scramble control shrna  (Genechem)

Journal: Cell Death Discovery

Article Title: SPINK2 silencing suppresses leukemic proliferation and restores myeloid commitment via MECOM downregulation in acute myeloid leukaemia

doi: 10.1038/s41420-026-02988-1

Figure Lengend Snippet: A Barplot showing the expression of SPINK2 measured by qPCR in a panel of AML cell lines. n = 6 for each cell line. B Bowtie plot representing SPINK2 mRNA quantification by q-PCR following SPINK2 shRNA silencing and normalized using GAPDH ( n = 6). C Lineplot displaying cell viability upon SPINK2 knock-down determined by counting cells every 24 h for four consecutive days post shRNA induction. The results are indicative of 3 independent experiments ( n = 6). D Two-dimensional flow cytometric dot plot showing BrdU incorporation 72 hours post doxycycline induction. The three gates indicate the G0/G1, S and G2/M phases of the cell cycle. The barplot on the righthand side represent an average of three independent experiments ( n = 6). E Bowtie plot showing the percentages of Annexin V + apoptotic/necrotic cells. F Representative two-dimensional flow cytometric contour plots showing the expression levels of CD14, CD64, CD35 and CD300E in FUJIOKA cells that have undergone SPINK2 silencing 96 hours post doxycycline induction ( n = 6). The statistical analysis reported in this figure have been performed using student’s t test (*** p < 0.001, * p < 0.05).

Article Snippet: A scrambled control shRNA plasmid (shCtr), Tet-pLKO-puro-Scrambled (Addgene plasmid #47541), was used as a non-targeting control [ ].

Techniques: Expressing, shRNA, Knockdown, BrdU Incorporation Assay

Journal: The Journal of Biological Chemistry

Article Title: Cytosolic nucleic acid sensing triggers type I interferon activation via the Hippo kinase LATS1

doi: 10.1016/j.jbc.2026.111204

Figure Lengend Snippet: LATS1 operates at the level of TBK1. A , immunoblot analysis of LATS1 expression in HEK 293T cells treated with control siRNA or siRNAs targeting LATS1 for 24 h. B , IFN-β luciferase reporter assay in HEK 293T cells treated with control siRNA or siRNAs targeting LATS1 as in A , followed by transfection of plasmids encoding RIG-I, MAVS, STING, TBK1, and IRF3. C , co-immunoprecipitation and immunoblot of FLAG-LATS1 and HA-TBK1 co-transfected in HEK 293T cells. D and E , immunoblot analysis of TBK1-LATS1 interactions in MEF cells transfected with pI:C ( D ) or ISD ( E ) (2.5 μg/ml each; 2 h). Statistical significance was determined using student’s t test (∗∗∗ p < 0.001, ∗∗ p < 0.01, and ∗ p < 0.05).

Article Snippet: For small interfering RNA (siRNA) studies, HEK 293T cells were transfected with 60 pmol of control scrambled siRNA or 3 different siRNA targeting human LATS1 (20 pmol each) (Bioneer) using Lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Expressing, Control, Luciferase, Reporter Assay, Transfection, Immunoprecipitation

Journal: Nature Communications

Article Title: Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

doi: 10.1038/s41467-026-69331-1

Figure Lengend Snippet: a Quantification of western blots to screen Kinase library regulated ATP6V1E1 phosphorylation. b In vitro phosphorylation assay of ATP6V1E1. The proteins of GFP-BMX, Flag-ATP6V1E1 and HA-ATP6V1E1 Y56/Y57A were overexpressed and purified in HEK293T cells. c–e Detection of ATP6V1E1 phosphorylation in macrophages transfected with Bmx siRNA ( c ), treated with BMX inhibitor (10 nM) ( d ) or in Bmx +/- macrophages ( e ), then infected with H37Rv (MOI = 5) for indicated times. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. f Subcellular fractionation of macrophages from WT or Bmx +/- mice or WT macrophages under the treatment with BMX-IN (10 nM) for 1 h. Cytosolic (C) and membrane (M) fractions were immunoblotted with antibodies against the V1 domain subunit ATP6V1E1 and the V0 domain subunit ATP6V0d1. α-Tubulin and V0d1 serve as loading controls for cytosolic and membrane fractions, respectively. g Representative LysoTracker staining of macrophages transfected with scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times. Scale bar, 10 μm. h Quantification of LysoTracker fluorescence intensity (mean ± SEM). i Intracellular CFU in macrophages transfected scrambled or Bmx -specific siRNA and then infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). j Intracellular CFU in macrophages from WT and Atp6v1e1 +/- mice treated with BMX inhibitor (10 nM) and infected with H37Rv (MOI = 5) for the indicated times (mean ± SEM). k–l Intracellular CFU and bacterial survival in macrophages from WT or Bmx +/- mice infected with H37Rv or H37RvΔChp2 for the indicated times (MOI = 5) (mean ± SEM). ( m )H&E staining of lung tissues from WT and Bmx +/- mice infected with H37Rv by intranasal infection ( ~ 200 CFU) for 4 weeks. (mean ± SEM, N = 3 mice) #1-3 indicate three representative lung tissues. Scale bar, 5000 μm. n Quantification of the histopathology score in (m) (mean ± SEM). o Bacterial CFUs in the lungs of WT and Bmx +/- mice in (m) (mean ± SEM). p Acid-fast staining of the lung tissues of WT and Bmx +/- mice in (m). Scale bar, 100 μm. Data in ( a–m ) are representative of one experiment with at least three independent biological replicates ( h–l , n , o , n = 3). Two-tailed unpaired Student’s t test (h-l and n) was used for statistical analysis. Two-sided Mann-Whitney U test ( o ) was used for statistical analysis.

Article Snippet: The siRNA targeting mouse Bmx (sc-38942) and control scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, and a siRNA pool specifically targeting mouse Atp6v1e1 was obtained from RiboBio (Guangzhou, China).

Techniques: Western Blot, Phospho-proteomics, In Vitro, Purification, Transfection, Infection, Fractionation, Membrane, Staining, Fluorescence, Histopathology, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Mycobacterium tuberculosis modulates phosphorylation of host ATP6V1E1 to promote intracellular survival

doi: 10.1038/s41467-026-69331-1

Figure Lengend Snippet: a Detection ATP6V1E1 phosphorylation in macrophages infected with H37Rv or H37RvΔChp2 (MOI = 5) for indicated times. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. b Immunoblotting of the lysates of macrophages transfected with scrambled or Bmx -specific siRNA and infected with H37Rv or H37RvΔChp2 (MOI = 5) for the indicated times by p-ATP6V1E1 Y56/Y57 antibody. ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. c Immunoblot analysis of ATP6V1E1 phosphorylation in peritoneal macrophages isolated from wild-type or Bmx +/- mice infected with H37Rv or H37RvΔChp2 for the indicated times (MOI = 5). ratio: p-ATP6V1E1 Y56/57 /ATP6V1E1. d In vitro phosphorylation assay of purified GFP-BMX and Flag-ATP6V1E1, which were overexpressed in HEK293T cells and treated with recombinant Chp2 protein(1 μg). e Peritoneal macrophages were infected with H37Rv or H37RvΔChp2 (MOI = 5) for 6 h, and changes of V-ATPase subunit abundance in the cytosolic (C) and membrane (M) fractions were analyzed by western blot. f Quantification of Western blots described in ( e ) (mean ± SEM). g , h Intracellular CFU and bacterial survival in macrophages transfected with scrambled or Bmx -specific siRNA and infected with H37Rv or H37RvΔRv1184c for the indicated times (MOI = 5) or then treat with NH 4 Cl (20 μM) (mean ± SEM), g P = 4.8736E-06 (Ctrl, si-Ctrl, 24 h). h P = 4.86583E-05 (Ctrl, si-Ctrl). ( i ) Surface plasmon resonance (SPR) assay of the direct interaction of ATP6V1E1 with BMX. BMX captured on a carboxy (COOH) chip can bind ATP6V1E1 with an affinity constant of 190 nM as determined in a localized surface plasmonic resonance (LSPR) assay. j Immunoblotting and immunoprecipitation of lysates from HEK293T cells transfected with plasmids for 48 h were performed and detected using the p-ATP6V1E1 Y56/Y57 antibody. k Endogenous interaction of ATP6V1E1 with BMX in mice peritoneal macrophages infected with H37Rv or H37RvΔRv1184c (MOI = 5) for the indicated times. l The predicted structural model of Rv1184c-BMX-V-ATPase complex by AlphaFold3 Server. Yellow represents ATP6V1G1, gray represents ATP6V1E1, blue represents Rv1184c, light purple represents BMX, dark purple represents the catalytic region of BMX, and red box represents the Y56/57 site of ATP6V1E1. Data in ( a–k ) are representative of one experiment with at least three independent biological replicates ( f–h , n = 3). Two-tailed unpaired Student’s t test ( f–h ) was used for statistical analysis.

Article Snippet: The siRNA targeting mouse Bmx (sc-38942) and control scrambled siRNA (sc-37007) were obtained from Santa Cruz Biotechnology, and a siRNA pool specifically targeting mouse Atp6v1e1 was obtained from RiboBio (Guangzhou, China).

Techniques: Phospho-proteomics, Infection, Western Blot, Transfection, Isolation, In Vitro, Purification, Recombinant, Membrane, SPR Assay, Immunoprecipitation, Two Tailed Test