Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: FFPE lung tissue sections from control and COPD donors were stained using immunohistochemistry for ECM and ECM-associated proteins with specific signals being detected with Nova red (red) and counterstained with hematoxylin (blue). Sections were scanned at 40x magnification using a digital slide scanner. Scale bar = 400um. Images are representative of protein detection patterns seen in control donors (n=18), SEO-COPD (n=12), and moderate COPD (n=14). ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Control, Staining, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Parenchyma was isolated and analyzed for percentage positive tissue area and mean intensity of positive pixels of expression for each protein. All 44 donors were available for analysis of COL1A1, COL6A2, FBLN2, LTBP4, LUM, DCN, VCAN, AND ELN, while for COL6A1, COL14A1, and FBLN5 43 donors were available. For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM proteins present in FFPE tissue sections from controls (n=18), SEO-COPD (n=12), and moderate COPD (n=14) were detected using immunohistochemistry. Airway walls were isolated and analyzed for percentage area and mean intensity of expression for each protein. The number of airway walls available for the analysis for each protein were COL1A1 (n= 155), COL6A1 (n=150), COL6A2 (n=149), COL14A1 (n=152), FBLN2 (n=158), FBLN5 (n=173), LTBP4 (n=163), LUM (n=158), DCN (n=156), VCAN (n=165), and ELN (n=158). For each protein, regression coefficients (± 95% CI) were obtained following linear regression and a p value of <0.05 was considered significant. Non-transformed variables are plotted first, followed by log transformed variables. The differences in ECM and ECM-associated proteins in COPD tissue were compared to control in terms of A-C) percentage area and D-F) mean intensity of positively stained pixels. Dark blue-colored bars in light blue colored boxes highlight significant differences. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; FFPE: formalin fixed paraffin embedded; COL1A1: collagen type I α chain 1; COL6A1: collagen type VI α chain 1; COL6A2: collagen type VI α chain 2; COL14A1: collagen type XIV α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Immunohistochemistry, Isolation, Expressing, Transformation Assay, Control, Staining, Formalin-fixed Paraffin-Embedded, Binding Assay

Journal: bioRxiv

Article Title: The lung extracellular matrix protein landscape in severe early-onset and moderate chronic obstructive pulmonary disease

doi: 10.1101/2023.10.20.562391

Figure Lengend Snippet: ECM differences noted in the different analyzes throughout this study have been summarized here. In the staining and ECM signatures, red or blue arrows indicate higher or lower proportional levels or component scores in COPD respectively, while they denote positive or negative associations with FEV1 respectively. ECM: extracellular matrix; SEO-COPD: Severe early-onset COPD patients; COL1A1: type I collagen α chain 1; COL6A1: type VI collagen α chain 1; COL6A2: type VI collagen α chain 2; COL14A1: type XIV collagen α chain 1; FBLN2: fibulin 2; FBLN5: fibulin 5; LTBP4: latent transforming growth factor binding protein 4; LUM: lumican; DCN: decorin; VCAN: versican; ELN: elastin; FEV1: forced expiratory volume in 1 second.

Article Snippet: Following PBS washes, primary antibodies diluted in 1% BSA/PBS for FBLN5 (1:8000, Mouse Anti-Fibulin 5/DANCE Antibody 1G6A4, Novus Biologicals), DCN (1:1500, Mouse Anti-Dermatan Sulfate Proteoglycan Antibody 6B6, Seikagaku), VCAN (1:200, Mouse Anti-Versican Antibody 2B1, Seikagaku), and ELN (1:400, Rabbit Anti-Elastin Antibody CL55011AP, Cedarlane Labs) were added to the respective sections for 1 hour at room temperature.

Techniques: Staining, Binding Assay

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin deficiency in human ccRCC is associated with poor clinical outcome, limited antigen presentation capabilities, and impacts immunotherapy efficacy. Parkin ( PRKN ) downregulation in patients with KIRC is significantly associated with overall survival and tumor stage. A, PRKN gene expression is significantly downregulated in patient tumor samples vs. matched controls ( N = 72 matches samples, P value = 3E−07 via paired Student t test; FPKMs mapped reads data from GDC HTSeq-FPKM pipeline was used). Dashed lines represent cutoffs used to bin patients with KIRC into low, mid, high PRKN gene expression levels. B, PRKN gene expression levels are significantly associated with overall survival ( N = 538 tumor samples, P < 0.0001 via log-rank test for trend). C, PRKN expression is significantly associated with overall survival in patients with stage 4 tumors (log-rank P < 0.007). D, mRNA expression levels of PRKN on HEK293, 786-O, CAKI-I, and A498 cells cultured overnight in growth media. E, mRNA and protein expression levels of A498 Parkin KO or EV cells. F, A498 Parkin KO or EV cells were stimulated with IFNγ (10 ng/mL) for 18 hours. Then, immunoblotting analysis was performed on Parkin, HLA-A, TAP1, and PSMB8. Data represent two to three independent experiments. Immunoblot data represent two to three independent experiments. G, IFNγ ELISA analysis was performed on donor-derived NY-ESO-1-specific T cells (5 × 10 4 ) cocultured in a 1:1 ratio overnight with A498 Parkin KO or EV cells. Tumor cells were prestimulated with IFNγ (10 ng/mL) or mock-treated for 18 hours before the assay. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Immunopeptidomics, Gene Expression, Expressing, Cell Culture, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Two Tailed Test

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates the MHC-I-associated tumor APM and fosters tumor progression. A and B, mRNA expression levels of Prkn , H2-k1 , Tap1 , Psmb8 , and B2m in RENCA cells stably transfected with sh Prkn _1 (sh#1), sh Prkn _3 (sh#3), and empty vector control (pLKO.1) lentiviral vectors. Data are represented as mean ± SD. ***, P < 0.001; unpaired, two-tailed t test. C, Protein expression analyses of Parkin, Tap1, Psmb8, and B2m (immunoblotting) and Parkin vs. MHC-I (intracellular staining, flow cytometry) of pLKO.1 controls, sh#1, and sh#3 RENCA cells. Data represent two to three independent experiments. D, IFNγ ELISPOT analysis was performed on T cells isolated from the spleens of pLKO.1 RENCA tumor-bearing mice at day 25 after challenge. T cells were cocultured overnight with stimulator Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (5:1 ratio). E, Parkin-positive (pLKO.1) or -negative (sh#1, sh#3) RENCA cells (10 6 ) were injected in the back of BALB/c mice and tumor growth was monitored. Data are represented as mean ± SEM. *, P < 0.05; ***, P < 0.001; two-way ANOVA.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Control, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Isolation, Injection

Journal: Cancer Research

Article Title: Parkin Deficiency Suppresses Antigen Presentation to Promote Tumor Immune Evasion and Immunotherapy Resistance

doi: 10.1158/0008-5472.CAN-22-2499

Figure Lengend Snippet: Parkin regulates cytosolic tumor antigen processing and presentation and facilitates effector CD8 + T-cell cancer immunity. A–C, Mouse melanoma B16-OVA Prkn knockout (KO, CRISPr/Cas9) or EV control cells were stimulated for 18 hours with IFNγ (10 ng/mL) or mock-stimulated before the analyses. A, mRNA expression levels of Prkn , H2-k1 , Psmb8 , and Tap1 in B16-OVA Parkin KO or EV cells. Data are represented as mean ± SD. ** P < 0.01; *** P < 0.001; unpaired, two-tailed t test. B, Protein expression analyses of Parkin, Tap1, and Psmb8 (immunoblotting) and SIINFEKL-bound to H-2Kb (MHCi-OVA; intracellular staining, flow cytometry) of B16-OVA KO and EV cells. Data represent two to three independent experiments. C, IFNγ ELISPOT analysis was performed on OT.1 T cells (10 5 ) cocultured overnight with B16-OVA KO and EV cells in a 5:1 ratio. D, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6 mice and tumor growth was monitored. Five mice per group were treated with anti-CD8 blocking antibody (aCD8) or isotype (ISO) control. E, Percentage of CD45 − /MHC-OVA + cells present in B16-OVA Parkin KO or EV isotype-treated tumors analyzed by flow cytometry. F, SIINFEKL-specific CD8 + T-cell tumor infiltration present in B16-OVA Parkin KO or EV isotype-treated tumors. G, mRNA expression levels of IFNγ in B16-OVA Parkin KO or EV isotype-treated tumors. E–G, Data are represented as mean ± SEM. ns, not significant; *, P < 0.05; **, P < 0.01; unpaired, two-tailed t test. H, B16-OVA Parkin KO or EV cells (10 6 ) were injected in the back of C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT.1) mice (5 per group) and tumor growth was monitored. I, Survival rate analysis (Kaplan–Meier, log-rank test) was performed by day 25 posttumor challenge, including five mice per group. D and H, Data are represented as mean ± SEM. ***, P < 0.001; two-way ANOVA.

Article Snippet: The primary antibodies for TAP1, PSMB8, β2-microglobulin (D8P1H), HSP90 (C45G5), β-actin (E4D9Z), Vinculin (VCL; E1E9V), Parkin, PTEN, AMPKa, phospho-AMPKa (Thr172; 40H9), Akt, phospho-Akt (Ser473; D9E), phospho-GSK-3β (Ser9, D85E12), and GSK-3β (D5C5Z) were purchased from Cell Signaling Technology; HLA-A was purchased from Thermo Fisher Scientific; and MHC-I (ER-HR52) was obtained from Santa Cruz Biotechnology.

Techniques: Knock-Out, CRISPR, Control, Expressing, Two Tailed Test, Western Blot, Staining, Flow Cytometry, Enzyme-linked Immunospot, Injection, Blocking Assay

Journal: bioRxiv

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1101/2020.04.10.036103

Figure Lengend Snippet: Upon exogenous stimulation of 5-ALA, cells generate PpIX via the heme biosynthesis pathway. PpIX is subsequently converted to heme by FECH or transported outside the cells through efflux receptors such as ABCB1. As oncogenic transformation activates enzymes of the heme biosynthesis pathway, cancer cells generate PpIX more efficiently than normal cells. As the Ras/MEK pathway promotes PpIX conversion to heme and PpIX efflux through ABCB1, MEK inhibition further enhances PpIX accumulation in cancer cells.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody (sc-55510) from Santa Cruz Biotechnology; anti-HIF-1α antibody (ab179483), and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Transformation Assay, Inhibition

Journal: bioRxiv

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1101/2020.04.10.036103

Figure Lengend Snippet: (A) Representative overlay histograms showing surface ABCB1 expression in NIH3T3 cells and RasV12 cells treated with or without different concentrations of MEK inhibitor, U0126. (B) Plot shows mean ± SD mean fluorescence intensity (MFI) from 3 independent experiments. (C) Mean ± SD relative FECH activity in NIH3T3 cells and RasV12 cells treated with or without different concentrations of U0126 from 3 independent experiments. (D) Representative western blot showing FECH expression and ERK phosphorylation levels in NIH3T3 and RasV12 cells treated with or without different concentrations of U0126. *p<0.01 by one-way ANOVA with Turkey’s posthoc test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody (sc-55510) from Santa Cruz Biotechnology; anti-HIF-1α antibody (ab179483), and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Expressing, Fluorescence, Activity Assay, Western Blot

Journal: bioRxiv

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1101/2020.04.10.036103

Figure Lengend Snippet: (A) Representative western blot showing RSK phosphorylation in RasV12 cells with or without MEK inhibition. (B) (Top) Representative gel image showing RSK1, RSK2, and RSK3 cDNA levels in RasV12 cells treated with or without various RSK siRNAs. (Bottom) Representative western blot showing RSK2 expression in RasV12 cells treated with or without various RSK siRNAs. (D, E) Fold change in PpIX accumulation in RasV12 cells (D) transfected with siRNA against RSKs and (E) treated with different concentrations of SL0101, a pan-RSK inhibitor. *p<0.01 by one-way ANOVA with Turkey’s posthoc test. (E) Representative western blots showing ABCB1 expression in RAS V12 cells treated with different concentrations of SL0101. p-s6 expression was used to confirm RSK inhibition, and GAPDH was used as the loading control.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody (sc-55510) from Santa Cruz Biotechnology; anti-HIF-1α antibody (ab179483), and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Western Blot, Inhibition, Expressing, Transfection

Journal: bioRxiv

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1101/2020.04.10.036103

Figure Lengend Snippet: Human cancer cell lines DLD-1, SNB-75, Hs 578T, and MDA MB 231 were pre-treated with or without SL0101 (RSK inhibitor), Zosuquidar (ABCB1 inhibitor), or HIF 1α inhibitor for 20 h and then with 5-ALA for 4 h. Mean ± SEM fold change in PpIX fluorescence in cell lysate compared to controls is shown. *p<0.01 by one-way ANOVA with Turkey’s posthoc test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody (sc-55510) from Santa Cruz Biotechnology; anti-HIF-1α antibody (ab179483), and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Fluorescence

Journal: bioRxiv

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1101/2020.04.10.036103

Figure Lengend Snippet: Ras/MEK activation increases HIF-1α expression, which in turn increases the activity of FECH, the enzyme that catalyzes the conversion of PpIX to heme. Ras/MEK activation also upregulates ABCB1 expression through RSKs, which promotes the rate of PpIX efflux.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody (sc-55510) from Santa Cruz Biotechnology; anti-HIF-1α antibody (ab179483), and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Activation Assay, Expressing, Activity Assay

Journal: Genes & Development

Article Title: ?3, a transposable element that promotes host sexual reproduction

doi: 10.1101/gad.557310

Figure Lengend Snippet: Mts1 expression was regulated by nutrients and cell type. (A, left panel) RNA blot analysis using total RNA from SAY45 and SAY45 × SAY119 (diploid) as indicated below the blot. Cells were grown in YPD, SC, or 0.5× YPD/SC using either glucose (glu) or galactose (gal) as a carbon source. The blot was hybridized with an MTS1-specific probe, stripped, and rehybridized with an ACT1-specific probe as a loading control. (Right panel) RNA blot analysis using total RNA from SAY45, SAY102 (sir2Δ), and SAY189 (sir2Δ hmlαΔp). Cells were grown in SC medium and the blot was hybridized with an MTS1-specific probe. As loading control, rRNA bands from the same strains are shown. (B) Protein blot analysis of total proteins (0.1 A600) from SAY988 (MTS1-TAP) grown in either SC or YPD. Anti-TAP (Invitrogen) was used as the primary antibody. Molecular weight markers (MW) are shown on the right.

Article Snippet: For protein blots, the anti-TAP primary antibody (Invitrogen) was diluted 1:500 and the anti-GFP antibody (Clontech) was diluted 1:1000.

Techniques: Expressing, Northern blot, Molecular Weight