Journal: Nucleic Acids Research

Article Title: Tyrosine kinase c-Abl couples RNA polymerase II transcription to DNA double-strand breaks

doi: 10.1093/nar/gkz024

Figure Lengend Snippet: Formation and p-Dicer-dependent turnover of dsRNA at DSBs. ( A ) ChIP analysis of GFP-RNaseH1 occupancy at DS1 using site-specific primers. ( B ) Quantitative real-time PCR (qRT-PCT) of DNA immunopurified from DNA–RNA hybrids (DRIP) or upon incubation with recombinant RNaseH at DS1 using S9.6 hybridoma supernatant and region-specific primers. ( C ) qRT-PCR analysis of transcripts associated with CTD Y1P and immunopurification (mNET-IP). Values were normalized to data in absence of GFP-RNaseH1. (A–E) Asterisk, P -value < 0.05, two-tailed t -test. Error bar: mean ± SEM, n = 3. ( D, E ) Imaging and RGB quantitation of CTD Y1P and p-Dicer (p-DCR-1). White box, 2.5× zoom; n , number of cells with shown phenotype in %. ( F ) qRT-PCR of cDNA after J2 immunoselection with (J2 RIP) and reverse transcription with forward (upper panel)- and reverse (lower panel)-oriented primers spanning a region up to 1000 nts distant from DS1 in presence (J2+) or absence (J2-) of 4OHT, or upon preincubation of lysed material with recombinant RNaseIII (J2+ RNaseIII) prior to J2 RIP. J2+ values were set to 1. Asterisk, p-value <0.05, two-tailed t-test. Error bar: mean ±SEM, n = 3. ( G ) Autoradiograph detecting J2 immuno-selected (RNA IP) or total (IN) RNA or pBR322 MspI digest (M) upon end-labeling and PAGE separation. AU, arbitrary units. ( H ) as in (D), but preincubated with Leptomycin B (LMB) and stained with J2. Representative images are shown.

Article Snippet: Cells were permeabilized with PBS/0.3% Tween-20 (10 min, RT), washed 1× in PBS and incubated for 20 min at RT with either BSA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris), RNaseA (Sigma, 0.2 μg/ml final conc., diluted in PBS containing 0.02 mM NaOAc and 0.2 mM Tris) or RNaseIII (NEB, 2U final conc., diluted in RNase-free H 2 O containing 1× commercial reaction buffer (NEB) prior to fixation.

Techniques: Real-time Polymerase Chain Reaction, Incubation, Recombinant, Quantitative RT-PCR, Immu-Puri, Two Tailed Test, Imaging, Quantitation Assay, Autoradiography, End Labeling, Staining

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: Chinese medicine

Article Title: Lycium barbarum polysaccharide inhibits ischemia-induced autophagy by promoting the biogenesis of neural stem cells-derived extracellular vesicles to enhance the delivery of miR-133a-3p.

doi: 10.1186/s13020-023-00831-8

Figure Lengend Snippet: Fig. 4 LBP pretreated NSC-EVs suppressed inflammatory response and reduced neuronal apoptosis after stroke. OGD-exposed neurons were incubated with PBS, NSC-EV, L-NSC-EV, and H-NSC-EV. Cells incubated under standard cell culture conditions were used as negative control. A– C The mRNA expression of TNFα, IL-6, and IL-1β in the sham and ipsilateral side of the infarcted brain was measured by RT-qPCR at post stroke day 3 D Representative images and E quantification of TUNEL+ cells in the infarct border. Data are shown as mean ± SD. Data are statistically different from each other with *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: The cell apoptosis of brain tissue was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay kit (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions.

Techniques: Incubation, Cell Culture, Negative Control, Expressing, Quantitative RT-PCR, TUNEL Assay

Journal: Frontiers in Oncology

Article Title: Novel Breast-Specific Long Non-coding RNA LINC00993 Acts as a Tumor Suppressor in Triple-Negative Breast Cancer

doi: 10.3389/fonc.2019.01325

Figure Lengend Snippet: LINC00993 suppresses the growth of triple-negative breast cancer (TNBC) cells in vitro . (A) Structure of LINC00993 expression plasmid for adenovirus. (B) LINC00993 expression adenovirus infection efficiency showed by fluorescence microscope in MDA-MB-231 cells. Original magnification, ×400. Scale bars, 50 μm. (C) Expression of LINC00993 detected by qRT-PCR. MDA-MB-231 cells were infected by adenovirus for 24 h, and RNA was extracted. (D) Image of clone formation assay. (E) Number of clones were counted 2 weeks after plantation. (F) MDA-MB-231 cells were planted into 24-well plates. Twenty-four hours later, adenovirus was added to each well. Three wells of cells were digested and counted every 24 h. (G) LINC00993 expression caused apoptosis shown by TUNEL assay. Green points reflected apoptosis, and we used DAPI to stain DNA. Positive control cells were treated with DNase I, negative control cells were collected without adding TUNEL reaction buffer. Original magnification, ×100. Scale bars, 50 μm. (H) Effect of LINC00993 on cell cycle detected by flow cytometry. (I) Flow cytometry cell cycle results shown in a bar plot. (J) Invasive ability tested by Transwell assay. Twenty thousand cells were plated in each well, cells were observed after 24 h of incubation. (K) Bar plot for the number of cells that migrated across the membrane. *** P < 0.001, based on Student's t -test. Data were presented as mean ± SEM.

Article Snippet: To analyze cell apoptosis, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were performed with One Step TUNEL Apoptosis Assay Kit (FITC, Beyotime, China) according to the manufacturer's instructions.

Techniques: In Vitro, Expressing, Plasmid Preparation, Infection, Fluorescence, Microscopy, Quantitative RT-PCR, Tube Formation Assay, Clone Assay, TUNEL Assay, Staining, Positive Control, Negative Control, Flow Cytometry, Transwell Assay, Incubation, Membrane

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 1. PWP1 Regulates Tissue Growth and Proliferation (A) dPWP1 RNAi in the posterior compartment of the developing wing (En-Gal4) leads to reduced compartment size. (B) Quantification of the ratio of posterior (P) (n = 10) and anterior (A) (n = 10) wing areas in (A). (C) Cell density ratio between posterior (P) (n = 10) and anterior (A) (n = 10) compartments in (A). (D) Pupation kinetics of control (n = 5) and dpwp1nclb1/2 (n = 5) larvae. dAEL, days after egg laying. (E) Representative images of control and dpwp1nclb1/2 pupae. (F) Quantification of pupal volumes of control (n = 4) and dpwp1nclb1/2 (n = 4) pupae. (G) Proliferation in Ctrl (Lac dsRNA) (n = 3) and dPWP1-specific dsRNA (n = 3) treated S2 cells. (H) Proliferation of HeLa cells after transfection with non-targeting (Ctrl) (n = 3) or PWP1-specific (n = 3) siRNAs. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S1.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Transfection

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 2. PWP1 Regulates Pol I-Mediated Ribosomal Gene Expression (A) dpwp1nclb1/2 mutants display short and thin bristles. (B) Pupation kinetics of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted larvae. dAEL, days after egg laying. (C) Representative images of control and dPWP1 fat-body (Cg-Gal4)-depleted pupae. (D) Quantification of volumes of control (n = 5) and dPWP1 (n = 5) fat-body (Cg-Gal4)-depleted pupae. (E) Representative immunofluorescent images of endogenous dPWP1 localization in comparison with fibrillarin in fat bodies of early third instar larvae. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, and 28S rRNA (RNA polymerase I targets) expression in control larvae (n = 3) and dpwp1 mutants (n = 3). cdk7 was used as a reference gene. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S2.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Gene Expression, Control, Comparison, Quantitative RT-PCR, Expressing

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 4. PWP1 Functionally Cooperates with MYBBP1A and Nucleolin (A) Summary of dPWP1 interacting ribosome biogenesis regulators in S2 cells. (B) Summary of PWP1 interacting ribosome biogenesis regulators in HEK293 cells. (C) Co-purification of HA-tagged dPWP1 with dMYBBP1A upon pull-down of V5-tagged dMYBBP1A from S2 cells. Tubulin serves as a loading control. (D) Representative immunofluorescent images of PWP1 and Nucleolin localization in U2OS cells. Scale bar, 5 mm. (E) Representative immunofluorescent images of Nucleolin localization in U2OS cells followed by PWP1 depletion. Scale bar, 5 mm. (F) qRT-PCR analysis of 5.8S rRNA, 18S rRNA, 28S, and 5S rRNA expression in control larvae (n = 3) and dmybbp1a mutants (n = 3). cdk7 was used as a reference gene. (G) Representative images of control (w-) and mybbp1a mutant larvae at 96 hr after egg laying. (H) Representative immunofluorescent images of dPWP1 localization in fat bodies of early third instar larvae. Depletion of dMYBBP1A from the fat body (Fb-GAL4) leads to the dissociation of nucleolar dPWP1. Scale bar, 5 mm. (I) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (H). A total of 15 nuclei from 3 independent fat bodies were quantified. (J and K) Gel electrophoresis analysis of total RNA (lower panel, visualized by Midori green or ethidium bromide staining) and newly transcribed RNA (upper panel, visualized by streptavidin-HRP detection) prepared from U2OS cells transfected with indicated siRNAs followed by 30 min of 4sU labeling. RNA from non-transfected U2OS cells cultured in the absence of 4sU (No 4sU) was used as a control for streptavidin-HRP signal specificity. *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S4.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Control, Quantitative RT-PCR, Expressing, Mutagenesis, Nucleic Acid Electrophoresis, Staining, Transfection, Labeling, Cell Culture

Journal: Developmental cell

Article Title: PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression.

doi: 10.1016/j.devcel.2017.09.022

Figure Lengend Snippet: Figure 5. mTORC1-Dependent Phosphorylation Regulates Nucle- olar PWP1 (A) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar fed and starved (6 hr) larvae. Scale bar, 5 mm. (B) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (A). A total of 15 nuclei from 3 independent fat bodies were quantified. (C) Representative immunofluorescent images of endogenous dPWP1 locali- zation in fat bodies of early third instar starved (4 hr) and re-fed (6 hr) larvae. Scale bar, 5 mm. (D) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (C). A total of 15 nuclei from 3 independent fat bodies were quantified. (E) Representative immunofluorescent images of dPWP1 localization in fat bodies dissected from early third instar larvae fed without or with rapamycin (14 hr). Scale bar, 5 mm. (F) Quantification of the immunofluorescence of the nucleolus/nucleoplasm localization ratio in (E). A total of 15 nuclei from 3 independent fat bodies were quantified. (G) Immunoblot of S2 cell lysates expressing a V5-tagged form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin alone (10 min) or in combination with rapamycin (2 hr). Phospho-dPWP1 species (anti-V5) are indicated by arrowheads. (H) Immunoblot of lysates of S2 cell expressing a V5-tagged form of dPWP1 together with RNAi against LacZ (ctrl), dS6K, or dRaptor resolved on Phos-tag SDS-PAGE. Cells were treated without or with insulin (10 min). (I) Quantification of phospho-dPWP1 species of (H). (J) Immunoblot of S2 cell lysates expressing a V5-tagged wild-type or S384 alanine mutated form of dPWP1 resolved on Phos-tag SDS-PAGE. Cells were treated with insulin (10 min). (K) Quantification of phospho-dPWP1 species of (J). (L) Immunofluorescent analysis of fat body expressing the wild-type and S384A form of dPWP1 dissected from early third instar larvae. Scale bar, 5 mm. (M) Quantification of the nucleolus/nucleoplasm localization ratio in (L). A total of 15 nuclei from 3 independent fat bodies were quantified. **p < 0.01, ***p < 0.001 (Student’s t test). Error bars indicate SDs. See also Figure S5.

Article Snippet: To knock down dPWP1 in S2 cells, a cDNA fragment of dpwp1 was amplified with primers flanked by the T7 promoter (Table S3), and dsRNA was produced using a TranscriptAid T7 High Yield Transcription Kit (Thermo) and annealed in distilled water, S2 cells were cultured with 5 mg/ml dsRNA in Schneider’s Drosophila Medium (LifeTechnologies) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies) and penicillin/streptomycin (LifeTechnologies) for 5 days.

Techniques: Phospho-proteomics, Western Blot, Expressing, SDS Page

Journal: eLife

Article Title: Genome-wide CRISPR screening reveals genetic modifiers of mutant EGFR dependence in human NSCLC

doi: 10.7554/eLife.50223

Figure Lengend Snippet: ( A ) Mass spectrometry analysis of global protein changes between control and ARIH2-deficient HCC827 cells. ( B ) Immunoblots of indicated proteins in control and ARIH2-deficient HCC827 cells. β-Actin was used as a loading control. ( C ) Quantitative RT-PCR analysis of relative mRNA levels of indicated genes in control or ARIH2-deficient HCC827 cells. Error bars represent mean ± SD; n = 4. ( D ) Immunoblots of indicated proteins showing ectopic expression of HA-METAP2 in HCC827 cells. GAPDH was used as a loading control. ( E ) Crystal violet staining colony formation assay of HCC827-Vector or HCC827-HA-METAP2 cell lines treated with DMSO, erlotinib (1 µM), or gefitinib (1 µM). ( F ) Quantification of colony formation in ( E ), shown as percentage of the HCC827-Vector sample. Mean (three biological replicates) ± SD is shown. ( G ) De novo protein synthesis of HCC827-Vector or HCC827-HA-METAP2 cells after treatment with DMSO or erlotinib (1 µM, 24 hr) as determined by L-azidohomoalanine (AHA) labeling. Cells were starved of methionine for 1 hr and incubated with AHA for 1 hr. Lysates were subjected to a Click-iT chemistry reaction to switch azido-modified nascent proteins to alkyne-biotin, and visualized by Streptavidin-HRP immunoblotting. β-Actin was used as a loading control. ( H ) De novo protein synthesis of control or ARIH2-deficient HCC827 cells after treatment with DMSO or erlotinib (1 µM, 24 hr) as determined by AHA labeling. β-Actin was used as a loading control. ( I ) Immunoblot of METAP2 in HCC827 cells upon proteasome inhibitor bortezomib treatment. β-Actin was used as a loading control. ( J ) Immunoblots of ARIH2 and METAP2 in control or ARIH2 knockout HCC827 cells upon bortezomib treatment. β-Actin was used as a loading control. ( K ) De novo METAP2 protein synthesis in control or ARIH2-deficient HCC827 cells as determined by AHA labeling and streptavidin pulldown. β-Actin was used as a loading control. ( L ) Immunoblots of indicated proteins showing ectopic expression of HA-ALDOA and HA-PSAT1 in HCC827 cells. GAPDH was used as a loading control. ( M ) Crystal violet staining colony formation assay of HCC827-Vector, HCC827-HA-ALDOA or HCC827-HA-PSAT1 cells treated with DMSO, erlotinib (1 µM), or gefitinib (1 µM). ( N ) Quantification of colony formation in ( M ), shown as percentage of the HCC827-Vector sample. Mean (three biological replicates) ± SD is shown. Statistical significance was tested using unpaired two-tailed t test ( C, F and N ); *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, not significant. Figure 6—source data 1. Raw data from .

Article Snippet: Samples were diluted with Triton-X lysis buffer to reduce the amount of SDS and same amount of proteins were then used for streptavidin magnetic beads (Thermo Fisher Scientific Cat# 88817) pulldown with incubation at 4°C for overnight.

Techniques: Mass Spectrometry, Control, Western Blot, Quantitative RT-PCR, Expressing, Staining, Colony Assay, Plasmid Preparation, Labeling, Incubation, Modification, Knock-Out, Two Tailed Test

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 2. LC-derived Exos induce macrophage polarization toward to M2 phenotype. Notes: After PMA induction, the morphology of THP-1 cells was observed under a microscope (A) (n = 3). qRT-PCR was applied to detect the expression of macrophage surface marker CD68 (B) and surface markers of M1 (iNOS and IL-1β) and M2 (CD206, CD163 and arginase-1) (D) (n = 3). The uptake of Exos in macrophages was inspected by fluorescence labeling of Exos (C) (n = 3). The protein levels of M2 markers CD206, CD163 and arginase-1 in macrophages were assessed by Western blot (E) (n = 3); **P < 0.01, ***P < 0.001, compared to the M group; LC, lung cancer; Exos, exosomes; PMA, phorbol-12-myristate-13-acetate; M, macrophage.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Derivative Assay, Microscopy, Quantitative RT-PCR, Expressing, Marker, Fluorescence, Labeling, Western Blot

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Exosomal circPVT1 derived from lung cancer promotes the progression of lung cancer by targeting miR-124-3p/EZH2 axis and regulating macrophage polarization.

doi: 10.1080/15384101.2021.2024997

Figure Lengend Snippet: Figure 5. Exosomal circPVT1 stimulates macrophage polarization to M2 type and enhances the biological function of LC cells. Notes: The A549 cells were transfected with oecircPVT1 or si-circPVT1. Exos were extracted from the transfected A549 cells (Exo- A-oecircPVT1 or Exo-A-si-circPVT1) and coincubated with macrophages (M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1). Then, qRT- PCR was utilized to measure the mRNA level of circPVT1 in A549 cells (A) and Exo-A (B) as well as the expressions of CD206, CD163 and arginase-1 in M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 (C) (n = 3). The protein expressions of M2 markers in M+ Exo- A-oecircPVT1 or M+ Exo-A-si-circPVT1 were detected by Western blot (D) (n = 3). Subsequently, the proliferation, invasion and migration abilities of A549 cells that coincubated with M+ Exo-A-oecircPVT1 or M+ Exo-A-si-circPVT1 were determined by CCK-8 assay (E), Transwell (F) and cell scratch assay (G), respectively (n = 3). The mRNA expressions of M2 markers and miR-124-3p in the transfected macrophages were inspected by qRT-PCR (H) (n = 3); *P < 0.05, ***P < 0.001, compared to the Blank group; *P < 0.05, **P < 0.01, ***P < 0.001, compared to the M+ Exo-A-si-NC or M+ Exo-A-vector group; *P < 0.05, **P < 0.01, compared to the M/ Blank group; M, macrophage; LC, lung cancer; Exos, exosomes.

Article Snippet: The membranes were incubated with the primary antibodies against rabbit anti-human GAPDH (5174S, 1:1000, Cell Signaling, Boston, USA), CD9 (sc-13,118, 1:500, Santa Cruz, Texas, USA), CD81 (sc-166,029, 1:500, Santa Cruz, Texas, USA), TSG101 (sc7964, 1:500, Santa Cruz, Texas, USA), CD206 (sc-376,108, 1:500, Santa Cruz, Texas, USA), arginase-1 (sc-166,920, 1:500, Santa Cruz, Texas, USA), CD63 (ab134045, 1:1000, Abcam, MA, USA), CD163 (ab182422, 1:1000, Abcam, MA, USA) and EZH2 (ab186006, 1:2000, Abcam, MA, USA) at room temperature in a shaking table for 1 h. Following primary incubation, the membranes were washed with the washing solution for 3 × 10 min and then incubated with the secondary antibody against horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000, Beijing ComWin Biotech Co., Ltd., Beijing, China) for 1 h at room temperature.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Migration, CCK-8 Assay, Wound Healing Assay, Plasmid Preparation

Journal: Experimental Physiology

Article Title: miR‐302a‐3p targets FMR1 to regulate pyroptosis of renal tubular epithelial cells induced by hypoxia–reoxygenation injury

doi: 10.1113/ep089887

Figure Lengend Snippet: FIGURE 1 Activation of NLRP3 inflammasome and pyroptosis of HK-2 cells are induced by H/R treatment. HK-2 cells were induced by H/R. (a) Lactate dehydrogenase (LDH) content determined with a kit. (b) Cell activity detected by CCK-8. (c) Pyroptosis detected by calcein AM/PI double staining; magnification: 100×, scale bar: 100 μm. (d) Protein levels of NLRP3 and GSDMD-N determined by western blot. (e) Caspase-1 activity. (f) IL-1β and IL-18 content determined by ELISA. (g) SOD and MDA content measured by kits. The data of (a–c, e, g) were tested by independent sample t-test. n = 3 repetitions of the experiment. The data of (d, f) were tested by two-way ANOVA, followed by Sidak’s multiple comparison test

Article Snippet: Calcein AM/propidium iodide (PI) double staining kit (Santa Cruz Biotechnology, Dallas, TX, USA) was used for quantitative detection of live and dead HK-2 cells.

Techniques: Activation Assay, Activity Assay, CCK-8 Assay, Double Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Experimental Physiology

Article Title: miR‐302a‐3p targets FMR1 to regulate pyroptosis of renal tubular epithelial cells induced by hypoxia–reoxygenation injury

doi: 10.1113/ep089887

Figure Lengend Snippet: FIGURE 2 Inhibition of NLRP3 inflammasome reduces H/R-induced HK-2 cell pyroptosis. HK-2 cells were treated with inflammasome inhibitor BAY11-7082, with DMSO treatment as the negative control, followed by H/R induction. (a) Lactate dehydrogenase (LDH) content determined with a kit. (b) Cell activity detected by CCK-8. (c) Pyroptosis detected by calcein AM/PI double staining; magnification: 100×, scale bar: 100 μm. (d) Protein levels of NLRP3 and GSDMD-N determined by western blot. (e) Caspase-1 activity determined with a kit. (f) IL-1β and IL-18 content determined by ELISA. (g) SOD and MDA content measured by kits. n = 3 repetitions of the experiment. The data of (a–c, e, g) were tested by one-way ANOVA; the data of (d, f) were tested by two-way ANOVA, followed by Tukey’s multiple comparison test. H/R+BAY: hypoxia/reoxygenation + BAY11-7082

Article Snippet: Calcein AM/propidium iodide (PI) double staining kit (Santa Cruz Biotechnology, Dallas, TX, USA) was used for quantitative detection of live and dead HK-2 cells.

Techniques: Inhibition, Negative Control, Activity Assay, CCK-8 Assay, Double Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Experimental Physiology

Article Title: miR‐302a‐3p targets FMR1 to regulate pyroptosis of renal tubular epithelial cells induced by hypoxia–reoxygenation injury

doi: 10.1113/ep089887

Figure Lengend Snippet: FIGURE 3 Inhibition of miR-302a-3p can inhibit NLRP3 inflammasome-mediated pyroptosis. HK-2 cells were treated with miR-302a-3p inhibitor, with inhibitor NC as control, and then induced by H/R. (a) RT-qPCR-detected miR-302a-3p expression. (b) Lactate dehydrogenase (LDH) content. (c) Cell activity detected by CCK-8. (d) Pyroptosis detected by calcein AM/PI double staining; magnification: 100×, scale bar: 100 μm. (e) Protein levels of NLRP3 and GSDMD-N determined by western blot. (f) Caspase-1 activity. (g) IL-1β and IL-18 content determined by ELISA. (h) SOD and MDA content. n = 3 repetitions of the experiment. The data of (a–d, f, h) were tested by one-way ANOVA; the data of (e, g) were tested by two-way ANOVA, followed by Tukey’s multiple comparison test. H/R+inhi-NC: hypoxia/reoxygenation + inhibitor NC; H/R+miR-inhi: hypoxia/reoxygenation + miR-302a-3p inhibitor

Article Snippet: Calcein AM/propidium iodide (PI) double staining kit (Santa Cruz Biotechnology, Dallas, TX, USA) was used for quantitative detection of live and dead HK-2 cells.

Techniques: Inhibition, Control, Quantitative RT-PCR, Expressing, Activity Assay, CCK-8 Assay, Double Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Experimental Physiology

Article Title: miR‐302a‐3p targets FMR1 to regulate pyroptosis of renal tubular epithelial cells induced by hypoxia–reoxygenation injury

doi: 10.1113/ep089887

Figure Lengend Snippet: FIGURE 5 Downregulation of FMR1 annulled the inhibition of miR-302a-3p inhibitor on H/R-induced pyroptosis of HK-2 cells. shRNA-FMR1 was used to treat HK-2 cells in H/R + miR-inhi group, and shRNA NC was used as control. (a) RT-qPCR detected FMR1 mRNA expression. (b) Lactate dehydrogenase (LDH) content. (c) Cell activity detected by CCK-8. (d) Pyroptosis examined by calcein AM/PI double staining; magnification: 100×, scale: 100 μm. (e) Protein levels of NLRP3 and GSDMD-N determined by western blot. (f) Caspase-1 activity. (g) IL-1β and IL-18 content determined by ELISA. (h) SOD and MDA content. n = 3 repetitions of the experiment. The data of (a–d, f, h) were tested by one-way ANOVA; the data of (e, g) were tested by two-way ANOVA, followed by Tukey’s multiple comparison test

Article Snippet: Calcein AM/propidium iodide (PI) double staining kit (Santa Cruz Biotechnology, Dallas, TX, USA) was used for quantitative detection of live and dead HK-2 cells.

Techniques: Inhibition, shRNA, Control, Quantitative RT-PCR, Expressing, Activity Assay, CCK-8 Assay, Double Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 2 Ginsenoside Rg3 induced immunogenic cell death in CRC cells. (A) IC50 of Rg3 for CT26 and HCT116 cells at 24 h. Data are presented as mean SD (n Z 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of Rg3 at 6, 12 and 24 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO. (C) The activity of UPR signaling pathways following treatment of Rg3 ([c] Z 30 mmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S1. (D) The characterization of ICD in CRC cells following treatment of Rg3 ([c] Z 30 mmol/L), including CRT exposure (6 h), ATP secretion (12 h) and HMGB1 release (12 h). Data are presented as mean SD (n Z 3). *P < 0.05 relative to DMSO; scale bar Z 20 mm. (E) The expression of CD11c and CD86 in DCs stimulated with the supernatant from Rg3 ([c] Z 30 mmol/L) -treated cells (24 h). Data are presented as mean SD (n Z 3). **P < 0.01 relative to DMSO. (F) The in vivo vaccination assay using BALB/C and nude mice. Data are presented as mean SD (n Z 4). ***P < 0.001.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Activity Assay, Protein-Protein interactions, Expressing, In Vivo

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 3 Quercetin caused reactive oxygen species in CRC cells. (A) IC50 of QTN for CT26 and HCT116 cells at 24 h. Data are presented as mean SD (n Z 3). (B) Apoptosis (%) in CT26 and HCT116 cells following treatment of QTN ([c] Z 80 mmol/L) at 24 h. Data are presented as mean SD (n Z 3). **P < 0.01 relative to DMSO. (C) The activity of Bcl-2/BAX/caspase 9/caspase 3 signaling pathways following treatment of QTN ([c] Z 80 mmol/L) at 6, 12 and 24 h. The quantification was demonstrated in Fig. S2. (D) The ROS level in CT26 and HCT116 cells following treatment of QTN ([c] Z 80 mmol/L) at 6, 12 and 24 h. Data are presented as mean SD (n Z 3). **P < 0.01 and ***P < 0.001 relative to DMSO. (E) Cell viability (%) of CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] Z 80 mmol/L) (24 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to untreated control. (F) Apoptosis (%) in CT26 and HCT116 cells with or without NAC prior to treatment of QTN ([c] Z 80 mmol/L) (24 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to untreated control.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Activity Assay, Protein-Protein interactions, Control

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 4 Synergistic effects of Rg3 and QTN in CT26 cells. (A) IC50 of drug combination at 24 h. Data are presented as mean SD (n Z 3). CI values at IC50 were shown in Fig. S3. (B) Apoptosis (%) caused by drug combination at 24 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO. (C) The CRT exposure with or without NAC before treatment of drug combination (6 h). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01 relative to DMSO; scale bar Z 20 mm. (D) The ATP secretion with or without NAC before treatment of drug combination at 12 h. Data are presented as mean SD (n Z 3).*P < 0.05 and **P < 0.01, between NAC and No NAC. (E) The HMGB1 release with or without NAC before treatment of drug combination at 12 h. Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01, between NAC and No NAC. (F) The expression of CD11c and CD86 in DCs stimulated (24 h) by the supernatant from Rg3- treated cells with or without pretreatment of NAC. Data are presented as mean SD (n Z 3). *P < 0.05, **P < 0.01 and ***P < 0.001, relative to DMSO.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Expressing

Journal: Acta pharmaceutica Sinica. B

Article Title: A cyclodextrin-based nanoformulation achieves co-delivery of ginsenoside Rg3 and quercetin for chemo-immunotherapy in colorectal cancer.

doi: 10.1016/j.apsb.2021.06.005

Figure Lengend Snippet: Figure 8 Combination therapy of targeted co-formulation and Anti-PD-L1 for CRC. (A) Treatment schedule and IVIS images. (B) The CRC progression over a 35-day period. Data are presented as mean SD (n Z 5). *P < 0.05 and **P < 0.01; NS, no significance. (C) Animal survival (median survival: PBS ~38 days, Anti-PD-L1 ~40 days, targeted co-formulation ~62 days, and combination z 96 days). Data are presented as mean SD (n Z 5). **P < 0.01 and ***P < 0.001. (D) Immunofluorescent staining assay (green Z DNA fragments and blue Z nuclei) on Day 20 to assess apoptosis in the tumor (scale bar Z 50 mm). Data are presented as mean SD (n Z 3). *P < 0.05 and **P < 0.01, relative to PBS. (E) Level of immune cells in the tumor on Day 20 was analyzed using flow cytometry (BD). Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance. (F) The mRNA expression of cytokines and chemokines in the tumor on Day 20 was analyzed using real time RT-PCR. Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance. (G) Orthotopic CRC mice treated with targeted co-formulation following the removal of CD4 þ or CD8þ T cells. Data are presented as mean SD (n Z 4). *P < 0.05 and **P < 0.01; NS, no significance.

Article Snippet: DNA fragments were detected using the TransDetect Fluorescein TUNEL Cell Apoptosis Kit (TransGen Biotech), and nuclei were stained with DAPI (Beyotime Biotech), for confocal microscopic analysis (Olympus); 2) Measurement of immune cells.

Techniques: Formulation, Staining, Cytometry, Expressing, Quantitative RT-PCR