human vac14  (Addgene inc)

Journal: NPJ Microgravity

Article Title: TRPML1 ion channel promotes HepaRG cell differentiation under simulated microgravity conditions

doi: 10.1038/s41526-025-00461-4

Figure Lengend Snippet: A DMSO-differentiated cells were pretreated with DHBP (an inhibitor of endoplasmic reticulum calcium release), ML-SI1 (a TRPML1 inhibitor) or BAPTA-AM (a calcium chelator) for 6 h before under RFC conditions, thereafter the total calcium ion (Ca²⁺) flux was determined by Flow cytometry analysis. Data are presented as mean ± SD. Statistical significance was determined using ANOVA. * P < 0.05, ** P < 0.01. B The effects of shRNA for TRPML1 on calcium flux in HepaRG cells (DMSO was used to induce differentiation) under RFC (10 rpm/min) for 72 h. Mean ± SD by Student’s t -test. C Immunofluorescence staining of AFP (red) and DAPI (blue) in HepaRG cells pretreated without or with BAPTA-AM (2.5 µM) under RFC or not. Scale bar: 80 μm. Mean ± SD by ANOVA. D The RT-qPCR analysis for mRNA levels of AFP in HepaRG cells pretreated without or with BAPTA-AM (2.5 µM) under RFC or not. Mean ± SD by ANOVA. E The western blot analysis for protein content of AFP in HepaRG cells pretreated without or with BAPTA-AM (2.5 µM) under RFC or not. Mean ± SD by ANOVA. F The effects of YM201636 on calcium flux in HepaRG cells under RFC (10 rpm/min) for 72 h. Mean ± SD by Student’s t -test. G , H The mRNA and protein levels of VAC14 in HepaRG cells were incubated without or with RFC (10 rpm/min) for 72 h as assessed by RT-qPCR assay and immunofluorescence staining. Scale bar: 80 μm. Mean ± SD by ANOVA. I The RT-qPCR analysis for mRNA level of AFP in HepaRG cells pretreated without or with YM201636 (1 µM) under RFC or not. Mean ± SD by Student’s t -test. J The immunofluorescence staining of AFP in HepaRG cells pretreated without or with YM201636 (1 µM) under RFC conditions or not. Scale bar: 80 μm. Mean ± SD by ANOVA. K The western blot analysis of AFP content in HepaRG cells pretreated without or with YM201636 (1 µM), under RFC or not. The data are based on three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used in this study were as follows: AFP (Proteintech, 14550-1-AP), ALB (Proteintech, 16475-1-AP), CK18 (Proteintech, 10830-1-AP), TRPML1 (Santa Cruz Biotechnology, sc-398868), VAC14 (Santa Cruz Biotechnology, sc-271831), F-actin (ThermoFisher, A30107), β-cantein (Proteintech, 51067-2-AP), FAK (ABclonal, A11131), β-actin (Proteintech, 81115-1-RR), FN1 (ABclonal, A12932), FN1 (ABclonal, A12932), VIM (ABclonal, A19607), α-SMA (Proteintech, 14395-1-AP), GAPDH (Proteintech, 60004-1-Ig), and all the Primary antibodies were at 1:1000 dulution.

Techniques: Flow Cytometry, shRNA, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Incubation

Journal: NPJ Microgravity

Article Title: TRPML1 ion channel promotes HepaRG cell differentiation under simulated microgravity conditions

doi: 10.1038/s41526-025-00461-4

Figure Lengend Snippet: A The RT-qPCR analysis for the mRNA levels of FAK, β-actin, FN1, VIM, and α-SAM in HepaRG cells, without or with RFC. Mean ± SD ( n = 3) by Student’s t -test. B The western blot analysis for the protein expression of FAK, β-actin, FN1, VIM, and α-SAM in HepaRG cells, without or with RFC. C Immunofluorescence staining of F-action (green) and DAPI (blue) in HepaRG cells incubated without or with RFC (10 rpm/min) for 72 h as assessed by confocal imaging. Scale bar: 80 μm. Mean ± SD by Student’s t -test. D The AFM analysis for the stiffness of HepaRG cells, without or with RFC. Mean ± SD ( n = 5) by Student’s t -test. E The effects of cytoskeletal remodeling on calcium flux in HepaRG cells treated without or with RFC (10 rpm/min) for 72 h. Mean ± SD by Student’s t -test. F The content of AFP in HepaRG cells incubated without or with Cyt B, under RFC conditions or not, as assessed by immunofluorescence staining (Con: the undifferentiated group, the other groups: differentiated with DMSO). Scale bar: 80 μm. Mean ± SD by ANOVA. G The content of VAC14 in HepaRG cells incubated without or with Cyt B, under RFC conditions or not, as assessed by immunofluorescence staining. Scale bar: 20 μm. Mean ± SD by ANOVA. The data are based on three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Antibodies used in this study were as follows: AFP (Proteintech, 14550-1-AP), ALB (Proteintech, 16475-1-AP), CK18 (Proteintech, 10830-1-AP), TRPML1 (Santa Cruz Biotechnology, sc-398868), VAC14 (Santa Cruz Biotechnology, sc-271831), F-actin (ThermoFisher, A30107), β-cantein (Proteintech, 51067-2-AP), FAK (ABclonal, A11131), β-actin (Proteintech, 81115-1-RR), FN1 (ABclonal, A12932), FN1 (ABclonal, A12932), VIM (ABclonal, A19607), α-SMA (Proteintech, 14395-1-AP), GAPDH (Proteintech, 60004-1-Ig), and all the Primary antibodies were at 1:1000 dulution.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining, Incubation, Imaging

Journal: bioRxiv

Article Title: LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase

doi: 10.1101/2023.10.19.563158

Figure Lengend Snippet: Genome-wide CRISPR screens identify the PIKFYVE lipid kinase complex as a mediator of LKB1-dependent tumor suppression. (A) Schematic representation of the CRISPR screen experiment. (B, C) Beta score plot of DNAs encoding sgRNAs from cultures of A549 EV and WT cells transduced with a genome-wide CRISPR library and grown in spheroid (B) or 2D (C) culture. Members of the PIKFYVE lipid kinase complex are labeled in red lettering. Known tumor suppressor genes are labeled in blue lettering. Beta score is a weighted score of the log 2 (fold-change) values of all sgRNAs for a given gene. (D) An enrichment analysis of the sgRNAs that were enriched in the WT spheroids (beta score >1) but not in the EV spheroids (beta score < 1). (E) A representation of the structure of the PIKFYVE lipid kinase complex . (F, G) Log 2 (fold-change) values of individual sgRNAs corresponding to FIG4 (E) or VAC14 (F) in EV and WT cells grown in 2D or spheroid culture. (H) Growth of A549 EV and WT spheroids treated with DMSO or 100 nM of the PIKFYVE inhibitor apilimod (API). (I, J) Growth of A549 EV and WT spheroids transduced with an empty lentiviral vector (CTRL) or a vector that expresses a PIKFYVE mutant (N1939K) that is API-resistant, and treated with DMSO or API 100 nM. The growth curve in (I) shows the growth of these spheroids in API over a 72-hour period, whereas the bar graph in (J) depicts the fold-change in the volume of the spheroids after 72 hours in culture in DMSO or API. (K, L) Growth of EV and WT spheroids derived from H2030 (K) or MOR (L) in the presence of DMSO or API. Error bars indicate standard deviation. n = 3-6. *, **, and **** indicate p<0.05, 0.01, and 0.0001. ns, non-significant. P-values indicate pairwise statistical comparisons to “WT + DMSO” in panels (H, K, and L) , and “WT + N1939K” in panel (I) .

Article Snippet: Proteins were separated on 4–12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen WG1402), transferred to a nitrocellulose membrane, and probed with antibodies against LKB1 (Santa Cruz sc-374334), GAPDH (CST 2118S), phospho-AMPKα T172 (CST 2535S), AMPKα (CST 5832S), phospho-ULK1 S555 (CST 5869S), ULK1 (CST 8054S), phospho-MEK1/2 S217/221 (CST 9154S), MEK1/2 (CST 8727S), phospho-ERK1/2 T202/Y204 (CST 4370S), ERK1/2 (CST 4695S), phospho-AKT T308 (CST 4056S), AKT1 (CST 2938S), phospho-S6 S240/244 (CST 5364S), S6 (CST 2217S), β-actin (CST 3700S), FIG4 (Novus NBP3-05130), and VAC14 (Sigma-Aldrich SAB4200074).

Techniques: Genome Wide, CRISPR, Transduction, Labeling, Plasmid Preparation, Mutagenesis, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: LKB1 suppresses growth and promotes the internalization of EGFR through the PIKFYVE lipid kinase

doi: 10.1101/2023.10.19.563158

Figure Lengend Snippet: Validation of the genome-wide CRISPR screens. (A) Log 2 (fold-change) values of individual sgRNAs corresponding to VAC14 in EV and WT cells – grown in 2D or spheroid culture – from a second replicate of the CRISPR screens. (B-E) FIG4 and VAC4 protein levels in EV and WT cells (derived from A549 or MOR lines) containing sgCTRL, or sgRNAs against FIG4 or VAC14 . (F-I) Spheroid growth of EV and WT cells (derived from A549 or MOR lines) containing sgCTRL, or sgRNAs against FIG4 or VAC14 . Error bars indicate standard deviation. n = 3-6. **** indicates p<0.0001. P-values indicate pairwise statistical comparisons to “WT sgCTRL.”

Article Snippet: Proteins were separated on 4–12% NuPAGE Bis-Tris polyacrylamide gels (Invitrogen WG1402), transferred to a nitrocellulose membrane, and probed with antibodies against LKB1 (Santa Cruz sc-374334), GAPDH (CST 2118S), phospho-AMPKα T172 (CST 2535S), AMPKα (CST 5832S), phospho-ULK1 S555 (CST 5869S), ULK1 (CST 8054S), phospho-MEK1/2 S217/221 (CST 9154S), MEK1/2 (CST 8727S), phospho-ERK1/2 T202/Y204 (CST 4370S), ERK1/2 (CST 4695S), phospho-AKT T308 (CST 4056S), AKT1 (CST 2938S), phospho-S6 S240/244 (CST 5364S), S6 (CST 2217S), β-actin (CST 3700S), FIG4 (Novus NBP3-05130), and VAC14 (Sigma-Aldrich SAB4200074).

Techniques: Genome Wide, CRISPR, Derivative Assay, Standard Deviation