Journal: Scientific Reports

Article Title: Kidney injury molecule-1 inhibits metastasis of renal cell carcinoma

doi: 10.1038/s41598-021-90919-8

Figure Lengend Snippet: Knockdown of human KIM-1 in 786-O cells and expression if murine KIM-1 in Renca RCC cells. ( A ) 786-O cell lines were transduced using lentivirus encoding either KIM-1 shRNA or a control shRNA plasmid. RT-qPCR of 786-O cell lines normalized to housekeeping gene (GAPDH), showing successful knockdown of KIM-1 mRNA in 786-O-shKIM-1, but not in 786-O-shControl (*p = 0.0248). ( B ) Western Blot analyses confirming KIM-1 protein knockdown in 786-O-shKIM-1, but not 786-O-shControl cell lines. ( C ) Renca cell lines were transduced using lentivirus encoding either KIM-1 expressing (KIM-1 pos ) or non-expressing (KIM-1 neg ) vectors. RT-qPCR of Renca cell lines normalized to housekeeping gene (GAPDH), showing successful upregulation of KIM-1 in Renca KIM-1 pos , but not Renca KIM-1 neg cell lines (**p < 0.001). ( D ) Western blot analyses confirming strong cellular KIM-1 protein expression in Renca KIM-1 pos , but not in Renca KIM-1 neg cell lines. The cropped Western blots images in ( A ) and ( B ) were obtained from the same gel but the resulting membranes were cut and probed with either anti-KIM-1 or anti-GAPDH antibodies for ( A ). The exposures was optimized by the digital imaging system.

Article Snippet: Control transduction was done using scrambled shRNA lentiviral particles (786-O-shControl) (sc-108080; Santa Cruz Biotechnology).

Techniques: Knockdown, Expressing, shRNA, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Imaging

Journal: The Journal of Biological Chemistry

Article Title: Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

doi: 10.1016/j.jbc.2022.101675

Figure Lengend Snippet: High-content imaging analysis of glucosomes from Hs578T cells. A , the percentages (%) of EGF-treated Hs578T cells displaying each size of PFKL-mEGFP assemblies were quantified from at least three independent imaging sessions in the presence of SCH772984 ( gray ), shERK1 ( yellow ), shERK2 ( blue ), or shControl Scrambled ( green ). Note that the previously reported distributions of glucosomes in various sizes in the absence and presence of 30 ng/ml EGF ( purple and red , respectively) are also graphed together for direct comparison. B , a table shows the average percentages (%) of Hs578T cells displaying the given sized glucosomes at each condition along with their SDs (±). Statistical analyses were performed using Tukey’s multiple comparison tests for two-way ANOVA analysis. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.0001. N.S.; not significant. EGF, epidermal growth factor; mEGFP, monomeric enhanced GFP; PFKL, liver-type phosphofructokinase 1.

Article Snippet: Hs578T cells were transfected with shRNAs targeting ERK1/2 (shERK1 and shERK2) and a control shRNA with a scrambled sequence (shControl Scrambled , CGCGAAGTCTGTACTCTTG, Addgene, Cat# 65232) ( ) using Lipofectamine 2000.

Techniques: Imaging

Journal: The Journal of Biological Chemistry

Article Title: Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

doi: 10.1016/j.jbc.2022.101675

Figure Lengend Snippet: Western blot analysis of ERK1/2 knockdown. Epidermal growth factor-treated Hs578T cells were transfected with shERK1, shERK2, or shControl Scrambled and subsequently selected in the presence of puromycin (1 μg/ml). A , Western blot analysis showed the knockdown of total ERK1/2, but no change was detected for pERK1/2 in the presence of shERK1/2. B and C , expression levels of total ERK1/2 and phosphorylated ERK1/2 (pERK1/2) were normalized based on load controls (β-actin), respectively. The error bars represent the SDs of at least three independent experiments. Statistical analyses were performed using two-sample two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01. N.S.; not significant. ERK, extracellular signal-regulated kinase.

Article Snippet: Hs578T cells were transfected with shRNAs targeting ERK1/2 (shERK1 and shERK2) and a control shRNA with a scrambled sequence (shControl Scrambled , CGCGAAGTCTGTACTCTTG, Addgene, Cat# 65232) ( ) using Lipofectamine 2000.

Techniques: Western Blot, Transfection, Expressing, Two Tailed Test

Journal: Molecular cell

Article Title: Glutaminolysis activates Rag-mTORC1 signaling.

doi: 10.1016/j.molcel.2012.05.043

Figure Lengend Snippet: Figure 3. Inhibition of Glutaminolysis Inhibits the Translocation of mTOR to the Lysosome (A) Lysosomal localization of mTORC1 in leucine and glutamine (LQ)- stimulated U2OS cells transfected with either nontargeting siRNA (siCtrl) or with siRNA against GLS or GDH.

Article Snippet: Plasmid expressing FLAG-Rheb-N153T (RhebGTP) (Addgene plasmid 19997), plasmid containing nontargeting shRNA (shCtrl, Addgene plasmid 1864), pRK5-HA GST RagB WT plasmid expressing wild-type RagB (Addgene plasmid 19301), and pRK5-HA GST RagB 99L plasmid expressing GTP-bound mutant of RagB (Addgene plasmid 19303) were obtained from Addgene.

Techniques: Inhibition, Translocation Assay, Transfection

Journal: Molecular cell

Article Title: Glutaminolysis activates Rag-mTORC1 signaling.

doi: 10.1016/j.molcel.2012.05.043

Figure Lengend Snippet: Figure 5. Glutaminolysis Increases GTP Loading of RagB (A and B) U2OS cells were cotransfected with a GST-RagB-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. After GST-RagB pull-down, radiolabeled GTP and GDP were analyzed by TLC (A) and quantified using ImageJ software (B). (C) HeLa cells were cotransfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. Phosphorylation of S6K was measured by immunoblotting. (D) HeLa cells were transfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP. Forty-eight hours later, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ) either in the presence or absence of DON, as indicated. Phosphorylation of S6K was then measured by immunoblotting. The mean is shown; error bars represent SEM (n > 3, *p < 0.05).

Article Snippet: Plasmid expressing FLAG-Rheb-N153T (RhebGTP) (Addgene plasmid 19997), plasmid containing nontargeting shRNA (shCtrl, Addgene plasmid 1864), pRK5-HA GST RagB WT plasmid expressing wild-type RagB (Addgene plasmid 19301), and pRK5-HA GST RagB 99L plasmid expressing GTP-bound mutant of RagB (Addgene plasmid 19303) were obtained from Addgene.

Techniques: Expressing, Plasmid Preparation, Transfection, Software, Phospho-proteomics, Western Blot

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