Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) MRI images showing brain abnormalities in human subjects with biallelic KIF26A variants (top) and plane-matched images from age-matched control brains(bottom). Sagittal (a) and axial (b) T2-weighted images of subject A01 at 3 months of age demonstrate reduced cerebral white matter volume (notably the corpus callosum (CC)) with supratentorial ventriculomegaly and cerebral atrophy. Sagittal (c) and axial (d) T2 weighted postmortem brain MRI of B01 (pregnancy ended at gestational week (GW) 21) demonstrate full thickness defects affecting the parietal lobes (black arrows). Note that tissue decomposition/swelling distorts brain morphology. Sagittal T1 (e) and axial T2 (f) images of subject C01 at 17 years of age demonstrate complete agenesis of the CC with preservation of the anterior commissure (white arrow) and colpocephaly. Mid-sagittal T2 weighted (g), axial T2 weighted (h), and coronal T1 weighted images (i) of subject E01 demonstrate increased thickness, gyral frequency, and haziness of the inferior right temporal cortex (circled in h and i) consistent with cortical dysplasia and polymicrogyria, with thinning of the CC (g). (B) Family pedigrees of affected individuals. Square, male; circle, female; black shading, affected (or presumably affected, not genotyped) individual. Those individuals who were genotyped are labeled. Double horizontal lines indicate consanguineous parents. “NA” indicates genotype information was not available. See also, . (C) Protein domains of KIF26A. Variants from affected individuals are annotated on the corresponding amino acid positions. (D) Western blot of transfected HEK293T lysate showing expression of KIF26A and mCherry tag upon transfection of WT and patient variant KIF26A expression plasmids. Note that the lower molecular weight band of KIF26A is of the correct molecular size of KIF26A protein (194.6kDa) and is therefore considered the authentic band used in quantification. (E) Quantification of relative expression levels of WT and variant KIF26A from Western blots on HEK293T cells transfected with expression plasmids. mCherry expressed from the same plasmids is used to normalize the differences in transfection efficiency. Dots represent independent biological replicates, bars represent Mean ± S.D. (n = 5 independent experiments. Student’s t-test: **, p < 0.005, ***, p < 0.0005). (F) Microtubule depolymerization assay on SHSY5H cells transfected with WT and variant KIF26A. Shown is the quantification for the percentage of transfected cells exhibiting Nocodazole (Noco)-induced microtubule depolymerization acutely after 15 minutes of 10μM Noco treatment. Values represent Mean ± S.D. (n = 10 areas of view for each condition. Student’s t-test: ***, p < 0.0005, N.S., not significant). See also , and .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Preserving, Labeling, Western Blot, Transfection, Expressing, Variant Assay, Molecular Weight

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) Relative expression of KIF26A throughout human embryonic and postnatal brain development. Development progresses temporally from left to right, with key developmental times annotated on the axis. GW, gestational week; yo, year old (after birth). Data obtained from BrainSpan Atlas of the Developing Human Brain . (B) Cell-type clusters (left) and feature plot showing KIF26A expression (right) in human GW17-18 fetal cortex single-cell RNA-seq. Dataset and cluster annotations obtained from . The clustering and annotation from the original publication are kept unchanged. End, endothelial cells; PgS, progenitors in S phase; PgG2M, progenitors in G2M phase; vRG, ventricular radial glia; oRG, outer radial glia; Per, pericytes; OPC, oligodendrocyte precursor cells; IP, intermediate progenitor; ExN, migrating excitatory neurons; ExM, maturing excitatory neurons; ExM-U, maturing upper layer excitatory neurons; ExDp, deep layer excitatory neurons; InMGE, medial ganglionic eminence interneurons; InCGE, caudal ganglionic eminence interneurons. (C) In situ hybridization for KIF26A and RBFOX3 (NeuN) on GW22 medial cortex. Bottom shows magnified view of the CP and the IZ indicated by the squares. Hybridization the CP and IZ is consistent with scRNAseq data suggesting expression in migrating and maturing excitatory neurons. Scale bars = 500μm (top), = 100 μm (bottom). MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone; oSVZ, outer subventricular zone; iSVZ, inner subventricular zone; VZ, ventricular zone.

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Expressing, RNA Sequencing Assay, In Situ Hybridization, Hybridization

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) Representative images of in utero electroporation (IUE) of control scrambled shRNA (left) and Kif26a shRNA (right) in mouse cortex at E13.5 and analyzed at E17.5 (E13.5-17.5, 4 days post electroporation). Bottom magnified view show electroporated cells are SATB2 + neurons. Scale bars = 100μm. (B) Quantification of the percentage of mCherry-labeled cells expressing neuronal marker SATB2 or IPC marker TBR2. Values represent Mean ± S.D. (n = 5 brains for scrambled, n = 4 for shRNA1, n=6 for shRNA2, n = 5 for shRNA3. Student’s t-test: N.S., not significant). (C) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-17.5. The cortex was evenly divided into 10 bins from basal (bin 1) to apical (bin 10) surfaces and the cell distribution was normalized by the total number of electroporated cells in the analyzed area. Only mCherry + , SATB2 + neurons are quantified. Values represent Mean ± S.D. (n = 7 brains for scrambled, n = 6 for shRNA1 and shRNA2, n = 5 for shRNA3). (D) Quantification of the percentage of labeled neurons displaying bipolar or multipolar morphology, showing loss of bipolar morphology with Kif26a deficient neurons. Values represent Mean ± S.D. (Same samples as (C) . Student’s t-test: **, p < 0.005; ***, p < 0.0005). (E) Representative images of IUE in mouse cortex at E13.5 and analyzed at E15.5 (E13.5-15.5, 2 days post electroporation). Scale bar = 100μm. (F) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-15.5. Similar to (C). Only mCherry+, SATB2+ neurons are quantified. Values represent Mean ± S.D. (n = 7 brains for scrambled, n = 4 for shRNA KD. Student’s t-test: ***, p < 0.0005). (G) Representative images of IUE in mouse cortex at E13.5 and analyzed at P5 (E13.5-P5, 10 days post electroporation). Scale bar = 100μm. (H) Quantification of the laminar distributions of electroporated neurons in the cortex for E13.5-P5. Similar to (C). Only mCherry+, SATB2+ neurons are quantified. Values represent Mean ± S.D. (n = 10 brains). (I) Representative magnified images showing morphology of electroporated neurons at P5. Scale bar = 100μm. (J) Quantification of the percentage of electroporated neurons exhibiting pyramidal morphology at P5. Pyramidal morphology is defined as neurons having 1 basal dendrite and at least 2 apical dendrites from the soma. Values represent Mean ± S.D. (n = 10 brains. Student’s t-test: ***, p < 0.0005). (K) Quantification of the percentage of P5 brains with electroporated axons crossing the corpus callosum (CC). All (10 out of 10) scrambled shRNA brains showed CC crossing, while none (0 out of 10) of Kif26a KD brains showed CC crossing. (L) Representative images showing electroporated neurons send axons across the corpus callosum to the contralateral hemisphere only in scrambled control (top), but not in Kif26a KD (bottom). Insets show magnified view of selected areas. Scale bar = 500μm. See also and .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: In Utero, Electroporation, shRNA, Labeling, Expressing, Marker

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) Representative images of neurons differentiated from control (WT) and KIF26A KO (KO) hiPSCs at Day 5 and Day 21 showing shorter neurites of KIF26A KO neurons. Scale bar = 100μm. (B) Quantification of the average neurite length of WT and KO neurons shows reduced neurite outgrowth in KIF26A KO neurons. Average neurite length is calculated by dividing the total neurite length within a field of view to the number of NeuN + neurons measured by an automated module. Values represent Mean± S.D. (n = 4 plates for WT, n = 8 plates for KO from two iPSC lines. Student’s t-test: **, p < 0.005; ***, p < 0.0005). (C) Phase contrast (top) and immunostaining (bottom) images of hiPSC-derived neurospheres 2 days after plating show reduced migration in KIF26A deficient neurons. Also shown on the right is KO neurosphere infected with lentivirus to express KIF26A-mCherry exogenously (rescue). Scale bars = 100μm (top), = 50μm (bottom). (D) Quantification of the distances migrating neuroblasts traveled away from the border of the neurosphere 2 days after plating. Values represent Mean± S.D. (n = 20 neurospheres from two pairs of WT and KO iPSC lines). See also .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Immunostaining, Derivative Assay, Migration, Infection

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A-C) Representative images showing KIF26A is expressed by neurons but not progenitors in control forebrain organoids at various developmental stages. Scale bars = 100μm. (D) Schematic of the EdU pulse-chase strategy to track neuronal migration in forebrain organoids. After initial EdU exposure (1μM for 1hr), the EdU bond to DNA in dividing progenitors got diluted with each cell division. As a result, the cells displaying strong EdU detection intensity 8 days later were post-mitotic neurons born at the first cell division at the time of EdU exposure. Also see . (E) Quantification of the percentage of EdU-labeled cells expressing neuronal marker CTIP2 or IPC marker TBR2. Values represent Mean ± S.D. (n = 20 organoids, Student’s t-test: N.S., not significant). (F) Representative images showing the laminar distribution of EdU labeled cells in WT (left) and KO (right) forebrain organoids. Scale bars = 50μm. (G) Quantification of the laminar distribution of EdU-labeled CTIP2 + TBR2 − neurons in WT and KO forebrain organoids shows defective migration in KIF26A KO organoids. The cortical structure of organoids is divided evenly into 10 bins from basal (bin 10) to apical (bin 1) surfaces. Only Edu + , CTIP2 + and TBR2 − cells are counted to ensure counted cells are migrating neurons exclusively. Values represent Mean ± S.D. (n = 20 organoids from two pairs of isogenic lines). (H) Quantification of the laminar distribution of EdU-labeled TBR2 + CTIP2 − IPCs in WT and KO forebrain organoids shows normal IPC localization. Values represent Mean ± S.D., same samples as in (G) . (I) Representative images showing 8 days of 0.5μM FAK inhibitor GSK2256098(GSK) treatment rescued the laminar distribution of EdU-labeled neurons in KO organoids to resemble the localization in WT organoids. WT and KO forebrain organoids were treated with either DMSO or GSK during the 8-day chase period after EdU labeling at Day 53. Scale bar = 50μm. (J) Quantification of the laminar distribution of EdU-labeled CTIP2 + TBR2 − neurons in WT and KO forebrain organoids with and without GSK treatment. Values represent Mean ± S.D. (n = 10 organoids). See also and .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Pulse Chase, Migration, Labeling, Expressing, Marker

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) Representative images showing the defective laminar organization in KIF26A KO forebrain organoids compared to WT. Dashed lines help to illustrate the borders between CP, SVZ and VZ. Scale bars = 50μm. (B) Quantitation of relative layer thickness in WT and KO organoids shows enlarged SVZ and reduced CP in KIF26A KO organoids. Left, schematics showing the measurement of layer thickness. For each cortical structure, three measurements were taken at 45° angle pointing towards the basal surface and taken average for the quantification. Values represent Mean ± S.D. (n=20 organoids from two pair of isogenic lines. Student’s t-test: N.S., not significant; ***, p < 0.0005). (C) KO organoids show elevated apoptosis at Day 60 and 80. Dashed lines delineate the borders between the CP, SVZ and VZ. Scale bar = 100μm. (D) Quantification of the density of apoptotic cells in the VZ, SVZ and CP layers of WT and KO organoids. Values represent Mean ± S.D. (n = 20 organoids from two pairs of isogenic lines. Student’s t-test, ***, p < 0.0005; N.S., no significant difference). See also .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Quantitation Assay

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: (A) Graph-based clustering of single cells from WT and KO brain organoids at Day 60 and Day 90 (n=46,776 cells for WT, n= 36,645 cells for KO). DivPg, dividing progenitors; vRG and oRG, ventricular and outer radial glia; IPC, intermediate progenitor cells; Inhib, inhibitory neurons; ImmatEx, immature excitatory neurons; MigEx, migrating excitatory neurons; MatEx, maturing excitatory neurons. (B) The expression of selected well-known marker genes used for cell type classification. (C) UMAP feature plot showing KIF26A expression is enriched in MigEx and MatEx clusters. (D) Pseudotime analysis showing the developmental trajectory (left) calculated using Monocle 3. Right, KIF26A expression ordered by pseudotime. Dots represent individual cells colored based on cluster. Trend-line shows KIF26A expression as function of pseudotime, calculated by fitting a quasipoisson model to the data. (E) Expression of well-known marker genes across the pseudotime trajectory. (F) Volcano plot showing differentially expressed genes (DEGs) between WT and KO cells in migrating excitatory neurons. Significant DEGs with adjusted p-value < 0.05, and Log 2 fold change > 0.25 or < −0.25, are shown in blue. Selected DEGs involved in neuronal survival and apoptosis are highlighed in red. (G) Immunostaining validating decreased NRP1 expression in KO organoid. Scale bar = 100μm. (H) Gene ontology (GO) analysis (FDR<0.05) of 348 significantly downregulated (adjusted p-value< 0.05, Log 2 fold change < −1) genes in KO migrating excitatory neurons compared to WT. Size and color of the bubbles represent the proportion of commonly dysregulated genes enriched in each pathway and the significance of enrichment, respectively. (I) Gene set enrichment analysis (GSEA) enrichment score curves showing downregulation of E2F and Myc pathways in KO migrating and maturing excitatory neurons compared to WT. Hallmark Gene Sets from MSigDB Collections were included for the analysis. (J) Schematic illustration of proposed molecular mechanisms, by which KIF26A modulates radial migration, dendritic and axonal development and apoptosis in excitatory neurons. See also .

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Inhibition, Expressing, Marker, Immunostaining, Migration

Journal: Developmental cell

Article Title: Loss of Non-motor Kinesin KIF26A Causes Congenital Brain Malformations via Dysregulated Neuronal Migration and Axonal Growth as well as Apoptosis

doi: 10.1016/j.devcel.2022.09.011

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CRISPR-Cas9-mediated KIF26A KO in PGP1 and 280 iPSC lines was performed by Synthego (Redwood City, CA).

Techniques: Recombinant, Plasmid Preparation, Lysis, Multiplex Assay, Imaging, In Situ, RNA Sequencing Assay, Derivative Assay, Mutagenesis, Knock-Out, CRISPR, Software

Journal: Genes & Development

Article Title: A C9orf72–CARM1 axis regulates lipid metabolism under glucose starvation-induced nutrient stress

doi: 10.1101/gad.315564.118

Figure Lengend Snippet: Loss of C9orf72 increases FA flux and biogenesis. ( A , B ) Wild-type and C9KO MEFs were starved with glucose-free medium for 16 h, and then the association of autophagy with LDs was monitored by immunostaining of BODIPY and lysosomal marker LAMP1. The number of BODIPY and LAMP1 double-positive puncta per cell was counted, and 30–40 cells in each group from three independent experiments were statistically analyzed. ( C ) Wild-type and C9KO MEFs were grown with or without glucose starvation for 16 h, and then LDs were isolated and subjected to SDS-PAGE and immunoblot against anti-LC3 and P62. ADRP served as a loading control. The ratios of LC3II/I and the amounts of P62 were calculated and statistically analyzed. n = 3. ( D – F ) Wild-type, C9KO, and C9/Atg5 double-knockout MEFs were starved with glucose-free medium for 16 h. The LDs were labeled with BODIPY and counted, and the association of autophagy and LDs was monitored as in A . Twenty-eight to 30 cells in each group from three independent experiments were statistically analyzed. ( G ) Wild-type and C9KO MEFs were cultured in CM or glucose-free medium with [1,2- 14 C] acetate at a final concentration of 0.3 µCi for 6 h. The incorporation of [1,2- 14 C] in total lipids was measured by a scintillation counter. Counts per minute (CPM) were normalized to the total amount of protein. n = 6. ( H – J ) Wild-type and C9KO MEFs were grown in glucose-free medium, and the protein levels of acetyl-CoA carboxylase (ACC) and NOX2 were determined at the indicated time points by immunoblotting. n = 3. (GS) Glucose starvation. Data are presented as mean ± SEM. (*) P < 0.05; (**) P < 0.01.

Article Snippet: The guide RNAs (gRNAs) for CRISPR–Cas9-mediated knockout of CARM1 or Atg5 were designed with Benchling as follows: CARM1 (oligo1: 5′-CACCGCTCACTATCGGCGACGCGAA-3′; oligo2: 5′-AAACTTCGCGTCGCCGATAGTGAGC-3′) and atg5 (oligo1: 5′-CACCGAAGAGTCAGCTATTTGACGT-3′; oligo2: 5′-AAACACGTCAAATAGCTGACTCTTC-3′).

Techniques: Immunostaining, Marker, Isolation, SDS Page, Western Blot, Control, Double Knockout, Labeling, Cell Culture, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis

doi: 10.1074/jbc.RA119.010821

Figure Lengend Snippet: Exocyst genes exhibit disparate phenotypes in a genome-wide CRISPR screen. A, illustration of Rab10 and exocyst anchored to membrane bilayers. The model is based on the structures of exocyst (PDB codes 5YFP and 3HIE) (22), and guanosine 5′-[β,γ-imido]triphosphate (GMP-PNP)-bound Rab10 (PDB code 5LPN) (67). The model was prepared using PyMOL (DeLano Scientific LLC, San Carlos, CA). Rab10, a known mediator of GLUT4 exocytosis (42, 51), interacts with the Exoc6/Sec15 subunit in yeast two-hybrid screens (36). The Exoc1 and Exoc7 subunits bind to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), whereas Rab10 is anchored to the membrane through prenyl groups. Because the structures of the Rab10–exocyst complex and exocyst–membrane association are still unavailable, the positions of exocyst and Rab10 in the model are arbitrary. The proteins are shown to scale whereas the lipids and membranes are not. Besides Rab GTPases, the exocyst also interacts with other molecules, such as RalA and SNAREs (not shown) (29,–31). B, ranking of exocyst genes in a genome-wide CRISPR screen of insulin-stimulated GLUT4 exocytosis in HeLa cells (42). The significance of a gene was calculated based on enrichment of its corresponding gRNAs in the screen using the MAGeCK algorithm (46). Genes above the horizontal cutoff line (p = 0.01) are significant hits. Only exocyst genes are shown. Of the 12 exocyst genes included in the CRISPR library, EXOC1, EXOC7, and EXOC8 were recovered as significant hits. C, a CRISPR score of a gene is calculated based on fold changes in the abundance of its corresponding gRNAs by comparing a passage control population of HeLa mutant cells (without stimulation or selection) with the initial CRISPR library. Genes with CRISPR scores below the horizontal cutoff line (CRISPR score, −0.25) are considered essential genes. Only exocyst genes are shown. CRISPR score ranking of all genes in the CRISPR library is shown in Table S2. D, diagram of the HA-GLUT4-GFP reporter used to monitor insulin-stimulated GLUT4 exocytosis. The GLUT4 reporter faithfully recapitulates trafficking of endogenous GLUT4 proteins (42, 68, 69). E, normalized surface levels of the GLUT4 reporter in WT and mutant HeLa cells. The cells were either left untreated or treated with 100 nm insulin for 30 min before surface GLUT4 reporters were labeled using anti-HA antibodies and APC-conjugated secondary antibodies. GFP and APC fluorescence was measured using flow cytometry. To inhibit insulin signaling, 100 nm wortmannin was added. Datasets were normalized to untreated WT cells. Data are presented as mean ± S.D. n = 3. ***, p < 0.001; n.s., not significant, p > 0.05.

Article Snippet: The enrichment of gRNAs was analyzed by comparing the sorted population with a passage control mutant population (without sorting) using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) algorithm ( https://sourceforge.net/projects/ mageck/ ).

Techniques: Genome Wide, CRISPR, Mutagenesis, Selection, Labeling, Fluorescence, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: Inducible Exoc7/Exo70 knockout reveals a critical role of the exocyst in insulin-regulated GLUT4 exocytosis

doi: 10.1074/jbc.RA119.010821

Figure Lengend Snippet: Development of an inducible CRISPR system for adipocyte genome editing. A, diagram of an inducible CRISPR KO system in which Cas9 expression is under the control of a tight tetracycline response element (TRE). Expression of the first single guide RNA is driven by a mouse U6 (mU6) promoter, whereas expression of the second sgRNA is driven by a human U6 (hU6) promoter. NLS, nuclear localization signal. B, timeline of adipocyte differentiation and dox-induced genome editing. Dox was added to preadipocytes at a final concentration of 2 μg/ml to induce Cas9 expression 24 h before addition of an adipocyte differentiation mixture. Unless indicated otherwise, all functional assays were performed using differentiated adipocytes. C, representative immunoblots showing the expression of the indicated proteins in WT or Exoc7 KO adipocytes. Two independent samples are shown for each cell line. M.W., molecular weight.

Article Snippet: The enrichment of gRNAs was analyzed by comparing the sorted population with a passage control mutant population (without sorting) using the Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) algorithm ( https://sourceforge.net/projects/ mageck/ ).

Techniques: CRISPR, Expressing, Concentration Assay, Functional Assay, Western Blot, Molecular Weight

Journal: Parasites & Vectors

Article Title: csi-miR-96-5p delivered by Clonorchis sinensis extracellular vesicles promotes intrahepatic cholangiocarcinoma proliferation and migration via the ferroptosis-related PTEN/SLC7A11/GPX4 axis

doi: 10.1186/s13071-023-06075-7

Figure Lengend Snippet: PTEN inhibited ICC proliferation and migration via a ferroptosis mechanism. a Stable PTEN overexpression (PTEN-EXO) or CRISPR/Cas9-based knockout (PTEN-KO) HuCCT1 cell lines were established; the scale shown is 100 um. b Cell proliferation was measured by CCK-8 assay in vitro. c Cell proliferation was measured by tumor xenograft in vivo. d Cell migration was measured by transwell assay. e Cell GSH/GSSG radio assay. f Cell Fe 2+ assay. g Cell MDA assay. h , i Cell WB assay of SLC7A11 and GPX4. ICC , intrahepatic cholangiocarcinoma; WB , western blotting; GSH , glutathione; GSSG , GSH disulfide; MDA , malondialdehyde. * P < 0.05, ** P < 0.01

Article Snippet: To create stable cell lines, recombinant lentivirus (LV) vectors containing csi-miR-96-5p mimics (Shanghai Genchem Co., Ltd.), a PTEN overexpression plasmid (PTEN-EXO) (Sino Biological, China), and a PTEN CRISPR/Cas9 based knockout (PTEN-KO) plasmid (Shanghai Genchem Co., Ltd.) were constructed.

Techniques: Migration, Over Expression, CRISPR, Knock-Out, CCK-8 Assay, In Vitro, In Vivo, Transwell Assay, Multiple Displacement Amplification, Western Blot