Journal: World Journal of Gastroenterology

Article Title: Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly

doi: 10.3748/wjg.v29.i35.5104

Figure Lengend Snippet: Effects of full-length regenerating gene 4 and nonsignal peptide regenerating gene 4 on phenotypes of DLD-1 cells. A: DLD-1 cells were incubated with increasing doses of recombinant human regenerating gene 4 ( REG4 ) (rh REG4 ) (0-500 nmol/L) for 48 h and subjected to cell proliferation assay by tetrazolium salt (MTT) assay; B: Treatment with anti- REG4 antibody produced a dose-dependent decrease in cell number of full-length (FL)- REG4 -overexpressing DLD-1 cells; C: MTT assay was used to detect the proliferation of DLD-1 cells transfected with FL- REG4 or nonsignal peptide (NSP)- REG4 , and treated with rh REG4 or REG4 antibody; D: Apoptosis of DLD-1 cells transfected with FL- REG4 or NSP- REG4 , and treated with rh REG4 or REG4 antibody detected by flow cytometry; E: Wound healing assay was used to detect migration of DLD-1 cells transfected with FL- REG4 or NSP- REG4 , and treated with rh REG4 or REG4 antibody; F: Transwell assay was used to detect migration and invasion of DLD-1 cells transfected with FL- REG4 or NSP- REG4 , and treated with rh REG4 or REG4 antibody; G: Compared with DLD-1 cells, western blotting showed that transfection with FL- REG4 and treatment with rh REG4 increased expression of epidermal growth factor receptor (EGFR)-Tyr992, Tyr1068, Tyr1148, Tyr1173, Akt, p-Akt, phosphorylated phosphoinositide 3-kinase (p-PI3K), nuclear factor (NF)-κB, p-NF-KB, Bcl-2 and Bcl-X/L. REG4 antibody inhibited the effect of FL- REG4 transfection; H: Western blotting showed that compared with parental cells, DLD-1 cells transfected with NSP- REG4 had no change in expression of EGFR-Tyr992, Tyr1068, Tyr1148, Tyr1173, AKT, p-AKT, p-PI3K, NF-KB, p-NF-KB, BCL-2, or BCL-XL. a P < 0.001; b P < 0.01; d No significance. Ab: Anti- REG4 antibody; REG4 : Regenerating gene 4; rh REG4: Recombinant human regenerating gene 4 ; FL: Full-length; NSP: Nonsignal peptide; EGFR: Epidermal growth factor receptor; p-PI3K: Phosphorylated phosphoinositide 3-kinase; NF: Nuclear factor.

Article Snippet: The cells were treated with high glucose (4.5 mg/L), recombinant human REG4 protein (rh REG4 ; R and D Systems, Minneapolis, MN, United States) or anti- REG4 antibody (R and D Systems).

Techniques: Incubation, Recombinant, Proliferation Assay, MTT Assay, Produced, Transfection, Flow Cytometry, Wound Healing Assay, Migration, Transwell Assay, Western Blot, Expressing

Journal: World Journal of Gastroenterology

Article Title: Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly

doi: 10.3748/wjg.v29.i35.5104

Figure Lengend Snippet: Effects of regenerating gene 4 on chemoresistance and droplet formation of DLD-1 cells. A: After treatment with cisplatin (DDP) or 5-fluorouracil (5-FU), the viability was measured in DLD-1 cells, DLD-1 cells treated with recombinant human regenerating gene 4 ( REG4 ) (rh REG4 ), DLD-1 cells transfected with full-length (FL)- REG4 plasmid, and DLD-1 cells treated with anti- REG4 antibody; B: The lipid droplet level was measured in DLD-1 cells, DLD-1 cells treated with rh REG4 , DLD-1 cells transfected with FL- REG4 plasmid, and DLD-1 cells treated with anti- REG4 antibody; C: REG4 expression was detected in DLD-1, chemoresistant DLD-1 cells to DDP and 5-FU by western blot, proteins related to de novo synthesis or assembly pathway of lipid droplets were also detected in DLD-1 cells and DLD-1 cells transfected with FL- REG4 ; D: Nile red staining was used to detect the level of lipid droplets in DLD-1 cells and DLD-1 cells transfected with FL- REG4 after treatment with high glucose (HG), acetyl-CoA carboxylase 1 (ACC1) inhibitor and ATP-citrate lyase (ACLY) inhibitor; E: Tetrazolium salt assay was used to detect half maximal inhibitory concentration of DLD-1 cells for 5-FU and DDP when treated with HG, ACC1 and ACLY inhibitors, alone or in combination with the above conditions. a P < 0.001; b P < 0.01. Ab: Anti- REG4 antibody; REG4 : Regenerating gene 4; rh REG4: Recombinant human regenerating gene 4 ; ACC1: Acetyl-CoA carboxylase 1; ACCY: ATP-citrate lyase; AC-H3: Acetyl-acetyl-histone 3; H4: Histone 4; ACC1: Acetyl-CoA carboxylase 1; HDAC: Si histone deacetylase; ING5: Inhibitor of growth protein 5; SREBP1: Sterol-regulatory element binding protein 1; ACAT: A-cholesterol acyltransferase; ADRP: Adipocyte differentiation-related protein; CIDE: Cell-death-inducing DFF45-like effector; TIP: Tail-interacting protein; DDP: Cisplatin; 5-FU: 5-fluorouracil; HG: High glucose.

Article Snippet: The cells were treated with high glucose (4.5 mg/L), recombinant human REG4 protein (rh REG4 ; R and D Systems, Minneapolis, MN, United States) or anti- REG4 antibody (R and D Systems).

Techniques: Recombinant, Transfection, Plasmid Preparation, Expressing, Western Blot, Staining, Concentration Assay, Histone Deacetylase Assay, Binding Assay

Journal: World Journal of Gastroenterology

Article Title: Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly

doi: 10.3748/wjg.v29.i35.5104

Figure Lengend Snippet: Full-length regenerating gene 4 weakened the transcription of acetyl-CoA carboxylase 1 and ATP-citrate lyase. A: DLD-1 cells and full-length-regenerating gene 4 (FL- REG4 ) transfectants were treated with suberoylanilide hydroxamic acid (SAHA) or small interfering RNA against histone deacetylase (siHDAC), and analyzed by chromatin immunoprecipitation; B: DLD-1 cells and FL- REG4 transfectants were treated with SAHA, or siHDAC, and analyzed by co-immunoprecipitation assay using anti-anti-acetyl (AC)-acetyl-histone 3-AC-histone 4, anti-HDAC, anti-sterol-regulatory element binding protein 1 or anti-inhibitor of growth protein 5 antibody; C: DLD-1 cells and FL- REG4 transfectants were treated with SAHA or siHDAC, and analyzed by quantitative reverse transcription polymerase chain reaction; D: DLD-1 cells and FL- REG4 transfectants were treated with SAHA or siHDAC, and analyzed by Nile red staining; E: DLD-1 cells and FL- REG4 transfectants were treated with SAHA or siHDAC, and analyzed by half maximal inhibitory concentration assay of 5-fluorouracil and cisplatin. a P < 0.001; b P < 0.01. REG4 : Regenerating gene 4; SAHA: Suberoylanilide hydroxamic acid; siHDAC: Small interfering RNA against histone deacetylase; IgG: Immunoglobulin G; ND: No DNA; ACC1: Acetyl-CoA carboxylase 1; ACCY: ATP-citrate lyase; AC-H3: Acetyl-acetyl-histone 3; H4: Histone 4; SREBP1: Sterol-regulatory element binding protein 1; ING5: Inhibitor of growth protein 5; Ctr: Control DLD-1 cells; PC: Positive control.

Article Snippet: The cells were treated with high glucose (4.5 mg/L), recombinant human REG4 protein (rh REG4 ; R and D Systems, Minneapolis, MN, United States) or anti- REG4 antibody (R and D Systems).

Techniques: Small Interfering RNA, Histone Deacetylase Assay, Chromatin Immunoprecipitation, Co-Immunoprecipitation Assay, Binding Assay, Reverse Transcription, Polymerase Chain Reaction, Staining, Concentration Assay, Control, Positive Control

Journal: World Journal of Gastroenterology

Article Title: Regenerating gene 4 promotes chemoresistance of colorectal cancer by affecting lipid droplet synthesis and assembly

doi: 10.3748/wjg.v29.i35.5104

Figure Lengend Snippet: Full-length regenerating gene 4 destablized acetyl-CoA carboxylase 1 and ATP-citrate lyase proteins via proteasomal degradation. A: DLD-1 cells and full-length-regenerating gene 4 (FL- REG4 ) transfectants were treated with cycloheximide (0.4 μg/mL), followed by western blotting to detect acetyl-CoA carboxylase 1 (ACC1) and ATP-citrate lyase (ACLY) expression; B: DLD-1 cells and FL- REG4 transfectants were treated with MG132 (5 μM, 9 h), followed by western blotting to detect ACC1 and ACLY expression; C: DLD-1 cells, FL- REG4 transfectants, and MG132-treated DLD-1 cells and FL- REG4 transfectants were subjected to proteasomal extract and western blotting to detect ACC1 and ACLY expression; D: Ubiquitin transferases (COP1 E3 ubiquitin ligase, synoviolin 1, Cbl proto-oncogene, NEDD4 like E3 ubiquitin protein ligase were detected by western blotting in DLD-1 cells and FL- REG4 transfectants; E: After co- immunoprecipitation, western blotting was performed in DLD-1 cells and FL- REG4 transfectants, with or without MG132 treatment; F: Nile red staining of DLD-1 cells, FL- REG4 transfectants, and MG132-treated DLD-1 cells and FL- REG4 transfectants; G: Tetrazolium salt assay was performed on DLD-1 cells, FL- REG4 transfectants and MG132- treated DLD-1 cells and FL- REG4 transfectants to detect half maximal inhibitory concentration of 5-fluorouracil and cisplatin. a P < 0.001; d No significance. REG4 : Regenerating gene 4; ACC1: Acetyl-CoA carboxylase 1; ACCY: ATP-citrate lyase; COP1: COP1 E3 ubiquitin ligase; SYVN1: Synoviolin 1; CBL: Cbl proto-oncogene; NEDD4L: NEDD4 like E3 ubiquitin protein ligase; IgG: Immunoglobulin G.

Article Snippet: The cells were treated with high glucose (4.5 mg/L), recombinant human REG4 protein (rh REG4 ; R and D Systems, Minneapolis, MN, United States) or anti- REG4 antibody (R and D Systems).

Techniques: Western Blot, Expressing, Ubiquitin Proteomics, Immunoprecipitation, Staining, Concentration Assay

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Impact of fibulin‐5 deficiency on the skin aging process. (A) Schematic representation of the interfollicular epidermis of mouse tail skin. Slow‐cycling epidermal stem cells (SCs) produce the K10 + interscale lineage (orange), and fast‐cycling epidermal SCs produce the K36 + scale lineage (blue). (B, C) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 2‐month‐old versus 30‐month‐old C57BL/6J mice and quantification (C). The white dashed line represents the epidermal–dermal boundary. Scale bars: 50 μm. (D, E) Immunostaining of fibulin‐5 (green) in sections of mouse tail skin from 3‐month‐old Fbln5 WT versus KO mice and quantification (E). The white dashed line represents the epidermal–dermal boundary and hair follicles. Scale bars: 50 μm. (F) Images of 12‐month‐old Fbln5 WT and KO mice. (G) The body weights of 12‐month‐old Fbln5 WT and KO mice. (H–K) Hematoxylin and eosin staining of sagittal sections of the skin of 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (I, K). Scale bars: 150 μm. Epidermal thickness was measured in interscale and scale regions. (L–O) Whole‐mount staining of BrdU (green, a proliferation marker) and Hoechst (blue) in 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (M, O). Scale bars: 200 μm. (P–U) Whole‐mount staining of K10 (green, interscale lineage), K36 (red, scale lineage), and Hoechst (blue) in 2‐month‐old versus 30‐month‐old C57BL/6J mice and 3‐ and 12‐month‐old Fbln5 WT versus KO mice and quantification (Q, S, U). Scale bars: 200 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (C, E, G, I, K, M, O, Q, S, U). *, p < 0.05; **, p < 0.01; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Immunostaining, Staining, Marker, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Changes in integrin and extracellular matrix expression due to the loss of fibulin‐5. (A) The heatmap shows changes in integrins and ECM proteins in 12‐month‐old Fbln5 WT and KO epidermal stem cells. Genes with a ≥ 2‐fold change are used for analysis. (B) Schematic representation of the epidermal–dermal junction and its associated proteins. (C–V) Immunostaining and quantification of the indicated proteins: Collagen XVII (C–F; green), integrin β1 (G–J; green), integrin α6 (K–N; red) integrin β3 (O–R; green), nectin‐3 (S–V; green), K5 (S–V; gray), and K36 (S–V; red, scale lineage). The white dashed lines represent the epidermal–dermal boundary. Scale bars: 50 μm. All data are presented as the mean ± SD. Each dot represents one mouse. Statistical significance is assessed using a two‐tailed unpaired t ‐test (D, F, H, J, N, P, R, T, V) or Mann–Whitney U test (L). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. The schematic in panel B is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Immunostaining, Two Tailed Test, MANN-WHITNEY

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Extracellular fibulin‐5 enhances YAP activity and fast‐cycling stem cell‐associated gene expression in human keratinocytes. (A–H) Immunostaining of YAP (A, green), SLC1A3 (C, red), Ki‐67 (E, gray), and ASS1 (G, green) in human keratinocytes and quantification (B, D, F, H). Cells are seeded at 150,000, 50,000, and 25,000 cells per well in 12‐well plates and cultured for 48 h before analysis. Scale bars: 50 μm. (I, J) Immunostaining of YAP in primary human keratinocytes and quantification (J). Cells are seeded at 50,000 cells per well in 12‐well plates and cultured for 24 h and then treated with verteporfin or vehicle control for 8 h. Nuclear YAP (%) was calculated as the proportion of cells with nuclear YAP localization among all Hoechst + nuclei. Scale bars: 50 μm. (K–M) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following 8 h of verteporfin treatment. (N, O) Immunostaining of YAP in primary human keratinocytes and quantification (O). Cells are seeded at 300,000 cells per well on collagen IV–coated plates with or without recombinant human fibulin‐5 and cultured to ~80% confluence. The medium is then replaced, and cells are analyzed 8 h later. Scale bars: 50 μm. (P–R) RT‐qPCR analysis of CTGF , SLC1A3 , and ASS1 following culture on plates coated with collagen IV ± fibulin‐5. All data are presented as the mean ± SD. Each dot represents one independent biological replicate. Statistical significance is assessed using a two‐tailed unpaired t ‐test (K, L, P, Q, R), Welch's t ‐test (J, M, O), or one‐way ANOVA (B, D, F, H). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, not significant.

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Activity Assay, Gene Expression, Immunostaining, Cell Culture, Control, Quantitative RT-PCR, Recombinant, Two Tailed Test

Journal: Aging Cell

Article Title: Integrin‐Binding Matricellular Protein Fibulin‐5 Maintains Epidermal Stem Cell Heterogeneity During Skin Aging

doi: 10.1111/acel.70483

Figure Lengend Snippet: Proposed model of cellular and molecular alterations associated with fibulin‐5 deficiency during skin aging. In young skin, slow‐cycling and fast‐cycling epidermal stem cells (SCs) are spatially compartmentalized and give rise to their respective lineages. During aging, decreased fibulin‐5 expression is associated with altered integrin and extracellular matrix (ECM) protein expression, potentially affecting intracellular signaling through fibulin‐5–integrin interactions. Reduced YAP activity is associated with a decrease in the fast‐cycling epidermal stem cell compartment in aged skin and human keratinocytes. The schematic is created with BioRender.com .

Article Snippet: For the fibulin‐5 coating assay, the 12‐well plates were coated overnight at 4°C with collagen type IV (50 μg/mL in PBS) either alone or with 90 ng/mL recombinant human fibulin‐5 (R&D Systems) in PBS.

Techniques: Expressing, Activity Assay

Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: The impairment can be mitigated by proteasome activators. (A) 20S Proteasome chymotrypsin-like peptidase activity is inhibited by oligomeric Aβ42, but not by Aβ42 monomers or fibrils. N = 4. Asterisks denote statistically significant differences (p<0.05). Right: atomic force microscopy (AFM) images of Aβ particles (tapping mode in air). The occasional larger particles in the “monomer” preparation are likely spontaneously forming oligomers. ( B ) Morphometric analysis of the 20S proteasome particles imaged by AFM (tapping mode in liquid) reveals shifts in the particles’ dimensions upon incubation with oligomeric Aβ42. 827 control 20S particles (incubated with a vehicle) and 1181 particles incubated with 2 µM oligomeric Aβ42 were analysed. Solid lines are fittings for the frequencies of control (black) and oligo-treated (red) particles. Since almost all particles are in to-view position, the “length” parameter generated during the particle analysis corresponds to the diameter of the 20S α face. The diameters are raw numbers without correction for tip broadening. When the correction of 2 pixels for SNL probe is applied, the diameter for peak 1 (raw: 14 - 15 nm) falls into 10 – 11 nm range, in excellent agreement with the crystal structure of the human 20S proteasome . See Results for putative assignment of proteasome forms to the numbered peaks. (C) Incubation with oligomeric Aβ42 shifts the conformational equilibrium of 20S core particles imaged by AFM (tapping mode in liquid) toward less open-gate and closed-gate forms, but more intermediate forms. (D) Oligomeric Aβ42 does not significantly affect degradation of oxidized hemoglobin. Degradation of hemoglobin is enhanced by a range of oligomeric Aβ42 concentrations. N=4 samples. ( E, F ) Treatment of the 20S proteasome with activators TAT1-DEN or TAT1-TOD partially protects from inhibition inflicted by the oligomeric Aβ42. ( G ) Incubation with the proteasome activator TAT1-DEN induces a dramatic shift toward open-gate forms, even in the presence of 2 µM of oligomeric Aβ42. The numbers in columns indicate percent of conformers. The number of particles analyzed: 733 (vehicle control), 843 (with oligo Aβ42), 270 (with 1 µM TAT1-DEN) and 171 (with oligo Aβ42and TAT1-DEN). Average ± SD, n= 5 to 9 fields.

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Activity Assay, Microscopy, Incubation, Control, Generated, Particle Size Analysis, Inhibition

Journal: bioRxiv

Article Title: β-Amyloid impairs Proteasome structure and function. Proteasome activation mitigates amyloid induced toxicity and cognitive deficits

doi: 10.1101/2024.10.23.619877

Figure Lengend Snippet: (A) Native page immunoblot depicting purified 20S and 26S proteasome under incubation with oligomeric Aβ42. Immunoblot performed against proteasome β5 subunit and accompanying total protein silver stain. Arrows depict 26S and 20S proteasome assemblages. (B) Native page immunoblot depicting purified 26S proteasome under incubation with varying concentrations of oligomeric Aβ42. Top image shows a representative set, histogram represents N=3 per condition. (C) Model for impact of Aβ on proteasome processes. *p < 0 . 05, Student’s t test was used unless otherwise stated. N represents the number of animals or samples per group .

Article Snippet: Purified 20S Proteasome (R&D Systems, Cat# E-360), purified 26S Proteasome (R&D Systems, Cat# E-365).

Techniques: Clear Native PAGE, Western Blot, Purification, Incubation, Silver Staining

Journal: Nature communications

Article Title: TRPV1 regulates excitatory innervation of OLM neurons in the hippocampus.

doi: 10.1038/ncomms15878

Figure Lengend Snippet: Figure 8 | TRPV1-induced excitatory synaptogenesis requires calcium influx, NGF and BDNF. (a) Immunostain of rat hippocampal cultures with TRPV1 and BDNF. (b) Quantitation of BDNF signal in somas of TRPV1-expressing neurons normalized to surrounding non-TRPV1-expressing cells (n ¼ 15 images from three cultures; error ¼ s.e.m., significance determined by unpaired Student’s t-test with Welch’s correction, **Po0.01). (c) Immunostain of WT and TRPV1 knockout mouse cultures with the C-terminal TRPV1 antibody (which detects a remainins splice isoform in the TRPV1 knockouts and marks TRPV1-expressing cells—Fig. 1e,f), BDNF, and DAPI. (d) Quantitation of BDNF puncta/mm (left panel) and BDNF puncta intensity (right panel) on OLM neurons marked by the C-terminal TRPV1 antibody, in WT and TRPV1 knockout cultures, normalized to WT. (e) Immunostains of TRPV1, vGluT and MAP2 in cultures in control conditions, and in cultures treated with capsaicin in the presence of absence of 2 mM EGTA to block calcium influx, 1 mg ml 1 TrkA-Fc to scavenge NGF, or 0.4mg ml 1 TrkB-Fc to scavenge BDNF. (f) Quantitation of excitatory synapse number (vGluT puncta number) on TRPV1-expressing hippocampal neurons in the indicated conditions. Images used for quantitation were: control n ¼ 28, 1 mM cap. n ¼ 14, 2 mM EGTA n ¼ 21, 2 mM EGTA þ 1 mM cap. n ¼ 21, 1 mgml 1

Article Snippet: Recombinant Human TrkA Fc and TrkB Fc Chimera Proteins were from R & D Systems.

Techniques: Quantitation Assay, Expressing, Knock-Out, Control, Blocking Assay