Journal: Advanced Science

Article Title: Tumor‐Derived CDC37 Inhibits Antigen Cross‐Presentation in Dendritic Cells and Impairs Anti‐Tumor Immunity in Breast Cancer

doi: 10.1002/advs.202506518

Figure Lengend Snippet: Antigen cross‐presenting was inhibited in breast cancers with high TMB and poor CTL infiltration. A,B). Representative immunofluorescence images of tumor‐specific CD8 + T cells in tumor site denoted by co‐staining of CD8 + and MUC1‐pentamer + (A) or GZMB + (B) in TNBC patients with high TMB and high CTL infiltration (TMB hi CTL hi , n = 21) or the ones with high TMB and low CTL infiltration (TMB hi CTL lo , n = 20). Asterisks denote the area of higher magnification images shown at the top right corner. Scale bar, 50 µm. C) The count of CD8 + MUC1‐pentamer + T cells (top) and CD8 + GZMB + T cells (bottom) in the tumor site of TNBC patients with different therapeutic responses to ICB. CR, complete response, n = 5; PR, partial response, n = 46; SD, stable disease, n = 21; PD, progressive disease, n = 12. D) Correlation between TMB and CD8 + GZMB + T cells in tumor biopsies of TNBC patients ( n = 84. Spearman's correlation coefficient r and two‐tailed P value). Cutoffs of median of CD8 + GZMB + T cells and median of TMB are given by dashed vertical and horizontal lines, respectively. E) Response (PR and CR) rates in percentages and 95% confidence intervals (CI) in subgroups defined by the cutoffs given as dashed lines in (D). F) Representative images and quantification of CD8 + GZMB + T cells in TdLN of TNBC patients with TMB hi CTL hi ( n = 21) or TMB hi CTL lo tumor ( n = 20). Asterisks denote the area of higher magnification images shown at the top right corner. Scale bar, 50 µm. G) Representative flow cytometric plots and quantification of GZMB staining in the CD8 + T cells from tumor site, TdLN and blood of TMB hi CTL hi ( n = 9) and TMB hi CTL lo ( n = 6) TNBC patients. H) Naive CD8 + T cells were cultured alone (‐) or primed by cDC1 isolated from tumor site or TdLN of TMB hi CTL hi ( n = 9) or TMB hi CTL lo ( n = 6) TNBC patients. Representative flow cytometric plots and quantification of percentages of GZMB staining in the in vitro primed CD8 + T cells. Results are mean ± s.d. of independent experiments producing similar results (C, F–H). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with indicated group, were calculated using two‐tailed one‐way analysis of variance (ANOVA) with Tukey's multiple‐comparisons test (C) or compared with TMB hi CTL lo group using two‐tailed Student's t test (F–H).

Article Snippet: DCs were treated with TCM, the soluble components or EVs of TMB high CTL high or TMB high CTL low tumor supernatants, respectively, for 16–24 h, followed by incubation with 200 μg mL −1 tumor lysates or 250 μg mL −1 OVA protein (Cat# 9006‐59‐1, MCE) or MUC1 recombinant protein (Cat# HY‐ P78740 , MCE) according to the experiment design.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Cell Culture, Isolation, In Vitro

Journal: Advanced Science

Article Title: Tumor‐Derived CDC37 Inhibits Antigen Cross‐Presentation in Dendritic Cells and Impairs Anti‐Tumor Immunity in Breast Cancer

doi: 10.1002/advs.202506518

Figure Lengend Snippet: CDC37 checked cytosolic antigen release by binding to HSP90/antigen complex in DC endosomes. A–C) DCs were pulsed with tumor lysates (A) or MUC1 peptides (B,C) and treated with PBS, EVs from His‐CDC37‐overexpressing SKBR3 tumor cells (A‐B) or EVs from wild type (CDC37 WT ) MDA‐MB‐468 tumor cells or CDC37 knockout (CDC37 KO ) MDA‐MB‐468 tumor cells (C), respectively. A,B) Proteins binding to His‐CDC37 in DCs was detected by co‐immunoprecipitation (Co‐IP) with anti‐His antibody. (A) Co‐IP followed by mass spectrometry (MS) identified HSP90 and MUC1 as internalized CDC37 binding peptides ( n = 3). (B) Binding of HSP90 and MUC1 to internalized His‐CDC37 in DCs was evaluated by Co‐IP with anti‐His antibody, followed by immunoblotting ( n = 3 independent experiments). C) Binding of HSP90 and MUC1 to total CDC37 in DCs was evaluated by Co‐IP with anti‐CDC37 antibody, followed by immunoblotting ( n = 3 independent experiments). D,E) DCs treated with EVs from CDC37 WT or CDC37 KO MDA‐MB‐468 tumor cells, respectively, were pulsed with MUC1 peptides for 1 h and then were washed and chased with medium for the indicated intervals. Representative images of immunofluorescence staining for CDC37, RAB5, HSP90, and MUC1 in DCs at 0 min (D) or 60 min (E) after being pulsed with MUC1 peptides. D) Scatterplots of CDC37 and RAB5 pixel intensities or CDC37 and MUC1 pixel intensities were shown with the Pearson correlation coefficient (PCC) above. E) Scatterplots of MUC1 and CDC37 pixel intensities or MUC1 and HSP90 pixel intensities were shown with the PCC above.

Article Snippet: DCs were treated with TCM, the soluble components or EVs of TMB high CTL high or TMB high CTL low tumor supernatants, respectively, for 16–24 h, followed by incubation with 200 μg mL −1 tumor lysates or 250 μg mL −1 OVA protein (Cat# 9006‐59‐1, MCE) or MUC1 recombinant protein (Cat# HY‐ P78740 , MCE) according to the experiment design.

Techniques: Binding Assay, Knock-Out, Immunoprecipitation, Co-Immunoprecipitation Assay, Mass Spectrometry, Western Blot, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Tumor‐Derived CDC37 Inhibits Antigen Cross‐Presentation in Dendritic Cells and Impairs Anti‐Tumor Immunity in Breast Cancer

doi: 10.1002/advs.202506518

Figure Lengend Snippet: Blocking CDC37/HSP90 interaction promoted cytosolic antigen release and enhanced ICB efficacy. A) Binding of CDC37 to HSP90 in endosomes of DCs treated with MDA‐MB‐468 tumor EVs and different concentration of DDO‐5936 were determined by Co‐IP followed by immunoblotting ( n = 3 independent experiments). B) DCs transfected with HSP90‐YFP and RAB5‐GFP plasmids were treated with CDC37‐CFP‐overexpressing SKBR3 tumor EVs and administrated with 25 µ m DDO‐5936. The interaction between CDC37 and HSP90 in DC endosomes was detected by confocal fluorescence microscopy. Representative live‐cell imaging for FRET signals arising from the binding of CDC37‐CFP to HSP90‐YFP in the region of interest (ROI), based on RAB5 expression, by monitoring the fluorescence signals in the range of 550−620 nm with excitation of YFP at 405 nm ( n = 3 independent experiments). Scale bar, 5µm. Quantification is shown in Figure (Supporting Information). C) Representative flow cytometric plots and quantification of early cell apoptosis induced by endosome‐cytosol export of internalized cytC in DCs pre‐treated with tumor EVs from MDA‐MB‐468 and different concentration of DDO‐5936 ( n = 3 independent experiments). Percentages of Annexin V + PI − DCs are shown. D) DCs were pre‐treated with MDA‐MB‐468 tumor EVs and different concentrations of DDO‐5936 for 16 h, followed by the pulse with OVA antigen. Representative images of immunofluorescence staining for OVA and HSP90 in DCs ( n = 3 independent experiments). Scale bar, 5µm. Quantification is shown in Figure (Supporting Information). E) DCs transfected with HSP90‐GFP were treated with MDA‐MB‐468 tumor EVs and 25 µ m DDO‐5936, followed by the pulse with mCherry‐OVA antigen. Representative live cell imaging for the binding of GFP‐HSP90 to mCherry‐OVA in DCs during OVA antigen treatment for 450 seconds ( n = 3 independent experiments). Scale bar, 5 µm. Quantification of co‐localization of HSP90 and OVA at indicated time point is shown. F) DCs pre‐treated with tumor EVs from MDA‐MB‐468 and different concentrations of DDO‐5936 were pulsed with tumor lysates ( n = 3 independent experiments). Representative flow cytometric plots and quantification of GZMB and IFN‐γ expression in the CD8 + T cells primed by DCs. G–J) Mice bearing EO771‐OVA orthotopic grafts were treated with anti‐PD‐L1 antibody and/or DDO‐5936 ( n = 5 per group). (G) Tumor growth curves of tumor‐bearing mice with indicated treatments. (H) Representative flow cytometric plots of SIINFEKL/H‐2K b expression on TiDCs from tumor‐bearing mice with indicated treatments. Quantification is shown in Figure (Supporting Information). I) Representative flow cytometric plots and quantification of the percentages of OVA‐tetramer staining in CD8 + T cells infiltrated in EO771‐OVA grafts with indicated treatment. J) Naive OT‐I CD8 + T cells were co‐cultured with TiDCs isolated from mouse breast tumor. Representative flow cytometric plots and quantification of the percentages of GZMB releasing CD8 + T cells. K–M) EO771‐OVA mouse breast cancer cells were inoculated to the mammary fat pads of immunocompetent syngeneic C57BL/6 mice or Batf3 −/− transgenic mice, and intraperitoneally administered the mice with anti‐PD‐L1 immunotherapy or IgG every three days along with daily DDO‐5936 treatment, initiated on the seventh day following tumor inoculation (n = 5 per group). K) Tumor volumes were monitored every 3 days after palpable tumor formation. L) Representative flow cytometric plots and quantification of tumor‐specific OVA‐tetramer⁺ CD8⁺ T cells. Quantification is shown in Figure (Supporting Information). M) Quantification of dead tumor cells denoted by co‐staining with CK and TUNEL in the harvested grafts. Representative immunofluorescence images are shown in . N–P) NSG mice bearing patient‐derived xenografts (PDXs) from TMB hi CTL lo patient with high CDC37 tumors were transfused with autologous tumor lysate‐pulsed DCs transduced with sgNC or sgHSP90 together with CD8 + T cells, followed by treatment with DDO‐5936, anti‐PD‐L1, or the combination ( n = 3 per group). N) Representative flow cytometric plots and quantification of tumor‐specific MUC1‐pentamer⁺ CD8⁺ T cells. Quantification is shown in Figure (Supporting Information). O) Quantification of dead tumor cells denoted by co‐staining with CK and TUNEL in the harvested grafts. Representative immunofluorescence images are shown in . P) Tumor volumes were monitored every 3 days after palpable tumor formation. Results are mean ± s.d. of independent experiments producing similar results. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with DCs without DDO‐5936 (0 µ m ) (C, F), or tumor‐bearing mice treated with IgG and DMSO (IgG+DMSO) ( I , J, L‐O), were determined by two‐tailed one‐way ANOVA with Dunnett's multiple‐comparisons test (C, F, I, J, L–O). * P < 0.05, ** P < 0.01, were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test (G, K, P). ## P < 0.01, ###P < 0.001, compared with WT (M), or sgNC group(O), under the indicated treatment, were determined by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test.

Article Snippet: DCs were treated with TCM, the soluble components or EVs of TMB high CTL high or TMB high CTL low tumor supernatants, respectively, for 16–24 h, followed by incubation with 200 μg mL −1 tumor lysates or 250 μg mL −1 OVA protein (Cat# 9006‐59‐1, MCE) or MUC1 recombinant protein (Cat# HY‐ P78740 , MCE) according to the experiment design.

Techniques: Blocking Assay, Binding Assay, Concentration Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Fluorescence, Microscopy, Live Cell Imaging, Expressing, Immunofluorescence, Staining, Cell Culture, Isolation, Transgenic Assay, TUNEL Assay, Derivative Assay, Transduction, Two Tailed Test

Journal: Advanced Science

Article Title: Tumor‐Derived CDC37 Inhibits Antigen Cross‐Presentation in Dendritic Cells and Impairs Anti‐Tumor Immunity in Breast Cancer

doi: 10.1002/advs.202506518

Figure Lengend Snippet: CDC37 expression was associated with ICB therapeutic response of breast cancer patients. A–C) TNBC patients who received ICB therapy were employed to detect CDC37 expression by immunohistochemical (IHC) staining in their pre‐treatment specimens and correlate CDC37 IHC scores to the infiltration of tumor‐specific CTLs and ICB efficacy ( n = 84). A) Representative images of IHC staining for CDC37 and immunofluorescence staining for GZMB + CD8 + and MUC1‐pentamer + CD8 + T cells in tumors. Boxes denote the area of higher magnification images shown at the top right corner. Scale bar, 50 µm. B) Correlation between CDC37 IHC scores and the count of CD8 + GZMB + T cells or CD8 + MUC1‐pentamer + T cells in tumor sites of breast cancer patients, respectively ( n = 84, Pearson's correlation coefficient r and two‐tailed P value are shown). C) The CDC37 IHC scores in the tumor site of TNBC patients with different therapeutic responses to ICB (mean ± s.d). CR, complete response, n = 5; PR, partial response, n = 46; SD, stable disease, n = 21; PD, progressive disease, n = 12. ** P < 0.01; *** P < 0.001 by two‐tailed one‐way ANOVA with Tukey's multiple‐comparisons test. D). Correlation between CDC37 IHC scores and the count of CD8 + GZMB + T cells in TNBC patients ( n = 138, Pearson's correlation coefficient r and two‐tailed P value are shown). E). Kaplan‐Meier curves for overall survival in TNBC patients with high (CDC37 hi , >50.5, n = 69) and low (CDC37 lo , <50.5, n = 69) CDC37 expression. Log‐rank P , hazard ratio (HR) and 95% confidence interval (95% CI) are shown. F). Kaplan‐Meier curves for overall survival in TNBC patients from TCGA dataset with high (>157.1, n = 71) and low CDC37 expression (<157.1, n = 72). Log‐rank P , HR and 95% CI are shown.

Article Snippet: DCs were treated with TCM, the soluble components or EVs of TMB high CTL high or TMB high CTL low tumor supernatants, respectively, for 16–24 h, followed by incubation with 200 μg mL −1 tumor lysates or 250 μg mL −1 OVA protein (Cat# 9006‐59‐1, MCE) or MUC1 recombinant protein (Cat# HY‐ P78740 , MCE) according to the experiment design.

Techniques: Expressing, Clinical Proteomics, Immunohistochemical staining, Immunohistochemistry, Immunofluorescence, Staining, Two Tailed Test

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 4 Activated EGFR upregulates MUC1-CT expression. a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1- CT in EGFR TL (T790M; L858R)-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL-induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox- impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL-induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL-induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test (c, d, e, g); one-way ANOVA (a, b); Brown-Forsythe and Welch ANOVA (f, h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1- CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c, d Growth of PDX (c) and transgenic mouse EGFR TL- induced lung tumour (d)-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA (b, c, d). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Blocking Assay, Transgenic Assay, Derivative Assay

Journal: Frontiers in Medicine

Article Title: Urinary CD8+HLA-DR+ T Cell Abundance Non-invasively Predicts Kidney Transplant Rejection

doi: 10.3389/fmed.2022.928516

Figure Lengend Snippet: Gating strategies for T cell subsets (A) and tubular epithelial cells (TEC) (B) . Isotype controls are displayed as blue, while full stains are represented in red. (C.1) Schematic overview of investigated subsets. Proximal TECs were defined CD10+ and CD13+, while distal TECs were characterized being CD227+ and CD326(EpCAM)+. (C.2) Maturation of naïve T cells into memory T cells. (D) Workflow for epigenetic analysis of urine samples. SSC, side scatter; FSC, forward scatter; TNV, naïve T cells; TEM, T effector memory cells; TCM, T central memory cells; TEMRA, T effector memory cells re-expressing CD45RA.

Article Snippet: The following antibodies were used: for T cells anti-CD3-APCeF780 (eBioscience, SK7, mo IgG1k), -CD4-PEVio770 (Miltenyi Biotec, REA623, REA) -CD8-APC (Biolegend, SK1, mo IgG1k) -CD45RO-PE (Biolegend, UCHL1, mo IgG1k2), -CD45-BUV805 (BD, 3D12, rat IgG1ak), -CCR7-BV421 (Biolegend, G043H7, mo IgG2ak), -HLA-DR-BUV395 (BD, G46-6, mo IgG2ak), -CD28-FITC (Biolegend, CD28.2, mo IgG1k) and for tubular epithelial cells anti-Cytokeratin-FITC (Miltenyi Biotec, CK3-6H5, mo IgG1k), -Vimentin-APC (Miltenyi Biotec, REA409, REA), -CD10-PeVio770 (Miltenyi Biotec, REA877, REA), -CD13-APCVio770 (Miltenyi Biotec, REA263, REA), -CD227-PE (Miltenyi Biotec, REA448, REA), -CD326-BV711 (Biolegend, 9C4, mo IgG2b).

Techniques: Expressing

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: Expression and purification of recombinant protein MUC1-PDL1-IgG1 Fc (MUC1-Vax). ( A ) Plasmid-transfected E . coli lysed protein fractions were run on a 12% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. Lane M is the prestained protein molecular weight marker, lane 1 is the bacterial protein before induction, lane 2 is the bacterial protein after isopropyl β-D-1-thiogalactoside (IPTG) induction, and lane 3 is the purified MUC1-Vax protein, and lane 4 is the MUC1-Vax protein after dialysis. Recombinant protein MUC1-Vax (arrow) is indicated. ( B ) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human His primary antibody. ( C ) Western blot analysis of purified recombinant protein MUC1-Vax with anti-human MUC1 antibody.

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Transfection, SDS Page, Staining, Molecular Weight, Marker, Western Blot

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: ( A ) C57BL/6 mice were subcutaneously inoculated with 2 × 10 5 luciferase expressing LLC (LLC-MUC1-PDL1-Luc) or 3 × 10 6 Panc02 (Panc02-MUC1-PDL1-Luc) cells by hypodermic injection (I.H.). The footpads were injected with 2 × 10 6 BMDCs loaded with different proteins on the 7th and 14th days after tumor cell injection, respectively. Splenocytes of immunized mice were isolated 4 days after immunization and the activation of CD4 + T cells and CD8 + T cells (3 per group) and cytokine secretion were detected by flow cytometry. The tumor size, growth, and survival time of the remaining mice were observed (4–5 in each group). ( B ) Activation of CD4 + T cells, data are expressed as mean ± SD. ( C ) Secretion of IL-2 by CD4 + T cells, data are expressed as mean ± SD. ( D ) Secretion of IFN-γ by CD4 + T cells, data are presented as mean ± SD. ( E ) Secretion of IFN-γ by CD8 + T cells, data are presented as mean ± SD. * p < 0.05.

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: Luciferase, Expressing, Injection, Isolation, Activation Assay, Flow Cytometry

Journal: Vaccines

Article Title: A Novel Therapeutic Tumor Vaccine Targeting MUC1 in Combination with PD-L1 Elicits Specific Anti-Tumor Immunity in Mice

doi: 10.3390/vaccines10071092

Figure Lengend Snippet: Cell bioluminescence monitoring was performed in vivo on tumor mice inoculated with LLC-MUC1-PDL1-Luc every 6 days, as shown in figure ( A ) Bioluminescence in vivo of mice inoculated with LLC ( n = 5), blank space means the mouse died; ( B ) In vivo bioluminescence of mice inoculated Panc02 ( n = 3).

Article Snippet: These tumor cells stably expressing human MUC1 and PD-L1 were treated with 5 μg/mL or 2.5 μg/mL puromycin (Solarbio, Beijing, China) in culture medium.

Techniques: In Vivo

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 4 Activated EGFR upregulates MUC1-CT expression. a Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1- CT in EGFR TL (T790M; L858R)-induced lung tissues from transgenic mice. The mice were fed doxycycline (Dox)-impregnated food pellets for 0, 1, and 2 weeks followed by whole lung-tissue extraction. N = 2 replicates. b qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL-induced lung tissues. Gapdh was used as an endogenous control. EG0, EG1, and EG2 represent tissues from the mice fed with dox- impregnated food pellets for 0, 1, and 2 weeks, respectively. N = 3 replicates. c Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT in EGFR TL-induced lung tissues after EGFR inactivation. The mice fed with Dox-impregnated food pellets for 8 weeks were given either the same Dox diet for an additional 2 weeks (EG10) or a regular diet for 2 weeks (EG8off2). Then the whole lung tissues were extracted for protein expression assay. N = 2 replicates. d qRT-PCR analysis of gene expression for EGFR and MUC1 in mouse EGFR TL-induced lung tissues after EGFR inactivation. The lung tissues from EG14 and EG8OFF2 mice were extracted for RNA analysis. GAPDH was used as an endogenous control. N = 3 replicates. e Western blotting and quantitative analysis for p-EGFR (Y1068) and EGFR expression in H1975 cells with MUC1 overexpression (OE). β-actin was used as a loading control. N = 3 replicates. f Western blotting and quantitative analysis for p-EGFR (Y1068), EGFR, and MUC1-CT expression in H1975 cells treated with 1 mM AICAR for one and 2 h. β-actin was used as a loading control. N = 3 replicates. g qRT-PCR analysis for MUC1 gene expression in H1975 cells with MUC1 knockdown. The cells were transfected with a lentiviral vector containing shRNA against MUC1 (shMUC1) or a scrambled control vector (sh-Control), followed by a 0.5 µg/ml puromycin selection. GAPDH was used as an endogenous control. N = 3 replicates. h Cell viability assay of H1975 cells treated with osimertinib and VX-509. 3000 cells with MUC1 knockdown (sh-MUC1) and a negative control vector (sh-control) were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), or both. The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated sh-control group. N = 4 replicates. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t-test (c, d, e, g); one-way ANOVA (a, b); Brown-Forsythe and Welch ANOVA (f, h). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Expressing, Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Gene Expression, Control, Over Expression, Knockdown, Transfection, Plasmid Preparation, shRNA, Selection, Viability Assay, Negative Control, Two Tailed Test

Journal: British journal of cancer

Article Title: An intrinsic purine metabolite AICAR blocks lung tumour growth by targeting oncoprotein mucin 1.

doi: 10.1038/s41416-023-02196-z

Figure Lengend Snippet: Fig. 6 AICAR combined with osimertinib and VX-509 block 3D structure formation in patient and transgenic mouse-derived tumours. a A diagram showing mechanisms of AICAR’s anticancer roles. In MUC1-dependent tumours, AICAR treatment directly binds and degrades MUC1- CT, increasing DNA damage in tumour cells. The degraded MUC1-CT de-stabilises p-EGFR and p-JAK1, further inactivating tumour-supportive signals. Created with BioRender.com. b Treatment response to VX-509 and osimertinib and AICAR in H1975 cells. 3000 cells were plated in a 96-well plate and treated with VX-509 (10 μM), osimertinib (0.5 μM), AICAR (1 mM), or a combination. The cell viability was measured 3 days after treatment. Values were normalised to a vehicle-treated group. N = 4 replicates. c, d Growth of PDX (c) and transgenic mouse EGFR TL- induced lung tumour (d)-derived organoids treated with AICAR, osimertinib, and VX-509. 2000 cells were plated in organoid-culture media followed by treatments with AICAR (1 mM), osimertinib (0.5 μM), VX-509 (10 μM), or combinations for 10 days. The media were replenished every three days. The 3D cultures’ size was measured on day ten by ImageJ. The organoid tumour area in the vehicle-treated group was normalised as 100%. Scale bar, 50 μm. N = 6–12 replicates. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA (b, c, d). *p < 0.05; **p < 0.01; ****p < 0.0001.

Article Snippet: To knock down or overexpress MUC1 expression, 10,000 H1975 cells were transfected with shRNAs against MUC1 (Origene, Cat #TL316625) or Lenti ORF clone of human MUC1 (Origene, Cat #RC221340L4) in a 96-well plate.

Techniques: Blocking Assay, Transgenic Assay, Derivative Assay

Journal: PloS one

Article Title: The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

doi: 10.1371/journal.pone.0042712

Figure Lengend Snippet: Figure 2. MUC1 extracellular domain antibody staining pattern in colonic tissue. Cryosections of rhesus monkey colonic tissues were processed for staining with B27.29 and HMFG1 antibodies as described in Methods. Nuclei were identified using DAPI. Arrows indicate examples of nucleus-associated staining and arrow heads indicate examples of apical plasma membrane staining of ductal epithelial cells. The white bar represents 10 mm. Images are representative of 3 independent experiments. doi:10.1371/journal.pone.0042712.g002

Article Snippet: Transfection with siRNAs Cells were transfected individually with 25 nM each of OnTARGET PLUS SMART pool MUC1 siRNA (L-004019-01-005, Dharmacon), MUC1 siRNA 599 (SR303004A, Origene, Rockville MD), MUC1 siRNA 600 (SR303004B Origene), MUC1 siRNA 601 (SR303004C, Origene), GAPDH siRNA (L-004253-01-000 Dharmacon) and On-TARGET PLUS non-targeting siRNA pool (D-001810-10-20 Dharmacon) using DharmaFECT siRNA Transfection Reagent 1 (Dharmacon, Thermo Scientific, Waltham MA).

Techniques: Staining, Clinical Proteomics, Membrane

Journal: PloS one

Article Title: The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

doi: 10.1371/journal.pone.0042712

Figure Lengend Snippet: Figure 3. Nuclear localization of MUC1 extracellular domain antibody-reactive proteins. (A) Trophoblasts (troph) and MCF-7 cells were stained with B27.29, HMFG1, or b1-integrin antibodies (each shown in red) and then examined by confocal microscopy. Nuclei were stained using DAPI (blue). The images show the staining patterns roughly midway through the respective z-series. Lateral projections (the plane of view is indicated by the yellow horizontal and vertical lines) of individual z-stack series are shown below and to the right of each image. The bars represent 5 mm. The asterisk indicates cytoplasmic/membrane staining. (B) Western blot analysis of total lysates obtained from Jar, MCF-7, and COS7.MUC1 cells using B27.29, DF3, and HMFG1 antibodies. (C) Western blot analysis of subcellular fractions prepared from Jar, MCF-7, and COS7.MUC1 cells using MUC1

Article Snippet: Transfection with siRNAs Cells were transfected individually with 25 nM each of OnTARGET PLUS SMART pool MUC1 siRNA (L-004019-01-005, Dharmacon), MUC1 siRNA 599 (SR303004A, Origene, Rockville MD), MUC1 siRNA 600 (SR303004B Origene), MUC1 siRNA 601 (SR303004C, Origene), GAPDH siRNA (L-004253-01-000 Dharmacon) and On-TARGET PLUS non-targeting siRNA pool (D-001810-10-20 Dharmacon) using DharmaFECT siRNA Transfection Reagent 1 (Dharmacon, Thermo Scientific, Waltham MA).

Techniques: Staining, Confocal Microscopy, Membrane, Western Blot

Journal: PloS one

Article Title: The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

doi: 10.1371/journal.pone.0042712

Figure Lengend Snippet: Figure 4. Effect of MUC1 siRNAs and shRNA on nuclear MUC1 expression. Jar cells were transfected independently with four different MUC1 siRNAs, non-targeting siRNA (NT), or GAPDH siRNA (see Methods). Five days after transfection cells were (A) stained using B27.29 or HMFG1 or (B) lysed and analyzed by Western blotting. In other experiments, Jar cells were stably transfected with MUC1 shRNA as described in Methods and stained using DF3 and HMFG1 antibodies (C) or lysed and analyzed for MUC1 expression by Western blotting (D) and RT-PCR (E). GAPDH was used as a loading control for Western blotting and RT-PCR. NT; non-targeting control. Reagent; transfection reagent alone. Medium; culture medium alone. doi:10.1371/journal.pone.0042712.g004

Article Snippet: Transfection with siRNAs Cells were transfected individually with 25 nM each of OnTARGET PLUS SMART pool MUC1 siRNA (L-004019-01-005, Dharmacon), MUC1 siRNA 599 (SR303004A, Origene, Rockville MD), MUC1 siRNA 600 (SR303004B Origene), MUC1 siRNA 601 (SR303004C, Origene), GAPDH siRNA (L-004253-01-000 Dharmacon) and On-TARGET PLUS non-targeting siRNA pool (D-001810-10-20 Dharmacon) using DharmaFECT siRNA Transfection Reagent 1 (Dharmacon, Thermo Scientific, Waltham MA).

Techniques: shRNA, Expressing, Transfection, Staining, Western Blot, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Control

Journal: PloS one

Article Title: The MUC1 extracellular domain subunit is found in nuclear speckles and associates with spliceosomes.

doi: 10.1371/journal.pone.0042712

Figure Lengend Snippet: Figure 6. Effect of RNase A and transcriptional inhibition on the intranuclear distribution of MUC1. (A) BeWo cells were fixed and permeabilized using methanol and then incubated with RNase A (100 mg/mL) for 2 h as described in Methods. The cells were then stained with antibodies against MUC1 (HMFG1 and B27.29), U2AF65, and spliceosomes as indicated. Nuclei were stained with DAPI. (B) BeWo cells were incubated in the presence of DRB (100 mM) for 2 h and then stained with antibodies against MUC1, U2AF65, and spliceosomes (Spl) as described in Methods. The bar represents 5 mm. Results are representative of 3 independent experiments. doi:10.1371/journal.pone.0042712.g006

Article Snippet: Transfection with siRNAs Cells were transfected individually with 25 nM each of OnTARGET PLUS SMART pool MUC1 siRNA (L-004019-01-005, Dharmacon), MUC1 siRNA 599 (SR303004A, Origene, Rockville MD), MUC1 siRNA 600 (SR303004B Origene), MUC1 siRNA 601 (SR303004C, Origene), GAPDH siRNA (L-004253-01-000 Dharmacon) and On-TARGET PLUS non-targeting siRNA pool (D-001810-10-20 Dharmacon) using DharmaFECT siRNA Transfection Reagent 1 (Dharmacon, Thermo Scientific, Waltham MA).

Techniques: Inhibition, Incubation, Staining

Journal: Cancers

Article Title: MUC1 Drives the Progression and Chemoresistance of Clear Cell Renal Carcinomas.

doi: 10.3390/cancers16020391

Figure Lengend Snippet: Figure 1. Distinct MUC1 protein abundance in genetically engineered ccRCC cell line models. Whole- cell protein extracts from either MUC1-overexpressing ACHN clones or MUC1-depleted RCC4 clones were analyzed through Western blotting using antibodies against MUC1-C and β-actin.

Article Snippet: RCC4 MUC1KO cells were obtained through a CRISPR/Cas9 strategy using the “EMA (MUC1) Human Gene Knockout Kit” (Origene, Rockville, MD, USA).

Techniques: Quantitative Proteomics, Clone Assay, Western Blot

Journal: Cancers

Article Title: MUC1 Drives the Progression and Chemoresistance of Clear Cell Renal Carcinomas.

doi: 10.3390/cancers16020391

Figure Lengend Snippet: Figure 2. Modulation of MUC1 expression influences the proliferation, migration, and invasiveness of ccRCC cells. Proliferation (A,B) and migration (C,D) were assessed using Incucyte technology, whereas cell invasion (E,F) was evaluated using 24-well Boyden chambers coated with Matrigel®

Article Snippet: RCC4 MUC1KO cells were obtained through a CRISPR/Cas9 strategy using the “EMA (MUC1) Human Gene Knockout Kit” (Origene, Rockville, MD, USA).

Techniques: Expressing, Migration

Journal: Cancers

Article Title: MUC1 Drives the Progression and Chemoresistance of Clear Cell Renal Carcinomas.

doi: 10.3390/cancers16020391

Figure Lengend Snippet: Figure 5. MUC1 increases the expression of numerous efflux pumps. Relative expression of ABCC1–6, ABCB1, and ABCG2 was determined using qPCR in ACHN (A) and RCC4 cells (B). Overexpression of MUC1 in ACHN cells increases the expression of many members of the ABC transporter family (A), whereas its depletion in RCC4 cells (B) has the opposite effect. PPIA was used as an internal control. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05 and ** p < 0.01).

Article Snippet: RCC4 MUC1KO cells were obtained through a CRISPR/Cas9 strategy using the “EMA (MUC1) Human Gene Knockout Kit” (Origene, Rockville, MD, USA).

Techniques: Expressing, Over Expression, Control

Journal: Cancers

Article Title: MUC1 Drives the Progression and Chemoresistance of Clear Cell Renal Carcinomas.

doi: 10.3390/cancers16020391

Figure Lengend Snippet: Figure 6. Modulation of MUC1 expression in ccRCC cells influences drug efflux pump activity. The intracellular fluorescence of the eFluxx-IDTM gold detection reagent in ACHN (A) and RCC4 (B) cells is shown. The multi-drug resistance activity factor (MRAF) represented the P-gP, MDR1, and BCRP activities in ACHN (C) and RCC4 (D) cells and was determined using the eFluxx-IDTM gold

Article Snippet: RCC4 MUC1KO cells were obtained through a CRISPR/Cas9 strategy using the “EMA (MUC1) Human Gene Knockout Kit” (Origene, Rockville, MD, USA).

Techniques: Expressing, Activity Assay, Fluorescence