Journal: Cell death & disease

Article Title: PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance.

doi: 10.1038/s41419-021-04036-5

Figure Lengend Snippet: Fig. 1 PIK3R3 and Nanog are upregulated in sarcoma CSCs and human sarcomas. A Heat map of expression of survival signaling-related genes in two sarcoma cell lines cultured as spheroids vs. monolayers as measured by RNA sequencing. Each sample was run in duplicate. Real- time RT-PCR (B) and (C) Western blot (C) for PIK3R3 and CD133 in three sarcoma cell lines grown as monolayers or as spheroids. D Proportion of CD133+ cells as measured by fluorescence-activated cell sorting (FACS) analysis in sarcoma cells grown as monolayers or as spheroids. E Western blot for PIK3R3, CD133, Nanog, Sox2, Oct-4 and c-Myc in CD133+ and CD133– populations of sarcoma cell lines cultured as spheroids. F Graph and immunostaining of commercially available tissue array slide containing 79 human sarcomas and 4 human normal tissues for CD133, Nanog, and PIK3R3. β-actin is the loading control. *p < 0.05 compared to control.

Article Snippet: Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol.

Techniques: Expressing, Cell Culture, RNA Sequencing, Quantitative RT-PCR, Western Blot, FACS, Immunostaining, Control

Journal: Cell death & disease

Article Title: PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance.

doi: 10.1038/s41419-021-04036-5

Figure Lengend Snippet: Fig. 2 Inhibition of PIK3R3 inhibits stemness of sarcoma CSCs. A–E Compare sh.PIK3R3- vs. sh.Scr-transduced sarcoma cells. All experiments except C were done in spheroid-cultured cells. Single-cell (A) and standard spheroid (>100 μm) formation (B), with quantitation of spheroid diameter at 7 days post-transduction. C Western blot for PIK3R3, CD133, Nanog, Sox2, Oct-4, and c-Myc. D Immunostaining for CD133 and Nanog (with DAPI counterstaining). E Western blot for PIK3R3, CD133 an Nanog in sorted CD133+ cells. F–G Compare WT PIK3R3 vs. carrier- transduced cells. F Western blot for PIK3R3, CD133 and Nanog. G Spheroid diameter. β-actin is the loading control. *p < 0.05 compared to control.

Article Snippet: Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol.

Techniques: Inhibition, Cell Culture, Quantitation Assay, Transduction, Western Blot, Immunostaining, Control

Journal: Cell death & disease

Article Title: PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance.

doi: 10.1038/s41419-021-04036-5

Figure Lengend Snippet: Fig. 3 PIK3R3 promotes migration and invasion in sarcoma CSCs. All panels compare sh.PIK3R3 vs. sh.Scr-transduced sarcoma cells. Invasion (A) and migration (B) assay for sarcoma spheroid cells. B Western blot and zymography for PIK3R3, N-cadherin, MMP-2, and MMP-9 in sarcoma spheroid cells. C Western blot and zymography for PIK3R3, N-cadherin, MMP-2, and MMP-9 in CD133+ cells. D Immunostaining for N-cadherin and Nanog, with DAPI counterstaining in CD133+ cells. E Tumor volume graph and representative photos of HT1080 CD133+ cells grown in athymic nude mice (n = 5 mice in each group).

Article Snippet: Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol.

Techniques: Migration, Western Blot, Zymography, Immunostaining

Journal: Cell death & disease

Article Title: PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance.

doi: 10.1038/s41419-021-04036-5

Figure Lengend Snippet: Fig. 4 PI3K activates the AKT/ERK signaling pathway in sarcoma CSCs. A–B Compare CD133+ vs. CD133–cells. A Quantitation of phosphorylation of indicated kinases in HT1080 cells as measured by human phospho-kinase array. B Western blot for CD133, total and phosphorylated AKT1/2, and total and phosphorylated ERK1/2. C–D Compare LY294002 (10 μM) vs. vehicle (DMSO)-treated CD133+ cells for 3 days. C Western blot and zymography for total and phosphorylated AKT1/2 and CD133, PIK3R3, Nanog, Sox2, Oct-4, N-cadherin, and MMP-2. D Spheroid formation. E Western blot and zymography for PIK3R3, total and phosphorylated ERK1/2, CD133, Nanog, N-cadherin, MMP-2, and MMP-2 in CD133+ spheroid cell lines transduced with sh.PIK3R3 or sh.Scr. β-actin is the loading control. *p < 0.05 compared to control.

Article Snippet: Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol.

Techniques: Quantitation Assay, Phospho-proteomics, Western Blot, Zymography, Transduction, Control

Journal: Cell death & disease

Article Title: PIK3R3, part of the regulatory domain of PI3K, is upregulated in sarcoma stem-like cells and promotes invasion, migration, and chemotherapy resistance.

doi: 10.1038/s41419-021-04036-5

Figure Lengend Snippet: Fig. 6 Eliminating PIK3R3 signaling regulates stemness, malignancy, and chemoresistance in mouse xenograft models. All experiments employ HT1080 fibrosarcoma cells. A Western blot confirming knockdown of PIK3R3. β-actin is the loading control. B Growth of xenografts in athymic nude mice following treatment with sh.Scr or sh.PIK3R3 and PBS or doxorubicin. C Photos of representative tumors. D Immunofluorescence of tumors for proliferation using Ki-67, apoptosis using cleaved caspase 3, Bcl-2, CD133, Nanog, and MMP-2, with quantitation at right. Scale bar, 50 µm. E A model of the function of PIK3R3 in sarcoma CSCs. *p < 0.01 compared to control.

Article Snippet: Silencing of PIK3R3, Nanog, and ERK1/2 and was achieved via lentiviral transduction of and human PIK3R3 shRNA (sc-39124-V; Santa Cruz), human Nanog shRNA (sc-43958-V; Santa Cruz), human ERK1 shRNA (sc-29307-V; Santa Cruz), human ERK2 shRNA (sc-35335-V; Santa Cruz) per the manufacturer’s protocol.

Techniques: Western Blot, Knockdown, Control, Quantitation Assay