scramble shrna shcontrol  (Addgene inc)

Journal: Journal of Pharmaceutical Analysis

Article Title: Oblique-incidence reflectivity difference technology identifies the antiviral drug Ribavirin as an inhibitor of lung tumor progression by targeting AMPK signaling

doi: 10.1016/j.jpha.2025.101306

Figure Lengend Snippet: Ribavirin has synergistic effects on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) downregulation. (A) Three AMPK-targeting short hairpin RNAs (shRNAs) were screened to evaluate their knockdown efficiency and suppress AMPK expression in lung cancer cells. The data shown are representative of n = 3 biological replicates. (B) Tumor anatomy of mouse Lewis lung carcinoma (LLC) cells subcutaneous tumor-bearing mice in different treatment groups. (C) Tumor volume of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± standard error of the mean (SEM); n = 8 mice per group, with differences denoted by ∗ P < 0.05 and ∗∗ P < 0.01. (D) Tumor weights of LLC subcutaneous tumor-bearing mice in different treatment groups. Statistical significance between datasets was assessed by one-way ANOVA, followed by Tukey’s multiple comparisons post hoc test between all groups. The values are the means ± SEM; n = 8 mice per group, with differences denoted by ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001. (E) Schematic illustration of the mechanism by which Ribavirin regulates AMPK activation. AMPK is phosphorylated in response to an increased AMP/adenosine triphosphate (ATP) or adenosine diphosphate (ADP)/ATP ratio, influencing cell growth and proliferation by inactivating mechanistic target of rapamycin complex 1 (mTORC1). Ribavirin exerts its regulatory effect by inhibiting AMPK phosphorylation. Created with www.BioRender.com . shControl: non-targeting control short hairpin RNA; shAMPKα: AMPKα-targeting short hairpin RNA; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; PBS: phosphate buffered saline; mTOR: mechanistic target of rapamycin; Raptor: regulatory-associated protein of mTOR; Deptor: DEP domain-containing mTOR-interacting protein; PRAS40: proline-rich Akt substrate of 40 kDa; mLST8: mammalian lethal with SEC13 protein 8; 4EBP1:eukaryotic translation initiation factor 4E-binding protein 1; P: phosphorylation.

Article Snippet: The mice were randomly divided into four groups ( n = 8): the non-targeting control short hairpin RNA (shControl)-LLC + phosphate buffered saline (PBS) (Sangon Biotech, Shanghai, China) group, shControl-LLC + Ribavirin group, shAMPKα-LLC + PBS group, and shAMPKα-LLC + Ribavirin group.

Techniques: Knockdown, Expressing, Activation Assay, Phospho-proteomics, Control, shRNA, Saline, Binding Assay

Journal: Journal of Translational Medicine

Article Title: CXCR4/CXCL12 axis promotes lymphatic metastasis in tongue squamous cell carcinoma via PI3K/AKT signaling pathway

doi: 10.1186/s12967-025-06707-9

Figure Lengend Snippet: CXCR4 regulates TSCC cell proliferation, migration, invasion, and EMT. a Cell proliferation assessed by CCK-8 assay in CAL-27 cells with different CXCR4 expression levels (Control, shControl, shRNA-CXCR4-KD, and shRNA-CXCR4-OE). b Colony formation assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). c Transwell migration assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. d Transwell invasion assay of CAL-27 cells with different CXCR4 expression levels. Representative images (left) and quantification (right). Scale bars, 100 μm. e Quantification of apoptotic cells measured by flow cytometry. f Representative flow cytometry plots showing apoptosis in CAL-27 cells with different CXCR4 expression levels. g Western blot analysis of EMT markers (E-cadherin, N-cadherin, and Vimentin) in CAL-27 cells with different CXCR4 expression levels. h qRT-PCR analysis of EMT markers in CAL-27 cells with different CXCR4 expression levels. i Wound healing assay showing the effect of CXCL12 on CAL-27 cell migration. Representative images (left) and quantification (right). Scale bars, 100 μm. j Real-time cell analysis (RTCA) showing the effect of CXCL12 on CAL-27 cell migration. Data are presented as mean ± SD from three independent experiments. *p < 0.05 , **p < 0.01 , ***p < 0.001 , ****p < 0.0001 (compared to Control or Serum-Free); # p < 0.05 , ## p < 0.01 , #### p < 0.0001 (compared to shControl or Blank-Serum); && p < 0.01 , &&&& p < 0.0001 (compared to shControl- CXCR4-KD)

Article Snippet: Lentiviral vectors expressing CXCR4 shRNA (shCXCR4) or non-targeting control shRNA (shControl) were constructed by cloning the target sequences into the pLKO.1 vector (Addgene, Cat10878).

Techniques: Migration, CCK-8 Assay, Expressing, Control, shRNA, Colony Assay, Transwell Migration Assay, Transwell Invasion Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Wound Healing Assay, Cell Analysis

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: Morphology, viability and proliferation of EA.hy926 cells after treatment with shHSPD1-A549 and shControl-A549 secretomes. (A) Morphology of EA.hy926 cells observed under an inverted microscope, captured at x100 magnification. (B) Viability of EA.hy926 cells after 24-h treatment with secretomes, determined by MTT assay. Data are presented as a percentage of the untreated control. (C) Proliferation of EA.hy926 cells measured using the BrdU assay after 24-h treatment with secretomes. Data are shown as a fold change relative to the untreated control. Bar graphs represent mean ± SD from three independent experiments. * P<0.05. sh, short hairpin.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques: Inverted Microscopy, MTT Assay, Control, BrdU Staining

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: Cell migration capability of EA.hy926 cells after treatment with shHSPD1-A549 and shControl-A549 secretomes. (A) Representative images of the wound area captured at 0, 8, 16 and 24 h after secretome treatment, captured at x100 magnification. (B) Percentage of wound healing. Data are presented as mean ± SD from three independent experiments. * P<0.05. sh, short hairpin; HSPD1, heat shock protein family D member 1.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques: Migration

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: Cell invasion of EA.hy926 cells after treatment with shHSPD1-A549 and shControl-A549 secretomes. (A) Representative images of crystal violet-stained EA.hy926 cells after 24-h treatment with secretomes, captured at x100 magnification. (B) The optical density of crystal violet-stained cells at 590 nm. Data are presented as mean ± SD from three independent experiments and expressed as a fold change relative to the untreated control. *** P<0.001. sh, short hairpin; HSPD1, heat shock protein family D member 1.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques: Staining, Control

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: Cell aggregation of EA.hy926 cells following treatment with shHSPD1-A549 and shControl-A549 secretomes. (A) Representative images of EA.hy926 cell aggregates after secretome treatment, captured at x100 magnification. (B) Size of cell aggregates. Data are presented as mean ± SD from three independent experiments. * P<0.05, ** P<0.01. sh, short hairpin; HSPD1, heat shock protein family D member 1.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques:

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: Endothelial tube formation of EA.hy926 cells after treatment with shHSPD1-A549 and shControl-A549 secretomes. (A) Representative images of tube formation in EA.hy926 cells after secretome treatment, captured at x40 magnification with additional zoomed-in areas. (B) Length of formed tubes. (C) Area of formed tubes. Data are presented as mean ± SD from three independent experiments. * P<0.05. sh, short hairpin; HSPD1, heat shock protein family D member 1.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques:

Journal: Biomedical Reports

Article Title: Heat shock protein family D member 1 mediates lung cancer cell‑induced angiogenesis of endothelial cells

doi: 10.3892/br.2025.1955

Figure Lengend Snippet: VEGF levels in the culture supernatant of EA.hy926 cells after treatment with shHSPD1-A549 and shControl-A549 secretomes. VEGF levels were measured by ELISA. The bar graph represents mean ± SD from three independent experiments. * P<0.05, ** P<0.01. VEGF, vascular endothelial growth factor; sh, short hairpin; HSPD1, heat shock protein family D member 1.

Article Snippet: The scrambled shRNA (shControl) with the oligonucleotide sequence: 5'-CCTAAGGTTAAGTCGCCCTCGCTCGAGCGAGGGCGACTTAACCTTAGG-3' inserted into the pLKO.1-puro plasmid (a gift from Dr David Sabatini, Whitehead Institute for Biomedical Research and Department of Biology, Massachusetts Institute of Technology, MA, USA; Addgene plasmid no. 1864) served as a negative control.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Galectin-1 Modulates Hepatocellular Carcinoma Response to Thermal Ablation Through Regulating Glycolysis

doi: 10.1101/2024.12.12.628238

Figure Lengend Snippet: A-B) SNU449 survival rates (n=3 each) (A) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell growth (B) . C) Western blot of Gal-1 in SNU449, SNU423, and HepG2/C3a (positive control). β-Tubulin was used to as loading control. D-E) SNU449 cell growth (n=3 each) (D) post-thermal exposure at 37°C and 47°C with Gal-1 inhibitor OTX or DMSO at 24, 48, and 72 hours. Corresponding cell survival rates (E) . F) Western blot of Gal-1 in shGal-1-SNU449 (SNU449 with Gal-1 knockdown) and respective shControl-SNU449. G-H) shControl and shGal-1-SNU449 cell growth (n=3) (G) post-thermal exposure at 37°C and 47°C at 24, 48, and 72 hours. Corresponding cell survival rates (H) . p-values were calculated using one-tailed-unpaired student’s t-test, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: The following day, cells were transduced with lentiviral shRNA particles carrying three to five expression constructs each encoding target-specific 19-25 nucleotides (plus hairpin) shRNA designed to silence the gene expression of galectin-1 (sc-35441-V, scbt) or respective shControl particles (sc-108080, scbt).

Techniques: Western Blot, Positive Control, Control, Knockdown, One-tailed Test

Journal: EMBO Reports

Article Title: Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

doi: 10.1038/s44319-024-00204-8

Figure Lengend Snippet: ( A ) Nup358-positive AL are present at ERMCSs. Confocal microscopic image of a U2OS cell displaying the relative localization of indicated proteins; Nup358 for AL (red), PDIA3 for ER (green) and MitoTracker for mitochondria (blue). Arrows show Nup358-positive AL associated with mitochondria. Scale bar, 10 µm. The proportion of Nup358-positive AL associated with mitochondria was 42.8 ± 6.6% ( n = 20 cells). ( B ) Association of Nup358-positive AL with mitochondria. Stimulated emission depletion (STED) super-resolution microscopy of a Huh7 cell immunostained with the AL marker (Nup358, green) and mitochondria marker (TOM20, red). Arrows indicate Nup358-positive AL associated with mitochondria. Scale, bar 5 µm. ( C ) Nup358, along with other AL-resident nucleoporins like Nup88 and Nup62, is present in the ERMCS fraction. HeLa cells were processed to obtain ERMCS (MAM) fractions, which along with other fractions were analysed for the presence of specific proteins by western blotting. PDIA3, ER marker; TOM20, mitochondrial marker; VAPB, ERMCS marker. ( D ) Increased ER–mitochondria contacts in Nup358-deficient cells. U2OS cells were initially transfected with control (siControl) or Nup358 (siNup358)-specific siRNA and later co-transfected with RFP-ER (red) and BFP-Mito (pseudo-coloured in green) constructs for labelling ER and mitochondria, respectively. Left: The co-localizing pixels are shown in grey. Scale bar, 10 µm. Right top: The extent of Nup358 depletion was evaluated by western blotting with Nup358-specific antibody. Vinculin was used as a loading control. Right bottom: Analysis of individual Manders’ overlap coefficient values of RFP-ER with BFP-Mito from siControl and siNup358-treated HeLa cells ( n = 25 cells from three independent experiments). Data are mean ± SD, Student’s t test. P value is indicated. ( E ) Extent of VAPB interacting with PTPIP51 as analysed by co-immunoprecipitation (co-IP) assay. Top: Extent of Nup358 depletion was assessed by western blotting. Bottom left: IP of endogenous PTPIPI51 from HeLa cells treated with siControl or siNup358 was performed and the extent of VAPB co-immunoprecipitated was assessed by western blotting. The arrow indicates PTPIP51 bands and asterisk indicates IgG heavy chain cross-reaction. Bottom right: Quantitation of the amount of VAPB associated with PTPIP51 ( n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated. ( F ) Enhanced in situ interaction between VAPB and PTPIP51 in the absence of Nup358. In situ proximity ligation assay (PLA) was performed for assessing the interaction between VAPB and PTIPI51 using specific antibodies. Left: Representative images showing PLA puncta (red) in HeLa cells treated with siControl or siNup358. DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. Right top: Extent of Nup358 depletion as analysed by western blotting, along with vinculin as loading control. Right bottom: Quantitation of the number of PLA puncta per cell from siControl and siNup358 HeLa cells ( n = 82 cells for siControl and 73 cells for siNup358 from three independent experiments). Data are mean ± SD, Student’s t test. P value is indicated. ( G ) Increased interaction between BAP31 and Fis1 in Nup358-deficient cells. The cells were treated and processed as described in ( F ). Left: PLA was performed for monitoring the in situ interaction between BAP31 and Fis1, components of another ER–mitochondria tethering complex. Right: Quantitation of the number of PLA puncta per cell from siControl and siNup358 HeLa cells ( n = 90 cells for siControl and siNup358 from three independent experiments). Data are mean ± SD, Student’s t test. P value is indicated. ( H ) Increased contacts between ER and mitochondria in Nup358-deficient cells as assessed by transmission electron microscopy (TEM). Left: TEM images displaying ER–mitochondria contacts in the presence (siControl) and absence of Nup358 (siNup358). Contact sites are highlighted with yellow arrows. Scale bar, 1 µm. Right: Quantitative data depicting percentage (%) of mitochondrial surface (perimeter) showing ≤30 nm proximity with the ER membrane ( n = 30 mitochondria for siControl and 35 mitochondria for siNup358-treated conditions). Data are mean ± SD, Student’s t test. P value is indicated. .

Article Snippet: Constructs harbouring inducible shRNA control (shControl) and shRNA against Nup358 (shNup358) were generated by cloning the following target sequences into the Tet-pLKO-puro (Addgene #21915). shControl (5′-GTGGACTCTTGAAAGTACTAT-3′) and shNup358 (5′-GGTGAAGATGGATGGAATA-3′) were cloned into the Tet-pLKO-puro vector (Addgene #21915) at the AgeI/EcoRI sites.

Techniques: Super-Resolution Microscopy, Marker, Western Blot, Transfection, Control, Construct, Co-Immunoprecipitation Assay, Immunoprecipitation, Quantitation Assay, In Situ, Proximity Ligation Assay, Staining, Transmission Assay, Electron Microscopy, Membrane

Journal: EMBO Reports

Article Title: Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

doi: 10.1038/s44319-024-00204-8

Figure Lengend Snippet: ( A ) Nup358 depletion leads to elevated mTORC2/Akt activation. HeLa cells were treated with Control (siControl) and Nup358 (siNup358) specific siRNA. Top: The cells were then analysed for the extent of mTORC2/Akt activation by western blotting using indicated antibodies. Vinculin was used as a loading control. Bottom: Quantitative representation of the relative levels of pAkt (S473) as compared to total Akt under the above-mentioned experimental conditions ( n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated. ( B ) Increased activation of mTORC2/Akt signalling occurs in Nup358 knockout (KO) HeLa cells as compared to wild-type (WT) cells. Top: Cells were lysed and subjected to western blotting with indicated antibodies. Bottom: Quantitative data depicting the relative level of pAkt (S473) as compared to total Akt under indicated conditions ( n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated. ( C ) Nup358 restricts mTORC2-mediated phosphorylation of Akt at S473 upon growth factor signalling. HeLa cells, transfected with indicated siRNAs, were serum starved for 3 h. The cells were then treated with (+) or without (−) insulin (1 nM for 20 min) and analysed for mTORC2/Akt activation by western blotting. Top: The extent of depletion of indicated proteins analysed by western blotting, with vinculin used as a loading control. Bottom left: The extent of Akt phosphorylation at S473 under indicated conditions was determined. Vinculin was used as a loading control. Bottom right: Quantitative representation of the relative levels of pAkt (S473) as compared to total Akt ( n = 3 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. ( D ) Nup358 depletion leads to mTORC1 activation, which was rescued by co-depletion of Rictor. HeLa cells, treated with indicated specific siRNAs, were analysed for the extent of protein depletion using western blotting, with mTOR being used as a loading control (left). Right top panels: mTORC2 activation was assessed by examining the phosphorylation of Akt at S473. mTORC1 activation was assessed by monitoring the phosphorylation of Akt at T308 and the mTORC1 target S6 at S235 and S236 using western blotting. Lower panels: Quantitation of the relative levels of phosphorylation of specific proteins normalised to GAPDH (for pAkt-S473) or respective total proteins as indicated ( n = 3 or 4 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. ( E ) Co-depletion of Rictor reverses the increase in ER–mitochondria connectivity in Nup358-depleted cells. HeLa cells were treated with specific siRNAs to deplete indicated proteins and analysed for the extent of ERMCSs present using in situ PLA (yellow) with VAPB and PTPIP51 antibodies (left). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. The extent of protein depletion (right top) and quantitation of PLA dots per cell (right bottom) are shown ( n = 72 cells for siControl; 59 cells for siNup358; 58 cells for siNup358 + siRictor from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. ( F ) The experiment was conducted as described in ( E ), except that instead of Rictor, as indicated, Raptor-specific siRNA was used. Left: Representative microscopic images displaying the PLA dots (yellow). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. The extent of protein depletion (right top) and quantitation of PLA dots per cell (right bottom) are shown ( n = 90 cells for siControl; 107 cells for siNup358; 98 cells for siNup358 + siRaptor from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. .

Article Snippet: Constructs harbouring inducible shRNA control (shControl) and shRNA against Nup358 (shNup358) were generated by cloning the following target sequences into the Tet-pLKO-puro (Addgene #21915). shControl (5′-GTGGACTCTTGAAAGTACTAT-3′) and shNup358 (5′-GGTGAAGATGGATGGAATA-3′) were cloned into the Tet-pLKO-puro vector (Addgene #21915) at the AgeI/EcoRI sites.

Techniques: Activation Assay, Control, Western Blot, Knock-Out, Phospho-proteomics, Transfection, Quantitation Assay, In Situ, Staining

Journal: EMBO Reports

Article Title: Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

doi: 10.1038/s44319-024-00204-8

Figure Lengend Snippet: Stable HEK293T cells with Inducible short hairpin RNA (shRNA) for control (shControl) or Nup358 (shNp358) were initially induced with doxycycline. Later the cells were transfected with GFP-MBP-control or GFP-siRNA-resistant (siRES)-Nup358 construct as indicated. Left: Expression levels of Nup358 in the indicated samples were monitored by western blotting with Nup358 antibodies. Vinculin was used as loading control. Middle: Levels of indicated proteins were assessed by western blotting. Vinculin was loading control. Right: Quantitative representation depicting relative levels of pAkt normalized to total Akt ( n = 4 independent experiments) under the indicated conditions. Data are mean ± SEM, Student’s t test. P values are indicated.

Article Snippet: Constructs harbouring inducible shRNA control (shControl) and shRNA against Nup358 (shNup358) were generated by cloning the following target sequences into the Tet-pLKO-puro (Addgene #21915). shControl (5′-GTGGACTCTTGAAAGTACTAT-3′) and shNup358 (5′-GGTGAAGATGGATGGAATA-3′) were cloned into the Tet-pLKO-puro vector (Addgene #21915) at the AgeI/EcoRI sites.

Techniques: shRNA, Control, Transfection, Construct, Expressing, Western Blot

Journal: EMBO Reports

Article Title: Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

doi: 10.1038/s44319-024-00204-8

Figure Lengend Snippet: ( A ) A schematics showing how F1 progeny was obtained by crossing parental (P) lines as indicated. The adult Drosophila flies (F1) were treated with 500 µM mifepristone (RU486) for 72 h to induce control shRNA (Luciferase, Luc) or dNup358-specific shRNA. ( B ) Brains lysates were assessed for dNup358 knockdown by western blotting. α-tubulin was used as loading control. ( C ) Left: Lysates were analysed for pAkt (S505) levels by western blotting. α-tubulin was used as loading control. Right: Quantitative data showing the levels of pAkt (normalized to total Akt) under indicated conditions ( n = 4 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P value is indicated.

Article Snippet: Constructs harbouring inducible shRNA control (shControl) and shRNA against Nup358 (shNup358) were generated by cloning the following target sequences into the Tet-pLKO-puro (Addgene #21915). shControl (5′-GTGGACTCTTGAAAGTACTAT-3′) and shNup358 (5′-GGTGAAGATGGATGGAATA-3′) were cloned into the Tet-pLKO-puro vector (Addgene #21915) at the AgeI/EcoRI sites.

Techniques: Control, shRNA, Luciferase, Knockdown, Western Blot

Journal: EMBO Reports

Article Title: Nup358 restricts ER-mitochondria connectivity by modulating mTORC2/Akt/GSK3β signalling

doi: 10.1038/s44319-024-00204-8

Figure Lengend Snippet: ( A ) Insulin-dependent increase in VAPB–PTPIP51 interaction is dependent on mTORC2. HeLa cells were transfected with control (siControl), Rictor (siRictor) or Sin1 (siSin1). Cells were later serum starved for 12 h and treated with (+) or without (−) 2 nM insulin for 20 min. Top: Cells were fixed and processed for PLA using VAPB and PTPIP51-specific antibodies (yellow dots). DNA was stained with Hoechst 33342 (blue). Scale bar, 10 µm. Bottom left: Depletion of Rictor and Sin1 was monitored by western blotting using specific antibodies GAPDH was used as loading control. Note that Rictor depletion led to co-depletion of Sin1 as reported earlier (Yang et al, ). Bottom right: Quantitation of PLA dots per cell are shown ( n = 90 cells for siControl, siRictor or siSin1 condition from three independent experiments). Data are mean ± SD, Student’s t test. P values are indicated. ( B ) Depletion of VAPB/PTPIP51 interferes with growth factor-stimulated activation of mTORC2/Akt signalling. HeLa cells were depleted of different proteins using specific siRNAs as mentioned. Left top: The extent of depletion was verified by western blotting using specific antibodies. Left bottom: The ability of PACS2 siRNA to deplete the human PACS2 was confirmed by co-transfecting HeLa cells with control (siControl) or PACS2 (siPACS2) siRNA along with HA-human PACS2 construct. The expression of HA-PACS2 was monitored by western blotting (WB) with a HA-specific antibody. Vinculin was used as a loading control. Right top: HeLa cells depleted of indicated proteins were serum starved for 3 h, and later treated with (+) or without (−) insulin (1 nM) for 20 min. The cells were then analysed for the specific proteins by western blotting. Right bottom: Quantitative analysis of the relative phosphorylation of Akt at S473 under the described conditions ( n = three independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. ( C ) Hyperactivation of mTORC2/Akt signalling upon Nup358 loss depends on the VAPB–PTPIP51 tethering complex. HeLa cells were depleted of indicated proteins and serum starved for 3 h. The cells were then treated with (+) or without (−) insulin (1 nM) for 20 min. Cells were analysed for insulin-induced mTORC2/Akt activation. Left: Western analysis to monitor the extent of protein depletion by siRNAs. Middle: Western blot showing the extent of phosphorylation of Akt at Ser473, along with total Akt levels, in the described conditions. Vinculin was used as a loading control. Right: Quantitative data depicting the change in a relative amount of pAkt (S473) under the indicated conditions ( n = 4 independent experiments). Data are mean ± SEM, unpaired Student’s t test. P values are indicated. .

Article Snippet: Constructs harbouring inducible shRNA control (shControl) and shRNA against Nup358 (shNup358) were generated by cloning the following target sequences into the Tet-pLKO-puro (Addgene #21915). shControl (5′-GTGGACTCTTGAAAGTACTAT-3′) and shNup358 (5′-GGTGAAGATGGATGGAATA-3′) were cloned into the Tet-pLKO-puro vector (Addgene #21915) at the AgeI/EcoRI sites.

Techniques: Transfection, Control, Staining, Western Blot, Quantitation Assay, Activation Assay, Construct, Expressing, Phospho-proteomics