Journal: bioRxiv

Article Title: Driving proteomic imbalance to combat Neurofibromatosis type I (NF1)-associated malignancy

doi: 10.1101/2024.05.24.595838

Figure Lengend Snippet: (A) Immunoblotting detection of JNK activation, mTORC1 activity, and protein polyubiquitination in S462 cells following stable TSC2 knockdown and/or HSF1 inhibition by 10 μM DTHIB for 3 days. (B) Quantitation of soluble AOs by flow cytometry using A11 antibody staining in S462 cells with and without TSC2 knockdown and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). (C) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown and DTHIB (10 μM) or combined DTHIB and LY2584702 (20 μM) treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). Cells were pre-treated with LY2584702 for 1 day followed by DTHIB treatment for another 3 days. (D) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown and DTHIB (10 μM) or combined DTHIB and CR (30 μM) treatment (mean ± SD, n=3 independent experiments, Two-way ANOVA). Cells were pre-treated with CR for 1 day followed by DTHIB treatment for another 3 days. (E) Immunoblotting detection of mTORC1 stimulation by 500 μM NV-5138 in S462 cells with and without 10 μM DTHIB treatment. (F) Quantitation of soluble AOs by flow cytometry using A11 antibody staining in S462 cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, One-way ANOVA). (G) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB or combined DTHIB and CR treatment (mean ± SD, n=5 independent experiments, One-way ANOVA). (H) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in immortalized human Schwann cells with and without 500 μM NV-5138 stimulation and 10 μM DTHIB treatment (mean ± SD, n=3 independent experiments, One-way ANOVA). (I) Immunoblotting detection of mTORC1 stimulation by 500 μM NV-5138 in immortalized human Schwann cells with and without 10 μM DTHIB treatment.

Article Snippet: The pLKO.1 shRNA plasmid targeting human TSC2 was a gift from Do-Hyung Kim (Cat#15478, Addgene).

Techniques: Western Blot, Activation Assay, Activity Assay, Knockdown, Inhibition, Quantitation Assay, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Driving proteomic imbalance to combat Neurofibromatosis type I (NF1)-associated malignancy

doi: 10.1101/2024.05.24.595838

Figure Lengend Snippet: (A) and (B) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains combined with cleaved caspase 3 (Asp175) antibody staining. S462 cells with and without stable TSC2 knockdown were treated with DMSO, 10 μM DTHIB, or combined DTHIB and 30 μM Q-VD-OPh (mean ± SD, n=3 independent experiments, Two-way ANOVA). (C) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown. Cells were treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 30 μM Necrostatin-1 or 20 μM Liproxstatin-1 for 3 days (mean ± SD, n=3 independent experiments, Two-way ANOVA). (D) Immunoblotting detection of ferroptosis markers in S462 cells with and without stable TSC2 knockdown treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 20 μM liproxstatin-1 for 3 days. Erastin was included as a positive control to induce canonical ferroptosis. (E) Immunoblotting detection of autophagy markers in S462 cells with stable TSC2 knockdown treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 3 μM wortmannin for 3 days. Rapamycin was included as a positive control to induce autophagy. (F) Quantitation of cytotoxicity by flow cytometry using Live-or-Dye stains in S462 cells with and without stable TSC2 knockdown. Cells were treated with 10 μM DTHIB alone or co-treated with 10 μM DTHIB and 3 μM wortmannin for 3 days (mean ± SD, n=3 independent experiments, Two-way ANOVA). (G) Schematic depiction of instigation of cell death by severe proteomic imbalance, owing to simultaneous mTORC1 stimulation ( ) and HSF1 inhibition ( ). In cancer cells, constitutive HSF1 activation provides extra chaperoning capacity to cope with elevated protein misfolding, partly due to enhanced protein synthesis and widespread genetic mutations. Nevertheless, amyloids still emerge, although at low levels. Importantly, HSF1 can neutralize highly toxic amyloid oligomers, averting lethal consequences. By contrast, HSF1 inhibition diminishes chaperoning capacity, insufficient to counterbalance the robust protein translation. mTORC1 stimulation further aggravates this proteomic imbalance, which, in turn, strongly promotes amyloidogenesis. In consequence, the amounts of amyloid oligomers exceed the neutralizing capacity of HSF1, leading to cell death; nonetheless, it remains unclear how this non-apoptotic, non-autophagic death occurs. (H) Schematic depiction of the concept of driving proteomic imbalance to combat malignancy. On the one hand, in cancer cells, mTORC1 is inevitably activated to stimulate protein translation, markedly augmenting protein quantity. On the other hand, the extra chaperoning capacity governed by HSF1, albeit dispensable for normal life, becomes necessary to ensure sufficient protein quality in cancer cells, thereby counterbalancing augmented protein quantity and suppressing proteomic instability. Thus, proteomic balance promotes malignant growth. By contrast, disrupting proteomic balance, through HSF1 inhibition, is sufficient to provoke proteomic instability and elicit tumor suppression. However, simultaneous mTORC1 stimulation can remarkably drive proteomic imbalance, causing severe proteomic instability and profound tumor suppression.

Article Snippet: The pLKO.1 shRNA plasmid targeting human TSC2 was a gift from Do-Hyung Kim (Cat#15478, Addgene).

Techniques: Quantitation Assay, Flow Cytometry, Staining, Knockdown, Western Blot, Positive Control, Inhibition, Activation Assay

Journal: STAR Protocols

Article Title: Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry

doi: 10.1016/j.xpro.2024.103082

Figure Lengend Snippet: Downstream analyses of transduction efficiency and gene knockdown effects (A) Schematic of lentiviral transfection of mammalian cell. (B) Representative flow cytometry scatterplot (left, 4 quadrants), GFP expression in non-target shRNA transfection: Q1-UL = dead cells not expressing GFP; Q1-UR = dead cells expressing GFP; Q1-LL = live cells, not expressing GFP; Q1-LR = live cells, expressing GFP. On right, a histogram depicting FITC intensity along X-axis and number of events at that intensity on the Y-axis. (C) Representative western blot images for control (non-target shRNA transfection) and TSC2 knockdown (20 MOI) in preterm human enteroids. S6, pS6, extracellular signal-regulated kinase 1/2 (ERK-1/2), and p-ERK- 1/2 are shown normalized to β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), respectively. Normalized phosphorylation S6 and ERK-1/2 is quantified in bar graphs. Enteroid TSC2 knockdown indicates an increase in phosphorylation of the ribosomal S6 protein, but no change was observed in the phosphorylation of ERK-1/2, an upstream target of mTOR.

Article Snippet: • MISSION shRNA lentiviral transduction particles ( TSC2 ; TRCN0000010454; Millipore Sigma, SHCLNV).

Techniques: Transduction, Transfection, Flow Cytometry, Expressing, shRNA, Western Blot

Journal: STAR Protocols

Article Title: Analyzing efficiency of a lentiviral shRNA knockdown system in human enteroids using western blot and flow cytometry

doi: 10.1016/j.xpro.2024.103082

Figure Lengend Snippet:

Article Snippet: • MISSION shRNA lentiviral transduction particles ( TSC2 ; TRCN0000010454; Millipore Sigma, SHCLNV).

Techniques: Recombinant, Membrane, Staining, Protease Inhibitor, Lysis, Extraction, Bicinchoninic Acid Protein Assay, Transduction, Positive Control, shRNA, Sequencing, Software, Flow Cytometry, Sterility, Transferring, Adhesive, Aerosol, Cell Counting, Electrophoresis, Microscopy, Purification

Journal: eLife

Article Title: mTOR signaling regulates the morphology and migration of outer radial glia in developing human cortex

doi: 10.7554/elife.58737

Figure Lengend Snippet: Figure 3. Manipulation of mTOR signaling results in migration defects. (A) shRNAs were delivered along and electroporated onto the ventricular surface of primary cortical tissue which was then acutely sectioned and cultured for six days prior to collection. (B) After electroporation of RPTOR or TSC2 shRNAs, GFP+ HOPX+ oRGs migrate less from the ventricular edge (n = 37 control, n = 33 Raptor and n = 27 TSC2-shRNA electroporated GFP +Hopx+ cells from three independent experiments; D’Agostino Pearson Normality Test: normally distributed; one-way ANOVA with multiple comparisons: Raptor shRNA: ****p<0.0001, TSC2 shRNA: ****p<0.0001, error bars represent SD). The distance of each HOPX+ GFP+ cell away from the VZ edge was measured as indicated by white brackets. (C) For dynamic imaging studies, primary cortical tissue was collected, dissociated, infected with a CMV::GFP adenovirus, and plated on glass-bottom 12 well plates. Small molecules were added one day later, two hours before the start of dynamic imaging. (D) GFP+ oRG cells undergo division via MST. Yellow arrowheads indicate cell body, white dot indicates initial position of cell body and pink arrowheads indicate two cell bodies after division. After inhibition of mTOR signaling oRG cells have shorter MSTs (n = 10 control and n = 8 rapamycin cells across two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0082, error bars represent SD). (E) oRGs migrate less from their original position after mTOR inhibition (n = 9 vehicle treated and n = 12 rapamycin treated cells from two independent experiments; D’Agostino Pearson Normality Test: normally distributed; unpaired two-tailed student’s t-tests: **p<0.0058 error bars represent SD). White, yellow, and pink arrowheads indicate cell bodies at starting time-point. Multiple arrowheads of the same color over time indicate daughter cells from the same parent cell. Figure 3 continued on next page

Article Snippet: Electroporation Organoids RPTOR shRNA, TSC2 shRNA (Addgene) and CAG-GFP plasmids were electroporated at 1 mg/ul using a BTX electroporator set at 5V for 5 milliseconds for three pulses.

Techniques: Migration, Cell Culture, Electroporation, Control, shRNA, Imaging, Infection, Inhibition, Two Tailed Test