Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Fluorescein punctate staining are the fluorescein-incorporated corneal epithelial cells of the superficial layer featured with decreased MUC1 expression. (A) The clinical or slit lamp view under cobalt blue light of the eyes of sham-operated rabbit model (lower panel) or dry eye rabbit model ( upper panel ) following fluorescein staining. (B) The fluorescein-stained corneal epithelia prepared as the frozen tissue sections were stained with MUC1 for immunohistochemistry ( brown , left panel ) and immunofluorescence ( middle and right panel ) analyses. For immunofluorescence, dual channels ( red and blue , middle panel ) or triple channels ( red , blue , and green , right panel ) were used. The cell nuclei were counterstained with hematoxylin ( purple , immunohistochemistry) or DAPI ( blue , immunofluorescence). (C) The frozen corneal epithelia section prepared from sham-operated and lacrimal gland-removed rabbits were immunostained with ZO-1 ( red ), nuclear counterstained with DAPI ( blue ), and the fluorescein ingress ( green ) were analyzed through a confocal microscopy. ( red and blue dual channels, middle panel ; red , blue , and green triple channels, right panel ).

Article Snippet: The expression of the truncated MUC1 was performed using MUC1 cDNA ORF Clone, human (HG12123-UT, SinoBiology) and Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: The fluorescein intensity is reversely correlated with the level of MUC1 in HCECs cultured in MPM. (A) The HCECs that have been grown in KSFM were cultured in MPM for two or five days, and the levels of MUC1, MUC4, and MUC16 mRNA were examined. (B) Representative flow cytometric analyses of MUC1 expressions on cell membranes of HCECs cultured in KSFM ( left ) or MPM ( right ) were presented in contour plots. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1 expression in HCECs cultured in KSFM ( left ) or MPM ( right ) followed by fluorescein staining were presented in contour plots. (D) The HCECs cultured in KSFM or MPM were stained with fluorescein, and the levels of fluorescence ( green ) and MUC1 ( red ) were examined using an immunofluorescence microscopy. Nuclei were stained with DAPI ( blue ). The images were obtained via a confocal microscopy.

Article Snippet: The expression of the truncated MUC1 was performed using MUC1 cDNA ORF Clone, human (HG12123-UT, SinoBiology) and Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Cell Culture, Fluorescence, Expressing, Staining, Immunofluorescence, Microscopy, Confocal Microscopy

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Diquafosol treatments preferentially promotes the expression of MUC1 on cell membranes. (A) The HCECs were grown in a culture media containing 0, 0.5, or 1.0 mM diquafosol for four days, and the levels of MUC1, MUC4, and MUC16 mRNA of treated cells were examined using qRT-PCR. (B) Representative flow cytometric analyses of MUC1, MUC4, and MUC16 expression on cell membranes of HCECs treated with 1 mM diquafosol ( red ) or control ( gray ) were presented in overlaid histograms of signal intensity. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1 expression in HCECs treated with sham ( left ) or 1 mM diquafosol ( right ) followed by fluorescein staining were presented in contour plots.

Article Snippet: The expression of the truncated MUC1 was performed using MUC1 cDNA ORF Clone, human (HG12123-UT, SinoBiology) and Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: Knockdown of MUC1 leads to an increase in fluorescein ingress. (A) The mRNA levels of MUC1, MUC4, and MUC16 in HCECs separately transduced with two lentiviral-based shRNA vectors (shMUC1-1 or shMUC1-2) were examined using RT-PCR. The β-actin gene was used as an internal control. (B) Representative flow cytometric analyses of fluorescence intensity and MUC1 expressions in transduced HCECs followed by fluorescein staining were shown in contour plots.

Article Snippet: The expression of the truncated MUC1 was performed using MUC1 cDNA ORF Clone, human (HG12123-UT, SinoBiology) and Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Fluorescence, Staining

Journal: Investigative Ophthalmology & Visual Science

Article Title: Transmembrane Mucin 1 Blocks Fluorescein Ingress to Corneal Epithelium

doi: 10.1167/iovs.63.2.31

Figure Lengend Snippet: The overexpression of MUC1 lacking the extracellular domain is unable to block fluorescein ingress. (A) Flow cytometric analyses of keratin 3, keratin 12, and vimentin expressions in HCECs cultured in KSFM ( gray ) or MPM ( red ) were presented in overlaid histograms of signal intensity. (B) Schematic diagrams of the wild-type and N-terminally truncated MUC1. (C) Representative flow cytometric analyses of fluorescence intensity and MUC1-C levels in HCECs transfected with the control vector ( left ) or the vector encoded with N-terminally truncated MUC1s ( right ) followed by fluorescein staining were shown in contour plots.

Article Snippet: The expression of the truncated MUC1 was performed using MUC1 cDNA ORF Clone, human (HG12123-UT, SinoBiology) and Lipofectamine 3000 Reagent (Thermo Fisher Scientific) according to the manufacturer's instructions.

Techniques: Over Expression, Blocking Assay, Cell Culture, Fluorescence, Transfection, Plasmid Preparation, 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: 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: a Diagram showing the strategy employed to shortlist AICAR-binding proteins. The three steps include in silico screening using FINDSITE comb2.0 , protein expression assay, and thermal stability assay. b Time-dependent western blotting and relative quantification of protein expression for AICAR-binding proteins. The treatment responses on H1975 cells treated with 1 mM AICAR for 1 and 2 h were grouped by strong inactivation (type 1) and weak response (type 2). GAPDH was used as a loading control. N = 2–3 replicates. c Dose-dependent western blotting and relative quantification of protein expression for MUC1-CT and TMEM70. H1975 cells were treated with increasing doses of AICAR (0, 0.4, 1.3, and 4.4 mM) for 22 h, followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. d Thermal stability assay for MUC1-CT and TMEM70. H1975 cells were treated with 1 mM AICAR for 15 min. The cell pellets were heated for 3 min at their respective temperature (37–55 °C), followed by a western blot assay. N = 2 replicates. e Immunofluorescence staining for MUC1-CT in H441 cells. The cells were treated with 0.3 mM AICAR for 4 h and then incubated with rabbit anti-MUC1-CT primary antibody followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. The nucleus was counterstained with DAPI. The images were taken using a Keyence fluorescent microscope. Scale bar, 50 μm. f qRT-PCR analysis for MUC1-CT targeting genes. Expression levels for CTGF , PGM2 , and ENO1 were analysed by qRT-PCR in H1975 cells treated with 0.3 mM AICAR for 4 h. GAPDH was used as an endogenous control. N = 3 replicates. g qRT-PCR analysis for MUC1 expression in H1975 cells with MUC1 overexpression (OE). The cells were transfected with a lentiviral vector containing MUC1 or scrambled control, followed by 0.5 µg/ml puromycin selection. The relative MUC1 expression level in scrambled control cells was calibrated as 1. GAPDH was used as an endogenous control. N = 3 replicates. h Organoid formation assay for AICAR treatment response in H1975 cells overexpressing MUC1 . The cells with MUC1 overexpression or a scrambled control vector were plated at 2000 cells per well and treated with vehicle or 0.3 mM AICAR continuously for nine days. Images were taken with an EVOS microscope, and the treatment responses were quantified using a 3D Celltiter-Glo assay. N = 4–5 replicates. Scale bar, 300 µm. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , f , h ); Welch’s t -test ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; 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: Binding Assay, In Silico, Expressing, Stability Assay, Western Blot, Control, Immunofluorescence, Staining, Incubation, Microscopy, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Selection, Tube Formation Assay, Glo Assay

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: a Top KEGG signalling pathways differentially expressed in H1975 cells treated with AICAR. The cells were treated with 1 mM AICAR for 4 h, followed by whole transcriptomic analysis. N = 3 replicates. b Enrichment plot by gene set enrichment analysis for the JAK-STAT signalling pathway in H1975 cells treated with AICAR. Profile of the running enrichment score (ES) (top) and positions of gene set members on the rank-ordered list (bottom) were shown. N = 3 replicates. c A heat map showing top enriched genes of the JAK-STAT signalling pathway in H1975 cells treated with AICAR compared with vehicle-treated cells. d Longitudinal analysis of p-JAK1, p-TYK2, and MUC1-CT expression. H1975 cells were treated with 1 mM AICAR for 0, 1, and 2 h followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. e Confocal images for co-localisation of MUC1-CT and p-JAK1 in lung cancer cells. H1975 cells were co-incubated with Armenian hamster anti-MUC1-CT and rabbit anti-p-JAK1 primary antibodies for 2 h. Then the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 555. The nucleus was counterstained with DAPI. The images (top: lower magnification; bottom: higher magnification) were taken using a Zeiss confocal fluorescent microscope. Scale Bar, 50 µm (top) and 20 µm (bottom). f Duolink ligation assay and confocal imaging for physical MUC1-JAK1 interactions. H441 cells were treated with vehicle or 1 mM AICAR for 1 h. After treatment, the cells were incubated with mouse anti-ZO-1 primary antibody overnight, followed by anti-mouse IgG conjugated with Alexa Fluor 488. Then the cells were co-incubated with anti-MUC1 and anti-JAK1 primary antibodies, followed by incubation with proximity ligation assay probes conjugated with Cy3, ligation, and amplification steps. The nucleus was counterstained with DAPI. The images were taken using a Zeiss confocal fluorescent microscope, and the Duolink dots were quantified using Image J. Scale Bar, 20 µm. g Cell viability assay of H1975 cells treated with AICAR and VX-509. Cells were plated in a 96-well plate and treated with AICAR (1 mM) with or without VX-509 (10 μM). The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated control group. N = 3–4 replicates. Data are mean ± s.e.m. and were analysed with Welch’s t -test ( a , b , f ); Brown-Forsythe and Welch one-way ANOVA ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; 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, Control, Incubation, Microscopy, Ligation, Imaging, Proximity Ligation Assay, Amplification, Viability Assay

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: 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: Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Expressing, 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: a qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues from transgenic mice. The far normal and tumour tissues from transgenic mice 14 weeks after Dox induction were extracted for RNA analysis. Gapdh was used as an endogenous control. N = 3 replicates. b Differential MUC1 gene expression in lung adenocarcinoma (LUAD) compared with tumour-adjacent tissues by analysing the TCGA_LUAD dataset. N = 59 (normal), and N = 517 (tumour). c Patients’ overall survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 135. d Patients’ disease-free survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 105. e Xenograft tumour growth in mice treated with AICAR. The xenograft tumour was pre-established by implanting 1 million H1975 cells subcutaneously. When the tumour reached 45 mm 3 , the mice were treated with 300 mg/kg/day AICAR in PBS or a vehicle subcutaneously for ten days. The tumour was measured with a digital caliper, and the tumour size was calculated. N = 7 replicates. f Xenograft tumour images and relative weight quantification from mice treated with AICAR or PBS. The average tumour weight from the PBS-treated group is normalised as 1. N = 7 replicates. g Mouse body weight after treatment with AICAR or PBS for ten days. N = 7 replicates. h , i H&E staining of subcutaneous tumours ( h ) and liver tissues ( I ) from H1975 cell line-derived xenograft (CDX) treated with PBS or AICAR. Scale bar, 125 μm. j – l Immunofluorescence staining for Ki-67 ( j ), γ-H 2 AX ( k ), and p21 Cip1 ( l ) in subcutaneous tumours from H1975 CDX treated with PBS or AICAR. Scale bar, 100 μm. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b ); log-rank test ( c , d ); Welch’s t -test ( e – g ). * p < 0.05; ** p < 0.01; **** 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: Quantitative RT-PCR, Expressing, Transgenic Assay, Control, Staining, Derivative Assay, Immunofluorescence, 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: 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: Transgenic Assay, Derivative Assay

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: a Diagram showing the strategy employed to shortlist AICAR-binding proteins. The three steps include in silico screening using FINDSITE comb2.0 , protein expression assay, and thermal stability assay. b Time-dependent western blotting and relative quantification of protein expression for AICAR-binding proteins. The treatment responses on H1975 cells treated with 1 mM AICAR for 1 and 2 h were grouped by strong inactivation (type 1) and weak response (type 2). GAPDH was used as a loading control. N = 2–3 replicates. c Dose-dependent western blotting and relative quantification of protein expression for MUC1-CT and TMEM70. H1975 cells were treated with increasing doses of AICAR (0, 0.4, 1.3, and 4.4 mM) for 22 h, followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. d Thermal stability assay for MUC1-CT and TMEM70. H1975 cells were treated with 1 mM AICAR for 15 min. The cell pellets were heated for 3 min at their respective temperature (37–55 °C), followed by a western blot assay. N = 2 replicates. e Immunofluorescence staining for MUC1-CT in H441 cells. The cells were treated with 0.3 mM AICAR for 4 h and then incubated with rabbit anti-MUC1-CT primary antibody followed by goat anti-rabbit IgG conjugated with Alexa Fluor 555. The nucleus was counterstained with DAPI. The images were taken using a Keyence fluorescent microscope. Scale bar, 50 μm. f qRT-PCR analysis for MUC1-CT targeting genes. Expression levels for CTGF , PGM2 , and ENO1 were analysed by qRT-PCR in H1975 cells treated with 0.3 mM AICAR for 4 h. GAPDH was used as an endogenous control. N = 3 replicates. g qRT-PCR analysis for MUC1 expression in H1975 cells with MUC1 overexpression (OE). The cells were transfected with a lentiviral vector containing MUC1 or scrambled control, followed by 0.5 µg/ml puromycin selection. The relative MUC1 expression level in scrambled control cells was calibrated as 1. GAPDH was used as an endogenous control. N = 3 replicates. h Organoid formation assay for AICAR treatment response in H1975 cells overexpressing MUC1 . The cells with MUC1 overexpression or a scrambled control vector were plated at 2000 cells per well and treated with vehicle or 0.3 mM AICAR continuously for nine days. Images were taken with an EVOS microscope, and the treatment responses were quantified using a 3D Celltiter-Glo assay. N = 4–5 replicates. Scale bar, 300 µm. Data are mean ± s.e.m. and were analysed with Brown-Forsythe and Welch ANOVA ( b , c , f , h ); Welch’s t -test ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; 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: Binding Assay, In Silico, Expressing, Stability Assay, Western Blot, Control, Immunofluorescence, Staining, Incubation, Microscopy, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Selection, Tube Formation Assay, Glo Assay

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: a Top KEGG signalling pathways differentially expressed in H1975 cells treated with AICAR. The cells were treated with 1 mM AICAR for 4 h, followed by whole transcriptomic analysis. N = 3 replicates. b Enrichment plot by gene set enrichment analysis for the JAK-STAT signalling pathway in H1975 cells treated with AICAR. Profile of the running enrichment score (ES) (top) and positions of gene set members on the rank-ordered list (bottom) were shown. N = 3 replicates. c A heat map showing top enriched genes of the JAK-STAT signalling pathway in H1975 cells treated with AICAR compared with vehicle-treated cells. d Longitudinal analysis of p-JAK1, p-TYK2, and MUC1-CT expression. H1975 cells were treated with 1 mM AICAR for 0, 1, and 2 h followed by a western blot assay. β-actin was used as a loading control. N = 3 replicates. e Confocal images for co-localisation of MUC1-CT and p-JAK1 in lung cancer cells. H1975 cells were co-incubated with Armenian hamster anti-MUC1-CT and rabbit anti-p-JAK1 primary antibodies for 2 h. Then the cells were incubated with secondary antibodies conjugated with Alexa Fluor 488 or 555. The nucleus was counterstained with DAPI. The images (top: lower magnification; bottom: higher magnification) were taken using a Zeiss confocal fluorescent microscope. Scale Bar, 50 µm (top) and 20 µm (bottom). f Duolink ligation assay and confocal imaging for physical MUC1-JAK1 interactions. H441 cells were treated with vehicle or 1 mM AICAR for 1 h. After treatment, the cells were incubated with mouse anti-ZO-1 primary antibody overnight, followed by anti-mouse IgG conjugated with Alexa Fluor 488. Then the cells were co-incubated with anti-MUC1 and anti-JAK1 primary antibodies, followed by incubation with proximity ligation assay probes conjugated with Cy3, ligation, and amplification steps. The nucleus was counterstained with DAPI. The images were taken using a Zeiss confocal fluorescent microscope, and the Duolink dots were quantified using Image J. Scale Bar, 20 µm. g Cell viability assay of H1975 cells treated with AICAR and VX-509. Cells were plated in a 96-well plate and treated with AICAR (1 mM) with or without VX-509 (10 μM). The cell viability was measured three days after treatment. Values were normalised to a vehicle-treated control group. N = 3–4 replicates. Data are mean ± s.e.m. and were analysed with Welch’s t -test ( a , b , f ); Brown-Forsythe and Welch one-way ANOVA ( d , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; 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, Control, Incubation, Microscopy, Ligation, Imaging, Proximity Ligation Assay, Amplification, Viability Assay

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: 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: Western Blot, Transgenic Assay, Extraction, Quantitative RT-PCR, Expressing, 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: a qRT-PCR analysis of gene expression for EGFR and MUC1 in EGFR TL -induced lung tissues from transgenic mice. The far normal and tumour tissues from transgenic mice 14 weeks after Dox induction were extracted for RNA analysis. Gapdh was used as an endogenous control. N = 3 replicates. b Differential MUC1 gene expression in lung adenocarcinoma (LUAD) compared with tumour-adjacent tissues by analysing the TCGA_LUAD dataset. N = 59 (normal), and N = 517 (tumour). c Patients’ overall survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 135. d Patients’ disease-free survival in lung adenocarcinoma patients at stages II–IV. The median expression levels of MUC1 were used for a cut-off of high and low expression for MUC1 . N = 105. e Xenograft tumour growth in mice treated with AICAR. The xenograft tumour was pre-established by implanting 1 million H1975 cells subcutaneously. When the tumour reached 45 mm 3 , the mice were treated with 300 mg/kg/day AICAR in PBS or a vehicle subcutaneously for ten days. The tumour was measured with a digital caliper, and the tumour size was calculated. N = 7 replicates. f Xenograft tumour images and relative weight quantification from mice treated with AICAR or PBS. The average tumour weight from the PBS-treated group is normalised as 1. N = 7 replicates. g Mouse body weight after treatment with AICAR or PBS for ten days. N = 7 replicates. h , i H&E staining of subcutaneous tumours ( h ) and liver tissues ( I ) from H1975 cell line-derived xenograft (CDX) treated with PBS or AICAR. Scale bar, 125 μm. j – l Immunofluorescence staining for Ki-67 ( j ), γ-H 2 AX ( k ), and p21 Cip1 ( l ) in subcutaneous tumours from H1975 CDX treated with PBS or AICAR. Scale bar, 100 μm. Data are mean ± s.e.m. and were analysed with unpaired two-tailed t -test ( a , b ); log-rank test ( c , d ); Welch’s t -test ( e – g ). * p < 0.05; ** p < 0.01; **** 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: Quantitative RT-PCR, Expressing, Transgenic Assay, Control, Staining, Derivative Assay, Immunofluorescence, 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: 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: Transgenic Assay, Derivative Assay

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: 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: 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: 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 ® with 10% fetal bovine serum as a chemoattractant. MUC1 overexpression promotes the proliferation, migration, and invasiveness of ACHN cells ( A , C , E ), whereas MUC1 depletion in RCC4 cells ( B , D , E ) has the opposite effect. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

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, Over Expression

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: Expression of MUC1 in ccRCC cells is associated with a multi-drug-resistant phenotype. MUC1 ACHN-overexpressing cells (MUC1FL) ( A ), MUC1 RCC4-depeleted cells (MUC1KO) ( B ), and their respective controls were exposed to increasing concentrations of cisplatin, oxaliplatin, etoposide, SN38, paclitaxel, or vinblastine. IC50 (half-maximal inhibitory concentration), a valid marker of drug sensitivity, was determined in each condition after 72 h of drug treatment using the MTS assay. The overexpression of MUC1 confers the ACHN cells a multi-drug-resistant profile ( A ), whereas the depletion of MUC1 in the RCC4 cells ( B ) has the opposite effect. Values are represented as the mean ± SEM and represent at least three separate experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001).

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, Concentration Assay, Marker, MTS Assay, Over Expression

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: The IC50 values of various conventional systemic therapies in ACHN and RCC4 cells.

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:

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: The expression of MUC1 in ccRCC cells is associated with their resistance to targeted therapies. MUC1-overexpressing ACHN cells (MUC1FL) ( A ), MUC1 RCC4-depeleted cells (MUC1KO) ( B ), and their respective controls were exposed to increasing concentrations of cabozantinib, crizotinib, dovitinib, or sunitinib. IC50 (half-maximal inhibitory concentration), a valid marker of drug sensitivity, was determined in each condition after 72 h of drug treatment using the MTS assay. While the modulation of MUC1 has no effect on sunitinib sensitivity ( A , B ), its overexpression impaired the responses of ACHN cells to the other tested targeted agents ( A ), whereas its depletion in RCC4 cells ( B ) has the opposite effect. 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, Concentration Assay, Marker, MTS Assay, Over Expression

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: The IC50 values of various targeted therapies in ACHN and RCC4 cells.

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:

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: 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: Modulation of MUC1 expression in ccRCC cells influences drug efflux pump activity. The intracellular fluorescence of the eFluxx-ID TM 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-ID TM gold detection reagent in the presence (or not) of verapamil (an ABCB1/P-gP inhibitor), MK-571 (an ABCC1/MRP1 inhibitor), and novobiocin (an ABCG2/BCRP inhibitor). Overexpression of MUC1 in ACHN cells promotes overall drug efflux pump activity ( A ) and ABCB1-, ABCC1-, and ABCG2-specific activity ( C ), whereas its depletion in RCC4 cells decreases the overall drug efflux pump activity ( B ) and ABCB1-specific activity ( D ). 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, Activity Assay, Fluorescence, Over Expression

Journal: Cancers

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

doi: 10.3390/cancers16020391

Figure Lengend Snippet: MUC1 increases the expression of cancer stem cell markers. Relative expression of Nanog and Sox2 was determined using qPCR in ACHN ( A ) and RCC4 cells ( B ). Overexpression of MUC1 in ACHN cells increases the expression of the stem cell markers Nanog and Sox2 ( 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, ** p < 0.01, and *** p < 0.001).

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: medRxiv

Article Title: Monitoring tubular epithelial cell damage in AKI via urine flow cytometry

doi: 10.1101/2022.01.31.22270101

Figure Lengend Snippet: The amount of urinary tubular epithelial cells (TEC) in patients with acute kidney injury (AKI) can be measured using flow cytometry. (A and B) Example dot-plots for the gating strategy used in the (A) exploratory study showing proximal (CD10 + ) and distal (CD326 + ) TEC, gated on cytokeratin positive cells using unstained controls, and in the (B) confirmatory study, where proximal (CD13 + CD10 + ) and distal (CD326 + CD227 + ) TEC can be found, each gated on cytokeratin positive cells using FMO controls. (C and D) Dot-plots of a suspended post-mortem (C) renal tissue sample, showing CD13 + CD10 + and CD326 + CD227 + cell populations, and of a post-mortem (D) ureteral tissue sample, indicating the absence of CD13 + CD10 + or CD326 + CD227 + cell populations. Both gated on cytokeratin positive cells using FMO controls. (E and F) Fluorescence microscopic image of (E) purified Cytokeratin + DAPI + CD13 + CD10 + cells and (F) purified Cytokeratin + DAPI + CD326 + CD227 + cells (blue = DAPI, green = cytokeratin). (G to J) Electron microscopic images of purified Cytokeratin + CD227 + cells using a cell sorter. (G and detail H) Apoptotic cell with characteristic marginalization of condensed chromatin (yellow arrows in H). (I and J) Necrotic cells with chromatin clumping (red arrow in J) and loss of plasma membrane integrity (yellow arrow in J).

Article Snippet: The complete staining protocol included pan-cytokeratin-APC (Clone: REA831, Isotype: Recombinant human IgG1) or, exclusively for fluorescence microscopy, pan-cytokeratin-FITC (Clone: CK3-6H5, Isotype: Mouse IgG1), CD13-PE (Clone: REA263, Isotype: Recombinant human IgG1), CD10-PE-Vio770 (Clone: 97C5, Isotype: Mouse IgG1), CD326-PE (Clone: HEA-125, Isotype: Mouse IgG1) and CD227-PE-Vio770 (Clone: REA448, Isotype: Recombinant human IgG1) (all Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and DAPI (Sigma-Aldrich, Hamburg, Germany).

Techniques: Flow Cytometry, Fluorescence, Purification, Clinical Proteomics, Membrane

Journal: medRxiv

Article Title: Monitoring tubular epithelial cell damage in AKI via urine flow cytometry

doi: 10.1101/2022.01.31.22270101

Figure Lengend Snippet: Using flow cytometry, the amount of urinary TEC functions as a biomarker for the detection of acute kidney injury (AKI). HC, healthy control; IPC, inpatient control; 1-3, KDIGO stage of patients with AKI. Control groups were pooled for comparison calculations. (A and B) Amount of proximal TEC (Cytokeratin + CD10 + ) and distal TEC (Cytokeratin + CD326 + ) in the exploratory cohort with n=21 patient samples, n=5 healthy control samples and n=5 control samples from inpatients without AKI. (C and D) Amount of proximal TEC (Cytokeratin + CD13 + CD10 + ) and distal TEC (Cytokeratin + CD326 + CD227 + ) in the confirmatory cohort with n=63 patient samples, n=5 healthy control samples and n=10 inpatient control samples. Comparisons calculated by Mann-Whitney test, for detailed results of statistical testing see Supplementary table S1 . (E) Receiver-operator characteristic (ROC) analysis for urinary proximal and distal TEC comparing patients with AKI and patients of both control groups used in the exploratory (blue) and confirmatory (black) study. Area under the curve (AUROC) for proximal TEC: 0.8619 (exploratory study); 0.9487 (confirmatory study). AUROC for distal TEC: 0.8095 (exploratory study); 0.9238 (confirmatory study). (F) Correlation of urinary proximal distal TEC counts per 100 mL urine each for exploratory (blue) and confirmatory (black) cohort. The cell counts were correlated with each other with relatively higher cell counts for distal TEC. (Exploratory cohort: Spearman r=0.6935, p=0.0005; confirmatory cohort: Spearman r=0.8262, p<0.0001).

Article Snippet: The complete staining protocol included pan-cytokeratin-APC (Clone: REA831, Isotype: Recombinant human IgG1) or, exclusively for fluorescence microscopy, pan-cytokeratin-FITC (Clone: CK3-6H5, Isotype: Mouse IgG1), CD13-PE (Clone: REA263, Isotype: Recombinant human IgG1), CD10-PE-Vio770 (Clone: 97C5, Isotype: Mouse IgG1), CD326-PE (Clone: HEA-125, Isotype: Mouse IgG1) and CD227-PE-Vio770 (Clone: REA448, Isotype: Recombinant human IgG1) (all Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and DAPI (Sigma-Aldrich, Hamburg, Germany).

Techniques: Flow Cytometry, Biomarker Discovery, Control, Comparison, MANN-WHITNEY