometry

Journal: bioRxiv

Article Title: IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

doi: 10.1101/2023.01.30.526266

Figure Lengend Snippet: Combination therapy with Fc-optimized anti-CD40 agonist antibody 2141-V11 and IL-15 enhances anti-tumor memory responses. ( A ) Schematic of subcutaneous rechallenge (ten-fold tumor cell dose) of humanized hCD40/hFcyR mice surviving long-term (>90 days after initial orthotopic MB49 bladder tumor implantation) following initial therapy with anti-CD40 antibody 2141-V11 with or without IL-15. ( B ) Tumor growth (in the absence of any additional therapy) of long-term survivors compared with naïve mice receiving an equivalent MB49 tumor cell implant (n = 2-5 mice per group; bars represent SD). ( C ) Representative flow cytometry plots and quantification across mice (n = 2-5 mice per group) of CD44 hi CD122 + CD8 T cells (top), CD44 hi CD122 + CD62L + CD8 T cells (middle), and CD11b + KLRG1 + CD27 − NK cells (bottom) in the peripheral blood of mice surviving long-term (day 110) after primary tumor implantation and tumor rechallenge. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Anti-human CD40 antibody 2141-V11 (derived from CP-870,893, clone 21.4.1 referenced in patent US7338660, ATCC accession number PTA-3605) containing a human IgG1 Fc domain carrying the G237D/P238D/H268D/P271G/A330R amino acid modifications was generated as previously described ( ).

Techniques: Tumor Implantation, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: (A) Immunohistochemical (IHC) staining of RABL6A in tissue microarrays (TMAs) of patient-matched NF (mainly plexiform NFs) and MPNSTs. Images at 10X magnification. Higher magnification images (400X) of RABL6A (B) and p27 (D) IHC staining of NF, ANNUBP and MPNST cores from the TMA. Stain intensity indicated in parentheses. H-scores, representing the stain intensity × percent of cells stained, of RABL6A (C) and p27 (E) in NF, ANNUBP and MPNST samples. Error bars, SEM. P-value, Student’s t-test. *, p<0.05; **, p<0.01. (F) Heat-map of RNA-Seq data with hierarchical clustering of a subset of functionally related differentially expressed genes (n=110) between paired patient NFs, ANNUBPs (designated A) and MPNSTs. Genes were categorized by IPA software. Red, relatively increased expression; blue, relatively decreased expression. Genes within each group are listed in Supplemental Table 2. (G) An MPNST gene activity wheel, depicting select genes associated with RABL6A expression, shows MPNSTs have an activated ‘RABL6A signature’ of gene expression (22, 23). Transcript levels of the indicated genes in MPNSTs: decreased (blue), increased (red), unchanged (black). Circled genes reflect pathway activation (red) or inhibition (blue). Expression of RABL6A is associated with increased (red arrows) or decreased (blue bars) mRNA levels of the displayed genes.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques: Immunohistochemical staining, Immunohistochemistry, Staining, RNA Sequencing, Software, Expressing, Activity Assay, Gene Expression, Activation Assay, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: S462 MPNST cells were transduced with empty vector (EV) or p27 shRNA viruses and subsequently infected with control (CON) or RABL6A shRNAs (KD1 and KD2). (A) Quantification of the fold change in cell death after RABL6A knockdown shows that p27 inactivation rescues cell death caused by RABL6A loss. (B) Representative westerns show partial (KD1) to full (KD2) restoration of RB1 phosphorylation (p-RB1) upon co-depletion of p27 with RABL6A. Below, cell ratio (relative to control cells) for the displayed experiment. (C) ImageJ quantification of western data from three or more experiments showing partial to full restoration of phospho-RB1 when p27 is depleted in tandem with RABL6A. (D) Representative images of EdU staining show p27 inactivation rescues DNA synthesis in RABL6A depleted cells. Percent EdU positive cells (cyan colored nuclei versus dark blue nuclei for EdU negative cells) are denoted. (E) Quantification of EdU positivity from three independent experiments. Data in A, C, and E are from three of more independent experiments. Error bars, SD from mean. P-value, Student’s t-test with Bonferroni’s correction. *, p<0.05; **, p<0.01.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques: Transduction, Plasmid Preparation, shRNA, Infection, Control, Knockdown, Phospho-proteomics, Western Blot, Staining, DNA Synthesis

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: (A) Western blots show elevated RABL6A levels in both NF1-associated (T265p21, 90–8, S520 and S462) and sporadic (STS26T [26T]c and YST-1) MPNST cell lines versus precursor, primary normal human Schwann cells (NHSC). (B) Representative westerns show that knockdown of RABL6A (KD1 and KD2) causes induction of cleaved PARP, a marker of apoptosis, in MPNST cells but not in NHSC. Loading control, GAPDH or γ-tubulin. Cell number relative to CON (cell ratio) for each cell line is shown. CON, empty vector control. (C) Quantification of cell death, as measured by trypan blue exclusion, shows RABL6A knockdown induces the death of MPNST cells but not NHSC. P-value, Student’s t-test with Dunnett’s correction for KD versus CON; **, p<0.01. ns, not significant. (D) RABL6A loss in both NHSC and MPNST cells causes reduced DNA synthesis, as measured by EdU positivity relative to CON cells (set at 1.0). **, P<0.001 for all samples compared to controls for each cell line. (E) Pie chart schematics of representative DNA content analyses show RABL6A loss causes G1 accumulation and decreased S phase. Data in B – E are from three of more independent experiments. C, D: Error bars, SD from mean.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques: Western Blot, Knockdown, Marker, Control, Plasmid Preparation, DNA Synthesis

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: (A) Representative westerns of MPNST cell lines expressing empty vector (CON) versus RABL6A shRNAs (KD1 and KD2). RABL6A loss causes p27 upregulation and CDK2 downregulation. (B) ImageJ quantification of western data from three or more experiments showing fold increase in p27 expression following RABL6A knockdown. (C) Representative westerns show loss of RABL6A in MPNST cells causes reduced phosphorylation of RB1 at CDK4/6 targeted sites (S807/811). (D, E) ImageJ quantification of western data from three or more experiments showing reduced levels of phospho-RB1 (D) and CDK2 (E) in RABL6A knockdown cells relative to CON cells (set at 1.0). (F) Proposed model of the RABL6A-p27-RB1 pathway and how it contributes to MPNST cell proliferation and survival. Dashed arrow indicates RABL6A may also act through RB1-independent mechanisms to promote MPNST pathogenesis. Data in B, D and E are normalized to loading control or total RB1, as indicated. Error bars, SD from mean. P-value, Student’s t-test with Dunnett’s correction for each KD versus the CON (set at 1.0) for each cell line. *, p<0.05; **, p<0.01.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques: Expressing, Plasmid Preparation, Western Blot, Knockdown, Phospho-proteomics, Control

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: (A) MPNST cells were treated for 3 days with varying concentrations of palbociclib and relative cell number assessed by CellQuant. IC50 values indicated at top right. (B) Left, MPNST cells were treated for 2 days with 10 μM palbociclib and viability assessed by trypan blue exclusion. Right, Representative westerns showing reduced p-RB1 at CDK4/6 sites after drug treatment. Below, cell ratios relative to untreated controls show palbociclib reduces MPNST cell number. (C) Relative number of primary human NHSCs after palbociclib treatment for 3 days, as measured by AlamarBlue, in low passage (≤ P4) or high passage (≥ P7) cells. IC50 value for low passage NHSC at top right. (D) Left, Percent cell death in low versus high passage NHSCs showing no induction of death by palbociclib. Right, Representative westerns verifying reduced p-RB1 S807/811 in drug treated cells. (E) S462 CON and RABL6A depleted cells (KD1 and KD2) were treated with palbociclib at varying concentrations for 3 days and relative cell number assessed by AlamarBlue. Loss of RABL6A reduces S462 cell sensitivity to palbociclib. (F) Left, Fold change in cell death reveals cells lacking RABL6A undergo reduced drug induced death compared to CON cells. Right, Representative westerns show that palbociclib effectively reduces p-RB1 807/811 in CON and KD cells. Below, ratio of p-RB1 to total RB1. Data in A-F obtained from three or more experiments. Error bars, SD from mean. B, D, F: P-value, Student’s t-test (F: with Dunnett’s correction); **, p<0.01. E: P-values determined by a generalized linear model to assess the difference between the curves.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques:

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: RABL6A is an essential driver of MPNSTs that negatively regulates the RB1 pathway and sensitizes tumor cells to CDK4/6 inhibitors

doi: 10.1158/1078-0432.CCR-19-2706

Figure Lengend Snippet: (A) MPNST cells were treated for 3 days with the indicated concentrations of dinaciclib and relative cell number quantified by AlamarBlue. IC50 values are shown. (B) Contour plot of interaction index (Bliss independence model) for the combination of low doses of CDK4/6 (palbociclib) and CDK2 (dinaciclib) inhibitors in sNF96.2 cells. Red, synergy; green, antagonism. (C) Colony formation assays in sNF96.2 cells show synergism between low concentrations of dinaciclib (2 or 3 nM) and palbociclib. Left, representative images. Right, quantification of percentage area covered by cells from 3 or more experiments. P<0.0001 for all comparisons except Dina vs Palbo-125. (D) Fold change in tumor volume for sNF96.2 orthotopic tumors treated daily with vehicle (water), palbociclib (0.5 mg/dose), dinaciclib (0.16 mg/dose) or the combination. P<0.05 for all comparisons except Dina vs Combo and Dina vs Palbo, which were non-significant. (E) IHC staining for Ki-67, S100+, and phospho-RB1 S807/811 in sNF96.2 tumor samples after treatment. H&E images at 400X magnification. IHC images at 200X magnification. Quantification of (F) Ki-67, (G) phospho-RB1 S807/811, and (H) CC3 in treated tumor samples. G: *, p<0.05 for Combo vs Veh and Combo vs Dina, other comparisons not significant. H: no comparisons significant. A, C: Error bars, SD from the mean. D, F, G, H: Error bars, SEM. C, F, G, H: P-value, Student’s t-test with Bonferroni correction. D: P-values determined by a generalized linear model to assess the difference between the curves.

Article Snippet: Three well characterized human MPNST cell lines were used for these studies: the sporadic human MPNST cell line STS26T (referred to as ‘26T’; NF1 wild-type; Cellosaurus RRID:CVCL_8917), NF1-associated human MPNST cell line S462 (Cellosaurus RRID:CVCL_1Y70), and NF1-associated human MPNST cell line sNF96.2 (ATCC CRL-2884).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: PAMR1 negatively impacts cell proliferation and migration of Human Colon Cancer HT29 Cell Line

doi: 10.1101/2022.09.07.506931

Figure Lengend Snippet: ( A) Expression of PAMR1 in normal and tumoral cancer samples from TCGA Firebrowse database ( http://firebrowse.org/ ). PAMR1’s expression is downregulated in Cervical and Endocervical Cancers (CESC), Colon Adenocarcinoma (COAD), Colorectal Adenocarcinoma (COADREAD) and Rectal Adenocarcinoma (READ) tumoral samples compared to normal ones. ( B, C, D and E) RNA Seq data Analysis showed a significant PAMR1 mRNA down expression in tumoral samples compared to normal samples in rectal, colon, colorectal adenocarcinomas and cervical cancer respectively. This downregulation is pronounced in all pathological stages (Stage I, Stage II, Stage III, Stage IV) and TNM stages, as early as stage I and T stage. The box plots represent the mean of Log2 RSEM ± SEM. The RNA Seq database analysis was carried out by PAST software. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Three human colorectal cancer cell lines were used in this study, namely HCT116 (ATCC CCL-247), HT29 (ATCC HTB38) and SW620 (ATCC CCL-227).

Techniques: Expressing, RNA Sequencing, Software

Journal: bioRxiv

Article Title: PAMR1 negatively impacts cell proliferation and migration of Human Colon Cancer HT29 Cell Line

doi: 10.1101/2022.09.07.506931

Figure Lengend Snippet: PAMR1 transcripts are significantly downregulated in colorectal cancer cell lines (HCT116, HT29, and SW620), compared to normal colon CCD841CoN cells. Similarly, to CRC cell lines, cervical cancer HeLa cells also exhibited very low PAMR1 expression with high CT values around 36 (normal cervix cell lines not available). The histogram represents mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Three human colorectal cancer cell lines were used in this study, namely HCT116 (ATCC CCL-247), HT29 (ATCC HTB38) and SW620 (ATCC CCL-227).

Techniques: Expressing

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) A provisional network was generated from integration of two microarray data sets. Node color represents increases (red), no significant changes (yellow), and decreases (green) in gene expression in murine prostate tissue after cholesterol alteration as ascertained by cDNA microarray. Changes in RNA expression levels of the corresponding nodes in LNCaP cells are shown as colored node boundaries (donut shape) and the color represents increases (red), no significant change (yellow), and decreases (green) in gene expression under CDM conditions compared to control. Arrows indicate direct activation, T-shaped lines direct repression, dashed arrows indirect activation, and lines physical interaction. ( B ) Gene expression under Normo and Hyper conditions ( in vivo ). To verify in vivo microarray data obtained from SCID experiments, mRNA levels of the indicated genes were determined. GAPDH expression was used to normalize gene expression. Error bars represent SD (n = 3). ( C ) Gene expression under Control and Cholesterol-depleted conditions ( in vitro ). LNCaP cells were incubated in CDM for 0, 3 or 16 h, and mRNA levels of the indicated genes were measured by RT-PCR analysis to validate cDNA microarray data. Error bars represent SD (n = 3). * p <0.05 (Student’s t-test).

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Generated, Microarray, Gene Expression, RNA Expression, Control, Activation Assay, In Vivo, Expressing, In Vitro, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: The Response of the Prostate to Circulating Cholesterol: Activating Transcription Factor 3 (ATF3) as a Prominent Node in a Cholesterol-Sensing Network

doi: 10.1371/journal.pone.0039448

Figure Lengend Snippet: ( A ) RT-PCR analysis in vivo . ATF3 levels are reduced in all prostatic lobes from Hyper mice, compared to those from the Normo group (AP = anterior prostate; VP = ventral prostate; DLP = dorsal prostate). ( B ) Immunoblot analysis. Immunoblot of PrEC lysates showed induction of ATF3 protein by CDM (left panel) and by β-cyclodextrin (right panel). MG132, a proteasome inhibitor, also increased ATF3 expression. ( C ) Immunofluorescence analysis. Induction of ATF3 protein by CDM in LNCaP cells as shown by IF. LNCaP cells were treated with CDM for 18 h, stained with anti-ATF3 antibody and nuclei were counterstained with DAPI (left panel: ATF3; middle panel: DAPI; right panel: overlay). ( D ) RT-PCR analysis. ATF3 mRNA levels in LNCaP cells treated with CDM were normalized to levels of GAPDH. RT-PCR analysis shows induction of ATF3 mRNA levels by CDM. ( E–F ) Promoter reporter analysis. A full-length ATF3 promoter was cloned into a luciferase reporter vector and transfected into LNCaP (D) or PrEC (E). Cells were then incubated in Control and CDM medium. ATF3 promoter activity was plotted as arbitrary units (± SD) after normalization with total protein concentration.

Article Snippet: LNCaP human prostate tumor cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% FBS and 1% Penicillin/Streptomycin at 37°C under 5% CO 2 .

Techniques: Reverse Transcription Polymerase Chain Reaction, In Vivo, Western Blot, Expressing, Immunofluorescence, Staining, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Incubation, Control, Activity Assay, Protein Concentration

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization of GCB‐resistant bladder cancer cells . (A) Schematic illustration of the establishment of GCB‐resistant bladder cancer cell lines (T24GCB and 5637GCB). (B) Cell viability of parental (T24, J82 and 5637) and GCB‐resistant (T24GCB and 5637GCB) bladder cancer cells was assessed using the MTT assay following 48 h treatment with a range of GCB concentrations (0, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1 and 3 µM; n = 6 per group). (C, F) Colony formation assays were used to evaluate the clonogenic survival of T24, J82, 5637, T24GCB and 5637GCB cells treated with GCB (0, 0.0001, 0.001 and 0.01 µM) for 14 days ( n = 3 per group). (D, G) Migration assay was used to evaluate the migratory ability of the indicated bladder cancer cells treated with GCB (0 and 0.1 µM) for 24 h ( n = 3 per group). (E, H) Invasion assay was used to evaluate the invasive potential of bladder cancer cells treated with GCB (0 and 0.1 µM) for 24 h ( n = 3 per group). (I) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, CMPK1, NME23, TK1, TK2, NT5C2, CNT3, ABCB1 and ABCG2), epithelial‐mesenchymal transition markers (E‐Cadherin and Vimentin) and the anti‐apoptotic protein BCL‐2 in parental and resistant bladder cancer cells following treatment with 0.01 µM GCB for 24 h. Data are presented as mean ± SEM. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: MTT Assay, Migration, Invasion Assay, Western Blot, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization and functional analysis of EVs in GCB‐resistant bladder cancer cells . (A) MTT assay was used to evaluate the viability of T24 and 5637 cells co‐cultured with T24‐ConMed and 5637‐ConMed or T24GCB‐ConMed and 5637GCB‐ConMed for 24 h, followed by GCB treatment, as described in panel E ( n = 6 per group). (B) Representative transmission electron microscopy images of EVs derived from bladder cancer cells (5637‐EVs, T24‐EVs and J82‐EVs) and their GCB‐resistant counterparts (5637GCB‐EVs and T24GCB‐EVs). Scale bar: 100 nm. (C) Nanoparticle tracking analysis (NS300) was used to evaluate the size distribution and concentration of EVs from 5637, 5637GCB, T24, T24GCB and J82 cells. (D) Imaging flow cytometry analysis of EVs stained with a lipid bilayer dye and labelled with EV surface markers CD9‐APC, CD63‐FITC and CD81‐PE. Each dot represents a single EV. (E) Representative fluorescence microscopy images of T24 cells following a 6‐h incubation with T24‐EVs, T24GCB‐EVs, or J82‐EVs pre‐labelled with CD9‐APC, CD63‐FITC and CD81‐PE. Scale bar: 10 µm. (F) Western blot analysis of EV markers (CD9, CD63, CD81, Alix and TSG101) and endosomal protein Rab27A in EVs from the indicated cell lines. (G) MTT assay evaluating the viability of T24 and 5637 cells co‐cultured with the indicated EVs for 24 h, followed by GCB treatment (0–3 µM) for 48 h ( n = 6 per group). (H and I) Colony formation assay was used to evaluate the clonogenic potential of T24 and 5637 cells after co‐culture with T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs, or J82‐EVs for 14 days ( n = 3 per group). (J) Western blot analysis of GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) and anti‐apoptotic protein BCL‐2 in T24 and 5637 cells co‐cultured with T24‐ConMed, T24GCB‐ConMed, or 5637‐ConMed, 5637GCB‐ConMed, or T24‐EVs, T24GCB‐EVs, or 5637‐EVs, 5637GCB‐EVs for 24 h. (K, M, N) Electron microscopy images of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs) in T24 and T24GCB cells. Quantification of MVBs per image (M) and ILVs per MVB (N). (L, O, P) Electron microscopy images of T24 cells incubated with T24‐EVs or T24GCB‐EVs for 24 h showing MVB and ILV structures. Quantification of MVBs per image (O) and ILVs per MVB (P). Data are presented as mean ± SEM. Statistical comparisons were performed using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were performed independently in triplicate. MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; GCB, gemcitabine; EVs, extracellular vesicles; MVBs, multivesicular bodies; ILVs, intraluminal vesicles.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, MTT Assay, Cell Culture, Transmission Assay, Electron Microscopy, Derivative Assay, Concentration Assay, Imaging, Flow Cytometry, Staining, Fluorescence, Microscopy, Incubation, Western Blot, Colony Assay, Co-Culture Assay, Two Tailed Test

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Identification of GCB resistance‐associated genes in GCB‐resistant bladder cancer cells and their EVs by RNA sequencing . (A) Schematic overview of total RNA sequencing comparing GCB‐sensitive bladder cancer cells (T24 and 5637) with their GCB‐resistant counterparts (T24GCB and 5637GCB), as well as EVs derived from GCB‐sensitive (T24‐EVs and 5637‐EVs) and GCB‐resistant cells (T24GCB‐EVs and 5637GCB‐EVs). (B) The heatmap illustrates DEGs between GCB‐sensitive and GCB‐resistant cells and EVs. DEGs were identified using a fold change > 2 (Log 2 FC > 1.5 or <–1.5) and adjusted p < 0.01. The colour scale represents relative gene expression levels (blue = low, red = high). (C) RT‐qPCR validation of selected DEGs in GCB‐sensitive and GCB‐resistant bladder cancer cells, as well as their corresponding EVs. (D) Pathway enrichment analysis of DEGs identified in GCB‐resistant EVs identified several significantly enriched pathways, including the ROS pathway, peroxisome, Myc targets V2 and angiogenesis. (E) Comparative heatmap of proteomic profiling illustrates differential protein expression in GCB‐resistant versus GCB‐sensitive EVs. The analysis identified overlapping candidates with RNA‐seq data from both cell (T24GCB vs. T24) and EV (T24GCB‐EVs vs. T24‐EVs) datasets. (F and G) Canonical pathway analysis using IPA identified overlapping enriched pathways between GCB‐resistant and GCB‐sensitive EVs in (F) 5637GCB‐EVs versus 5637‐EVs and (G) T24GCB‐EVs versus T24‐EVs. GCB, gemcitabine; EVs, extracellular vesicles; DEGs, differentially expressed genes; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; ROS, reactive oxygen species; IPA, Ingenuity Pathway Analysis.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: RNA Sequencing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Biomarker Discovery, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 in GCB resistance in bladder cancer cells . (A) Western blot analysis of histone H3.2 (H3C14) protein expression in GCB‐sensitive bladder cancer cells (T24 and 5637) versus their GCB‐resistant counterparts (T24GCB and 5637GCB) and J82 cells. (B) Western blot analysis of histone H3.2 (H3C14) protein levels in EVs isolated from 5637, 5637GCB, T24, T24GCB and J82 cells. (C) Western blot analysis of histone H3.2 (H3C14) in T24 and 5637 cells after 24 h treatment with conditioned media (ConMed) or EVs derived from T24, T24GCB and 5637, 5637GCB cells. (D) RT‐qPCR analysis was used to evaluate the knockdown efficiency of H3C14 RNA in T24 and 5637 cells transfected with H3C14 siRNA (siH3C14#1 and siH3C14#2). (E) MTT assay was used to evaluate cell viability in T24 and 5637 cells transfected with scramble siRNA (Vector) or siH3C14 following treatment with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (F and I) Colony formation assays were used to evaluate measuring clonogenic survival clonogenicity of T24 and 5637 cells transfected with Vector or siH3C14 over 14 days ( n = 3 per group). (G and J) Migration assays were used to evaluate the migratory capacity of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (H and K) Invasion assays were used to evaluate the invasive ability of T24 and 5637 cells transfected with Vector or siH3C14 ( n = 3 per group). (M and N) Flow cytometry analysis of apoptosis in T24 and 5637 cells transfected with Vector or siH3C14 following treatment with 0.01 µM gemcitabine. Apoptosis was assessed using PI and Annexin V staining ( n = 3 per group). (L) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, DCK, TK1, TK2, NT5C2 and CNT3) in T24 and 5637 cells transfected with Vector, siH3C14#1, or siH3C14#2. For all panels, data are presented as mean ± SEM. An unpaired two‐tailed Student's t ‐test was used for comparisons between groups. * p < 0.05, ** p < 0.01 and *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PI, propidium iodide.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, Western Blot, Expressing, Isolation, Derivative Assay, Quantitative RT-PCR, Knockdown, Transfection, MTT Assay, Plasmid Preparation, Migration, Flow Cytometry, Staining, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Functional characterization of H3C14 gain‐of‐function in GCB‐resistant bladder cancer cells and its role in GCB sensitivity . (A) RT‐qPCR analysis was conducted to confirm H3C14 overexpression in T24GCB, 5637GCB and J82 cells transfected with H3C14‐GFP plasmid. (B) Representative fluorescence microscopy images show subcellular localization of H3C14‐GFP in T24GCB, 5637GCB, and J82 cells. Scale bar: 20 µm. (C) MTT assays were used to evaluate cell viability in T24GCB‐H3C14‐GFP, 5637GCB‐H3C14‐GFP and J82‐H3C14‐GFP cells after 48 h treatment with GCB concentrations ranging from 0 to 3 µM ( n = 6 per group). (D) Western blot analysis of histone H3.2 (H3C14) and GCB‐metabolizing enzymes and transporters (CNT1, ENT1, NT5C2 and CNT3) in T24GCB, 5637GCB and J82 cells transfected with either vector or H3C14‐GFP, with or without 0.01 µM GCB treatment. (E and F) Western blot analysis of histone H3.2 (H3C14) protein stability in T24 and T24GCB cells treated with cycloheximide (CHX, 50 µg/mL) for 0, 2, 4 or 6 h, and with MG132 (10 µM) for 6 h. (G) Schematic diagram of xenograft tumour model in nude mice—T24GCB‐Vector or T24GCB‐H3C14 cells were subcutaneously injected, followed by GCB treatment (1 mg/kg every 2 days for 28 days). (H) Representative tumour images from the four treatment groups: Vector, Vector + GCB, H3C14 and H3C14 + GCB ( n = 3 mice per group). (I) Tumour weight comparison among the four groups ( n = 3 mice per group). (J) Tumour growth curves present changes in tumour volume over time across treatment groups ( n = 3 mice per group). (K) Histogram of final tumour weights from each treatment group ( n = 3 mice per group). (L–O) IHC staining of H3C14, CNT3 and TK1 in xenograft tumours from the four groups. H‐scores were calculated to quantify expression levels ( n = 3 mice per group). (P–R) Kaplan–Meier survival analysis of patients with bladder cancer from TCGA datasets based on high versus low expression of H3C14, CNT3 and TK1. (S) TCGA survival analysis based on high H3C14 and low CNT3 expression revealed the highest survival probability in this subgroup. Data are presented as mean ± SEM. Statistical comparisons were made using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were independently repeated at least three times. GCB, gemcitabine; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; IHC, Immunohistochemical; TCGA, The Cancer Genome Atlas.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Functional Assay, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Fluorescence, Microscopy, Western Blot, Injection, Comparison, Immunohistochemistry, Expressing, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction, Immunohistochemical staining

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Regulation of H3C14 Contributes to Gemcitabine Resistance in Bladder Cancer

doi: 10.1002/jev2.70179

Figure Lengend Snippet: Characterization of H3.2 (H3C14)‐carrying EVs in GCB Resistance . (A) Schematic illustration of the isolation of EV subpopulations from T24GCB cells using MicroBeads Pan, targeting CD9, CD63 and CD81. (B) MTT assay was used to assess the viability of T24 and 5637 cells treated with PBS, ultracentrifugation‐derived T24GCB‐EVs (or 5637GCB‐EVs), or MicroBeads Pan‐isolated EVs (+MicroBeads Pan), or EVs not captured by MicroBeads Pan (–MicroBeads Pan). Cells were subsequently treated with a series of GCB concentrations (0–3 µM) for 48 h ( n = 6 per group). (C) Mass spectrometry‐based comparative proteomic profiling of Transport‐EVs and Excretion‐EVs. The heatmap illustrates differentially expressed proteins, with red indicating higher expression and blue indicating lower expression levels. (D) Western blot analysis of EV‐associated markers (CD9 and CD81), histone H3.2 (H3C14), LAMB1 and CD147 in EVs derived from ultracentrifugation, MicroBeads Pan‐enriched (+MicroBeads Pan), or non‐captured (–MicroBeads Pan) fractions from T24GCB‐EVs and 5637GCB‐EVs. EVs may be broadly classified into Transport‐EVs (either CD9+, CD63+, or CD81+) and Excretion‐EVs (lacking CD9, CD63 and CD81). (E) Western blot analysis of histone H3.2 (H3C14), Alix, LAMB1 and CD147 in EVs isolated via ultracentrifugation or immunoprecipitation using magnetic beads conjugated with IgG, anti‐CD147, or anti‐LAMB1 antibodies from T24GCB‐EVs. (F–I) Imaging flow cytometry analysis of EVs derived from T24‐EVs, T24GCB‐EVs and T24GCB‐H3C14‐EVs (TG‐H3C14 EVs). EVs were stained with a lipid bilayer dye and labelled with CD9‐APC, CD63‐FITC, CD81‐PE, LAMB1‐AF568 and CD147‐Cy7 antibodies. Percentage gated values represent the proportion of EVs co‐expressing the indicated surface markers: (F) CD63⁺CD9⁺, CD63⁺CD81⁺, CD63⁺LAMB1⁺ and CD63⁺CD147⁺ (G) CD9⁺CD81⁺, CD9⁺LAMB1⁺ and CD9⁺CD147⁺ (H) CD81⁺LAMB1⁺ and CD81⁺CD147⁺ (I) CD147⁺LAMB1⁺ For all panels, data are presented as mean ± SEM. Statistical significance was determined using an unpaired two‐tailed Student's t ‐test. * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were repeated independently at least three times. GCB, gemcitabine; EVs, extracellular vesicles; RT‐qPCR, reverse transcription quantitative polymerase chain reaction; MTT, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide; PBS, paraformaldehyde.

Article Snippet: Human bladder cancer cell lines T24, J82 and 5637 were obtained from the American Type Culture Collection and the Bioresource Collection and Research Centre.

Techniques: Isolation, MTT Assay, Derivative Assay, Mass Spectrometry, Expressing, Western Blot, Immunoprecipitation, Magnetic Beads, Imaging, Flow Cytometry, Staining, Two Tailed Test, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 1. Cyanidin chloride (CyCl) inhibits cell proliferation in colon cancer cell lines. The antitumor effect of CyCl on HCT116, HT29, and SW620 cells was measured by trypan blue staining. Cells were treated with 0, 10, 25, and 50 µM of CyCl for 24, 48, and 72 h. Each experiment was performed in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared with control.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Staining, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 3. CyCl induces apoptosis in colon cancer cell lines. (A) HCT116, HT29, and SW620 cells were treated with indicated concentrations of CyCl and incubated for 24 h. Protein extracts were separated by SDS-PAGE, and Western blot analysis was conducted for the levels of B-cell lymphoma 2-associated X protein (Bax), X-linked inhibitor of apoptosis protein (XIAP), cleaved caspase-3, and poly-ADP-ribose polymerase (PARP) cleavage. (B) Colon cancer cells were treated with 50 µM of CyCl and incubated for 24 h. RNA was extracted from the cells and mRNA expression of Bax, B-cell lymphoma 2 (Bcl2), cellular inhibitor of apoptosis protein (cIAP)1, and cIAP2 was measured by qRT-PCR analysis. All experiments were carried out in triplicate. * p < 0.05 and ** p < 0.01, significantly different compared with control.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Incubation, SDS Page, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 4. CyCl suppresses the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway in colon cancer cells. (A) HCT116, HT29, and SW620 cells were transiently transfected with NF-κB-Luc plasmids for 24 h and treated with 0, 25, and 50 µM of CyCl for 24 h. Cell extracts were harvested, and luciferase assays were performed. (B) HCT116, HT29, and SW620 cells were treated with tumor necrosis factor-alpha (TNF-α) and 50 µM of CyCl and incubated for 24 h. Protein extracts were separated by SDS-PAGE, and Western blot analysis was conducted for the expressions of p-inhibitor of kappa B alpha (IκBα), IκBα, p-inhibitor of nuclear factor kappa-B kinase subunit alpha/beta (IKKα/β), IKKα, IKKβ, p65, and p50 protein. (C) HCT116, HT29, and SW620 cells were treated with TNF-α and 50 µM of CyCl and incubated for 24 h. RNA was extracted from the cells and mRNA expression of TNF-α and IL-6 was measured by qRT-PCR analysis. (D) The inhibitory effect of CyCl (10 µM) on IKKβ kinase activity in vitro was analyzed by the SelectScreen™Biochemical Kinase Profiling Service. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared with control; # p < 0.05, ## p < 0.01, and ### p < 0.001, significantly different compared with TNF-α-treated cells.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Transfection, Luciferase, Incubation, SDS Page, Western Blot, Expressing, Quantitative RT-PCR, Activity Assay, In Vitro, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 5. CyCl induces Nrf2 activation and the expression of antioxidant enzymes. (A) 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) radical scavenging activity (%) of CyCl. (B) HCT116, HT29, and SW620 cells were treated with TNF-α and 50 µM of CyCl and incubated for 24 h. RNA was extracted from the cells and mRNA expression of NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase-1 (HO-1) was measured by qRT-PCR analysis. (C) HCT116, HT29, and SW620 cells were treated with TNF-α and 50 µM of CyCl and incubated for 24 h. Protein extracts were separated by SDS-PAGE and Western blot analysis was conducted for the expression of NQO1 and HO-1 protein. (D) Representative immunoblots of nuclear and cytosolic proteins demonstrating changes in the subcellular localization of Nrf2 following CyCl treatment in TNF-α-stimulated cells. The quantitative results are expressed as the mean ± SD of three independent experiments. (E) HCT116, HT29, and SW620 cells were transiently transfected with Nrf2-Luc plasmids for 24 h and treated with 50 µM of CyCl for 24 h. Cell extracts were harvested, and luciferase assays were performed. All experiments were carried out in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared with control; # p < 0.05 and ## p < 0.01, significantly different compared with TNF-α-treated cells.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Activation Assay, Expressing, Activity Assay, Incubation, Quantitative RT-PCR, SDS Page, Western Blot, Transfection, Luciferase, Control

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 6. Knockdown of Nrf2 by Nrf2 small interfering RNA (siRNA) abolishes CyCl-induced apoptosis. (A) HCT116, HT29, and SW620 cells were transfected with 15 nM of control or Nrf2 siRNA. After 24 h, cells were treated with 50 µM of CyCl for 24 h and harvested. The expression of Nrf2, Bax, and Bcl2 mRNA was measured by qRT-PCR. (B) HCT116, HT29, and SW620 cells were transfected with 15 nM of control or Nrf2 siRNA for 24 h and treated with 50 µM of CyCl for 24 h. After protein isolation, Western blot analysis was conducted for the levels of cleaved caspase-3, Bax, and Nrf2 protein. (C) HCT116 cells were transfected with control or Nrf2 siRNA for 24 h and treated with 50 µM of CyCl for 24 h. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All experiments were carried out in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared with empty vector-transfected control; # p < 0.05 and ### p < 0.001, significantly different compared with siNrf2-transfected control.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Knockdown, Small Interfering RNA, Transfection, Control, Expressing, Quantitative RT-PCR, Isolation, Western Blot, MTT Assay, Plasmid Preparation

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 7. Nrf2 knockdown abrogates the suppression of NF-κB signaling induced by CyCl. (A) The NF-κB-response-element-Luc reporter was cotransfected with Nrf2 siRNA into colon cancer HCT116, HT29, and SW620 cells. After 24 h, cells were treated with or without 50 µM of CyCl. After 24 h, cells were lysed for the luciferase assay. (B) HCT116 cells were transfected with control or Nrf2 siRNA. After 24 h, cells were treated with 50 µM of CyCl for 24 h. Western blot analysis was performed for the levels of p-IκBα, IκBα, p-IKKα/β, and IKKβ protein. All experiments were carried out in triplicate. * p < 0.05, ** p < 0.01, and *** p < 0.001, significantly different compared with empty vector-transfected control.

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Knockdown, Luciferase, Transfection, Control, Western Blot, Plasmid Preparation

Journal: Antioxidants (Basel, Switzerland)

Article Title: Cyanidin Chloride Induces Apoptosis by Inhibiting NF-κB Signaling through Activation of Nrf2 in Colorectal Cancer Cells.

doi: 10.3390/antiox9040285

Figure Lengend Snippet: Figure 8. CyCl inhibits colon cancer foci formation. Colony formation assay shows a significant reduction in colony formation in HCT116, HT29, and SW620 cell lines treated with 50 µM of CyCl. Quantitative analyses are given in the form of bar graphs for HCT116, HT29, and SW620 cells, respectively. A dramatic decrease in colony formation is evident from the figure. Statistically significant values are marked with an asterisk. (*** p < 0.001).

Article Snippet: Human colon cancer HCT116, HT29, and SW620 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in accordance with ATCC’s guidelines.

Techniques: Colony Assay