Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A The expression of USP39 mRNA was analyzed by RT-PCR in SK-hep-1 and HepG2 HCC cells infected with shRNAs. B Western blotting analysis of USP39 protein level in SK-hep-1 and HepG2 cells infected with shRNAs. C – E The effect of USP39 on HCC cells (SK-hep-1) proliferation was determined by MTT assays ( C ) at different time points and colony formation ( D ). The colony counts were normalized to the control and expressed as a percentage, and results are represented in the bar graph ( E ). F – I Representative images of HCC cell migration ability as shown by wound-healing assays ( F – G ) and migration assay ( H – I ). Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection, Western Blot, Control, Migration

Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A – D RT-PCR and western blotting indicated that the mRNA expression levels of USP39 and TRIM26 ( A , C ) and the protein level of ZEB1 ( B , D ) in SK-hep-1 cells co-translated with USP39 and TRIM26 expressing plasmids. ( E , G ) The mRNA levels of USP39 and TRIM26 were analyzed by RT-PCR in SK-hep-1 HCC cells. F , H Effect of USP39-knockdown and TRIM26 silencing on the protein level of ZEB1 in SK-hep-1 HCC cell determined by western blotting. I Effect of overexpression of USP39 and TRIM26 on the ZEB1 ubiquitination in SK-hep-1 cells. J Effect of USP39-knockdown and TRIM26 silencing on the cell proliferation of SK-hep-1 assessed by MTT assay. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Knockdown, Over Expression, Ubiquitin Proteomics, MTT Assay

Journal: Cell Death and Differentiation

Article Title: Deubiquitinase USP39 and E3 ligase TRIM26 balance the level of ZEB1 ubiquitination and thereby determine the progression of hepatocellular carcinoma

doi: 10.1038/s41418-021-00754-7

Figure Lengend Snippet: A The effect of USP39 and TRIM26 in HCC cell proliferation in vivo was determined by xenograft assays. USP39 and TRIM26 knockdown SK-hep-1 cells were respectively injected into flanks of BALB/c nude mice. After 30 days, tumors were isolated and photographed. B Tumor volumes were calculated. C Tumor weight. D , E Levels of USP39, TRIM26, and ZEB1 were analyzed by RT-PCR ( D ) and western blotting ( E ). F The expression levels of USP39, TRIM26, ZEB1 and Ki67 in tumors of different groups by IHC (original magnification, ×40; inlet, ×10). G , H Representative images showed the tumors metastasis of different groups by whole-body bioluminescence imaging ( G ) and lung metastases ( H ). The number of nodules in the lung was counted and statistically analyzed. Student’s t test: * P < 0.05; ** P < 0.01; *** P < 0.001. All the data are representative of at least three independent experiments and presented as the means ± SD.

Article Snippet: HCC cell viability was detected using a cell proliferation assay kit (Beyotime, China).

Techniques: In Vivo, Knockdown, Injection, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Imaging

Journal: Nature Communications

Article Title: An ex vivo uterine system captures implantation, embryogenesis, and trophoblast invasion via maternal-embryonic signaling

doi: 10.1038/s41467-025-60610-x

Figure Lengend Snippet: A A representative 3D image of whole-mount immunofluorescence for FOXA2, a marker of uterine glands as well as embryonic PrE. See also Movie S . B Representative 3D images of whole-mount immunofluorescence for CD45 and F4/80, markers of leukocytes and macrophages, respectively. White arrows indicate the presence of leukocytes and macrophages. See also Movie S . C Slices from a representative 3D image of whole-mount immunofluorescence for a decidualization marker KLF5. White arrows indicate the induction of KLF5 in the embryo-attached subluminal stroma. Dotted line indicates the boundary between LE and stroma. D Representative 3D images of whole-mount immunofluorescence for FLK-1 and αSMA, markers of VE and VSM, respectively. White dotted areas indicate the avascular region in the vicinity of the embryo attachment site corresponding to in vivo implantation. See also Movie S . E A slice from representative 3D images of whole-mount immunofluorescence for a proliferation marker Ki67. The cultured ex vivo uterus sustains an appropriate proliferation-differentiation switching (PDS) pattern, an index of uterine receptivity to the embryos. Note the cessation of proliferation in LE essential for ensuing trophoblast invasion. See also Movie S . Dotted line indicates the boundary between LE and stroma. F Immunofluorescence of CK8 on histological sections at 36 h shows the onset of trophoblast invasion (arrowheads) into the stromal layer of the endometria. G A slice from a representative 3D image of whole-mount immunofluorescence for E-cadherin at 48 h shows the breach and elimination of uterine LE. tdTomato-negative uterine E-cadherin breaks at the boundary with the embryo indicated by white arrows. H H&E staining at 48 h exhibits enlarged nuclei in invading trophoblasts. White and orange dotted lines represent the surface of LE and the boundary between LE and stroma, respectively. PrE, primitive endoderm; Em, embryo; LE, luminal epithelium; GE, glandular epithelium; Str, stroma; Dec, decidua. DsRed2 or tdTomato indicates the mitochondria or cell membrane of all embryonic/extraembryonic cell lineages. Scale bars: 50 μm. Three independent experiments with different recipient animals were performed for all the data.

Article Snippet: For CD45, F4/80, KLF5, αSMA, FLK-1, Ki67, and E-cadherin, primary antibodies for CD45 (1:300, rat, #103101, BioLegend, USA), F4/80 (1:300, rat, MCA497, BIORAD), KLF5 (1:300, rabbit, #21017-1-AP, Proteintec, USA), αSMA (1:300, rabbit, #19245, Cell Signaling, USA), FLK-1 (1:300, rat, #550549, BD Biosciences, USA), Ki67 (1:300, rabbit, MA5-14520, Thermo Fisher Scientific, USA), and E-cadherin (1:300, rabbit, #3195, Cell Signaling, USA) were used with the appropriate secondary antibodies with the same protocol as pAKT/AKT staining.

Techniques: Immunofluorescence, Marker, In Vivo, Cell Culture, Ex Vivo, Staining, Membrane

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Sonidegib treatment reduces taste buds (TB), SHH ligand and proliferation in rat fungiform papilla (FP) while innervation is retained. ( a ) Immunofluorescent antibody detection of SHH ligand (red) and K19 (green) for TB cells; K18 (red) for TB cells and Ki67 (green) for cell proliferation, after Vehicle, 16d or 28d Sonidegib treatments. SHH is reduced in association with TB, K19+ cell loss. Asterisks (*) indicate nonspecific SHH immunoproduct in cornified surface cells in 16d Sonidegib image. The Vehicle, K18/Ki67 image shows 3 regions positive for Ki67+ cells (Apical, Basal and Perigemmal). Proliferating cells are lost in Apical FP region after 16–28d Sonidegib. ( b ) Number of Ki67+ cells in Apical and Basal regions of FP in Vehicle- and Sonidegib-treated mice. Numbers of tongues analyzed are in parentheses. For each tongue 8–10 FP were analyzed. Sonidegib treatment reduces apical epithelial cell proliferation in FP compared to Vehicle. Statistical analysis was one-way ANOVA with Tukey HSD posthoc comparisons (*p ≤ 0.05, compared to Vehicle, APICAL). ( c ) Immunofluorescent antibody detection of K19 or K18 (red) for TB cells and NF (green) for lingual and CT innervation or P2X3 (green) for CT nerve fibers. Innervation was retained after Sonidegib exposure. Asterisks (*) indicate nonspecific P2X3 immunoproduct in surface layer in Vehicle image. ( d ) Enlarged images from 28d Sonidegib papillae. Arrows point to NF+ or P2X3+ fibers in the FP epithelium. Throughout, white dotted lines indicate the basal lamina. Yellow dotted lines indicate surface of epithelium. ( a , c ) Scale bar: 50 μm, applies to all images. ( d ) Scale bar: 25 μm, applies to both images.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques:

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Sonidegib treatment in rat reduces taste buds (TB) and SHH ligand in circumvallate papilla (CV) whereas cell proliferation and GL innervation are retained. Immunofluorescent antibody detection of SHH ligand (red) and K19 (green) for TB cells; K18 (red) for TB cells and Ki67 (green) for cell proliferation; K19 (red) for TB cells and NF (green) for innervation; and, K18 (red) for TB cells and P2X3 (green) for taste nerve fibers, after Vehicle or 36d Sonidegib treatment. White dotted lines outline the epithelium. Asterisk in SHH/K19, 36d Sonidegib indicates nonspecific K19 immunostaining. Arrow points to the P2X3+ nerves extending into CV epithelium after Sonidegib treatment. SHH is reduced in association with TB cells. Cell proliferation is maintained and nerves fibers are retained after Sonidegib treatment. Scale bar: 50μm, applies to all images, except K19/NF.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques: Immunostaining

Journal: Scientific Reports

Article Title: Species generalization and differences in Hedgehog pathway regulation of fungiform and circumvallate papilla taste function and somatosensation demonstrated with sonidegib

doi: 10.1038/s41598-018-34399-3

Figure Lengend Snippet: Long term Sonidegib treatment in mouse reduces taste buds (TB) and SHH ligand in circumvallate papilla (CV) whereas proliferation and innervation are retained. Immunofluorescent antibody detection of SHH ligand (red) and K8 (green) for TB cells; K8 (red) for TB cells and Ki67 (green) for cell proliferation; and K8 (red) with NF (green) for GL innervation or P2X3 (green) for GL taste fibers, after Vehicle or 48d Sonidegib treatment. For SHH/K8, large dotted lines indicate the basal lamina. Small dotted lines outline the surface epithelium. Inset (K8/P2X3) shows an image of nerves extending into CV epithelial basal lamina (arrow). Scale bar: 50 μm, applies to all images. Inset at 2×.

Article Snippet: Antibodies included: SHH ligand: goat anti-SHH (AF464, 0.1 μg/ml, R&D Systems for mice), mouse anti-SHH (sc-365112, 1:500, Santa Cruz Biotechnology for rat); taste cell markers K8, K18, K19 – , with different cytokeratins for optimal immunoreactions in double labeling, K8 (rat TROMA-1, 1:1000, Developmental Studies Hybridoma Bank) for mice, K18 (mouse anti-cytokeratin 18, sc-51582, 1:100, Santa Cruz Biotechnology) or K19 (rabbit anti-keratin 19, ab52625, 1:5000, Abcam) for rat; cell proliferation, Ki67 (rabbit anti-Ki67, NB110-89717SS, 1:5000, Novus Biologicals); innervation, NF (chicken anti-neurofilament heavy, ab4680, 1:5000, Abcam), P2X3 (rabbit anti-P2X3, APR-016, 1:1000, Alomone Labs).

Techniques:

Journal: Science signaling

Article Title: Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles.

doi: 10.1126/scisignal.ade8744

Figure Lengend Snippet: Fig. 6. Matrigel-embedded spheroids and clinical data highlight replication stress tolerance features of escapees. (A) Spheroid growth of the indicated cells after the indicated treatments. Across all spheroid panels: KRASi, 100 nM AMG510; BRAFi, 1 μM encorafenib; ATRi, 5 μM AZD6738; FANCi, 250 nM PIP199. Data are means ± SEM of 10 spheroids per condition, representative of two experiments; P values specified, determined by unpaired t test of the final time point. (B) Representative maximum intensity projections of 3D spheroid clusters stained for p-Rb and cleaved PARP (c-PARP, apoptotic marker) after 7 days of the indicated treatments. Scale bar, 20 μm. Images are representative of two experiments. (C) Quantification of cell count at day 7 in each individual spheroid. Each stacked bar represents one spheroid, where the cell count is broken down by p-Rb status. Combination treatment groups often resulted in spheroids with zero p-Rb+ cells. n = 5 spheroids per condition, representative of two experiments. (D) Percentage of c-PARP–positive cells per spheroid in (C). P values specified, determined by unpaired t test. (E) Formalin-fixed paraffin-embedded (FFPE) tissue sections longitudinally biopsied before and after treatment from two patients with BRAFV600E melanoma (see fig. S12A for patient and tumor details). Immunofluorescence of Ki67 (proliferation marker) and γ-H2AX (DNA damage marker) is displayed. Scale bars, 25 μm (MB2282) and 50 μm (MB3022). (F) Quantification of γ-H2AX intensity (blue coloring) in all cells and in Ki67+ cells (red coloring) from data in (E). Line drawn at the mean; n > 235 cells per ungated condition, n > 45 cells per Ki67-gated condition; P value as indicated or **P < 0.01 and ***P < 0.001, determined by permutation test. (G) Kaplan-Meier survival analyses based on data generated from the Harmonized Cancer Datasets within the Genomic Data Commons (GDC) research database (as of 28 April 2022; https://portal.gdc.cancer.gov). Regardless of treatment type, all patients with various tumor types harboring ≥1 EGFR, KRAS, or BRAF mutation and ≥1 TP53 mutation were included; N = 33 to 829 patients in each group. Cohort comparisons were made between tumor subgroups containing wild-type or mutant forms of the indicated genes. Leftmost plot examines a cohort that has ≥1 mutation among a list of FA-associated genes (fig. S13A). Displayed P values were determined by log-rank test.

Article Snippet: 16, eade8744 (2023) 1 August 2023 12 of 18 (D3U1W) mouse mAb (1:1000, Cell Signaling Technology, no. 86298); Ki67 sheep pAb (1:200, R&D Systems, no. AF7617); ERK1/2 mouse mAb (1:1000, Cell Signaling Technology, no. 4696); p-ERK1/2 (T202/Y204) mouse mAb (1:1000, MilliporeSigma, no. M9692); histone H3 mouse mAb (1:2000, Cell Signaling Technology, no. 3638); and cleaved PARP (Asp214) mouse mAb (1:500, BD Biosciences no. 552596).

Techniques: Staining, Marker, Cell Counting, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Generated, Mutagenesis