high throughput small interfering rna rnai screening assay  (Revvity)

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: Localization of GPR123 in hESCs and its role in colony morphology. ( A ): Immunofluorescence observation of the GPR122 expression in hESCs. Scale bar 100 μm. ( B ): TEM observation of the GPR123 localization in hESCs. Immunogold labeling (arrows) of the nucleolus ( B ) and nuclear membrane ( B’ ) with specific antibody against GPR123 (10 nm gold particles). Abbreviation: n stands for nucleus, nl—nucleolus, and nm—nuclear membrane. Inserts at the bottoms ( B , B’ ) represent GPR123 immunogold labeling at a higher resolution. Scale bar 1 μm. ( C ): Representative Western blot analyses of the GPR123 expression in hESCs (H9) and in embryonic bodies (EB) at day 1 (D1), day 10 (D10), and at day 14 (D14) EBs differentiation. n = 3. ( D ): Real-time quantitative polymerase chain reaction analysis of the relative expression of GPR123 versus GAPDH under GPR123 RNAi in hESCs. Data are shown as mean ± SEM, n = 3, with significance difference indicated with asterisks (***, p < 0.001). ( E ): Representative Western blot analysis of GPR123 in hESCs transfected with control and GPR123 RNAi, n = 3. GAPDH serve as a loading control. ( F ): Representative images depicting typical colony morphology at phase-contrast observation for the control and GPR123 RNAi–treated colonies at day 1, day 2, day 3, and day 4 of transfection. Scale bar 400 μm. ( G ): Representative images of the alkaline-phosphatase staining of control and GPR123 RNAi hESCs (H9). Scale bar 100 μm. Abbreviations: DAPI—4′,6-diamidino-2-phenylindole; hESC—human embryonic stem cell; GAPDH—glyceraldehyde-3-phosphate dehydrogenase; and RNAi—RNA interference.

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Immunofluorescence, Expressing, Labeling, Membrane, Western Blot, Real-time Polymerase Chain Reaction, Transfection, Control, Staining

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: Downregulation of GPR123 abrogates human-induced pluripotent stem cells generation. ( A ): Phase-contrast observation of the typical hiPSC colonies at day 12 of the reprogramming process under the control RNAi ( A ) and GPR123 RNAi ( B ). Arrows poined to hiPSCs colonies. Scale bar 100 μm. ( C ): Representative image of the typical colonies at day 18 of the reprogramming process under the control RNAi and GPR123 RNAi. Arrows poined to hiPSCs colonies. Scale bar 100 μm. ( D ): Representative images of the alkaline-phosphatase (AP) staining of the control and GPR123 RNAi hiPSCs at day 18 of reprogramming. ( E , E’ , F , F’ ): Flow cytometry analysis of different subpopulations during the time course (at day 12 and day 18) of reprogramming in the control and GPR123 RNAi-treated groups. ( G ): Real-time quantitative polymerase chain reaction analysis of the relative expression of GPR123 in TRA-1-60+/CD44- populations (true iPSCs) and in partly reprogrammed cells (TRA-1-60+/CD44+) at day 14 in the control and GPR123 RNAi groups. Data are shown as mean ± SEM, n = 3. (H): Real-time quantitative polymerase chain reaction analysis of the GPR123 expression normalized to expression at the neo1 fibroblasts during the time-course of transduction. Data are shown as mean ± SEM, n = 3, with the significance difference indicated with asterisks (***, p < 0.001). Abbreviations: AP—alkaline phosphatase.

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Control, Staining, Flow Cytometry, Real-time Polymerase Chain Reaction, Expressing, Transduction

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: Downregulation of GPR123 results in loss of pluripotency and increased expression of differentiation marker genes in hPSCs. ( A ): Real-time quantitative PCR analysis of OCT4 , NANOG , SOX2 , KLF4 , and c-MYC expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean ± SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.01 as **, p < 0.001 as ***). ( B ): Representative Western blot analyses of the pluripotent markers OCT4 and NANOG expression in hESCs (H9) treated with the control and GPR123 RNAi. ( C ): Real-time quantitative PCR analysis of the differentiation markers expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean ± SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.01 as **, p < 0.001 as ***). ( D ): Representative Western blot analyses of the differentiation markers NESTIN and VIMENTIN expression in hESCs (H9) treated with the control and GPR123 RNAi. ( E ): Real-time quantitative PCR analysis of MET genes expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean ±SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.05 as *, p < 0.001 as ***). ( F ): Representative Western blot analyses of the E-cadherin and N-cadherin expression in hESCs (H9) treated with the control and GPR123 RNAi. ( G , G’ ): Confocal immunofluorescence observation of the NANOG and E-cadherin expression in the control and GPR123 RNAi hESCs (H9). Scale bar 50 μm. ( H ): Representative confocal immunofluorescence images of E-cadherin expression in the control and GPR123 RNAi hiPSCs at day 12 of the reprogramming. Scale bar 50 μm.

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Expressing, Marker, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunofluorescence

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: Downregulation of GPR123 leads to accumulation of cells at the G2 phase of the cell cycle. ( A ): MODFIT analysis of the cell cycle of hESCs (H9) treated with the control and GPR123 RNAi. ( B ): Real-time quantitative PCR analysis of the cell cycle genes expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean–SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.05 as *). ( C ): Representative Western blot analyses of the CYCLIN D1, CYCLIN E, CYCLIN B1, and CYCLINA1 in control and GPR123 RNAi hESCs (H9). ( D ): Real-time quantitative PCR analysis of the CDC25A , CDC25B, and CDC25C genes expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean –SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.05 as *). ( E , F ): Flow cytometric analysis of apoptosis in hESCs (H9). ( E , F ): The hiPSCs under treatment with Control and GPR123 RNAi. ( G ): Real-time quantitative PCR analysis of the apoptosis genes expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean –SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.01 as **, p < 0.001 as ***).

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Control, Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: Expression and co-localization of Gαi and GPR123 in hESCs. ( A ): Real-time quantitative PCR analysis of the AC5 , PKA, CREB , STAT3 , RhoA , MAPK14 , and SMAD3 genes expression in hESCs (H9) control and GPR123 RNAi groups. Data represent relative expression to GAPDH and were normalized against the control RNAi. Results are presented as mean –SEM ( n = 3), with significance difference indicated with asterisks ( p < 0.01 as **, p < 0.001 as ***). ( B ): Representative Western blot analyses of the p-ERK (Thr202/Tyr204) and p-CREB (Ser133) in the control and GPR123 RNAi hESCs (H9). ( C ): Real-time quantitative PCR analysis of the Gα subunits expression in GPR123 RNAi hESCs (H9). Data represent relative expression to RPL13A and were normalized against expression of the Gα subunits in the control RNAi hESC line (H9). Results are presented as mean ± SEM ( n = 3), statistical significance was analyzed using Student’s t -test, p < 0.05 was considered significant and is denoted as *, p < 0.01 as **, p < 0.001 as ***. ( D ): Representative images of the Confocal immunofluorescence observation of the co-localization between Gαi with GPR123 in hESCs (H9) treated with the control RNAi (upper panel) and with GPR123 RNAi (bottom panel). Scale bar 50 μm. ( E ): TEM observation of the Gαi and GPR123 localization in hESCs. Immunogold labeling in the nucleus ( n ), nuclear membrane (nm), and in the nucleolus (nL) with specific antibodies against the Gαi (15 nm gold particles, black thick arrows) and GPR123 (10 nm gold particles, black thin arrows). An inset in the lower left corner depicts the area with both labels. Scale bar 1 μm.

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Western Blot, Immunofluorescence, Labeling, Membrane

Journal: Cells

Article Title: Essential Role of Adhesion GPCR, GPR123, for Human Pluripotent Stem Cells and Reprogramming towards Pluripotency

doi: 10.3390/cells12020304

Figure Lengend Snippet: GPR123 is important for wound healing of hiPSCs and ACTIN reorganization during the reprogramming process. ( A ): Graphical representation of the wound healing of hiPSCs treated with the control and GPR123 RNAi. Human iPSCs were subjected to scratch wounding from 0 h until 24 h ( n = 5). ( A’ ): Representative images of the time-lapse phase-contrast observation of the wound healing at time 0 and 24hrs. Scale bar 200 µm. ( B ): Representative images of the confocal immunofluorescent staining with Rhodamine phalloidine in the control and GPR123 RNAi hiPSCs. Scale bar 50 μm. ( C ): Representative Western blot analyses of β-ACTIN and p-FAK(Tyr 397) in hiPSCs treated with the control and GPR123 RNAi.

Article Snippet: In agreement with the data obtained by us previously by high-throughput small interfering RNA (RNAi) screening assay, which allowed specific knockdown of the 784 members of the different kinases and phosphatases from the Dharmacon library during the initiation phase of reprogramming [ ], we observed significant downregulation in the number of the hiPSCs colonies from day 12 to day 18 of the reprogramming period ( A–C) with complete absence of the AF+ colonies at day 18 in the GPR123 RNAi group ( D).

Techniques: Control, Staining, Western Blot

Journal: bioRxiv

Article Title: The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

doi: 10.1101/2022.02.10.479964

Figure Lengend Snippet: (A) Immunofluorescence staining of dynein intermediate chain (DIC, green), and Ndc80 (red), in early mitotic prophase (top panels) or prometaphase (bottom panel), with the chromosomes counterstained using DAPI. Bars, 5 μm. (B) Western blot analysis of HeLa cells treated with siRNAs for control and Ndc80 (Bi), of Hela cells of parental and Nuf2 CRIPR/Cas9 KO (Bii), and of RPE1 cells treated with siRNAs for control and Nuf2 (Biii). α-tubulin was used as a loading control. (C) Cells were subjected to the above-mentioned perturbations, then treated with the indicated drugs/chemicals and fixed according to the scheme. (D and F) Immunofluorescence staining of STLC treated mitotic HeLa cells depleted of Ndc80 (D) or knocked out of Nuf2 (F) in comparison to respective control RNAi or parental control cells, and stained for α-tubulin (green), a kinetochore marker Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs in the conditions indicated. (E and G) Quantification of the status of kMT attachments in cells from D and F. Error bars represent S.D. from three independent experiments. For each experiment, on average ~ 20 kinetochore pairs from 10 different monopolar cells were examined. ****P < 0.0001 (Student’s t test). (H and I) Trajectories of two representative sister kinetochore pairs in HeLa cells transfected with siRNAs for control and Ndc80. 50 kinetochore pairs were analyzed for each condition. Also see Supplemental Videos 1 and 2. (J) HeLa cells treated with control RNAi or Ndc80 RNAi were fixed 0 or 10 min after nocodazole washout. The cells were then immunostained for α-tubulin (green), a kinetochore marker Zwint1 (red), with chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs at these two time points under either condition. Also see . (K) Quantification of the status of kMT attachments in cells from J. Error bars represent S.D. from three independent experiments. For each experiment, 10 mitotic cells were examined. ****P < 0.0001 (Student’s t test).

Article Snippet: For RNA interference (RNAi) experiments, cells were transfected at 30-50% confluence using Dharmafect 2 (Dharmacon) according to the manufacturer’s instructions and analyzed 48-72 h after transfection.

Techniques: Immunofluorescence, Staining, Western Blot, Control, Comparison, Marker, Transfection

Journal: bioRxiv

Article Title: The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

doi: 10.1101/2022.02.10.479964

Figure Lengend Snippet: (A) Cells were subjected to the indicated perturbations, treated with the indicated drugs/chemicals and fixed according to the scheme. (B) Immunofluorescence staining of early prometaphase HeLa cells depleted of Ndc80 (top two panels) or knocked out of Nuf2 (bottom two panels) in comparison to control RNAi or parental cells respectively and stained for α-tubulin (green) and a kinetochore marker, Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs in the indicated conditions. (C and D) Quantification of the status of kMT attachments in cells from B. Error bars represent S.D. from three independent experiments. For each experiment, on average ~20 kinetochore pairs from 10 early prometaphase cells were examined. ****P < 0.0001 (Student’s t test). (E) The distance measured between kinetochore and nearest microtubule in cells from B. Error bars represent S.D. from three independent experiments. For each experiment, on average 20 kinetochore pairs from 5 early prometaphase cells were examined. ****P < 0.0001 (Student’s t test). (F) Immunofluorescence staining of early prometaphase RPE1 cells depleted of Nuf2 in comparison to control RNAi cells (top panel) and stained for α-tubulin (green), a kinetochore marker Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. Inset shows the kMT attachment status of individual kinetochore pairs. (G) Quantification of the status of kMT attachments in cells from F. Error bars represent S.D. from three independent experiments. For each experiment, on average ~20 kinetochore pairs from 10 early prometaphase cells were examined. ****P < 0.0001 (Student’s t test). (H) Selected frames from live imaging of double-thymidine synchronized HeLa cells stably expressing mCherry-Histone H2B to visualize the chromosomes, in addition to GFP-α-tubulin and GFP-CENPA to visualize kMT attachment and treated with control RNAi (top two panels) or Ndc80 RNAi cells (bottom two panels). Images were captured every 1 min interval for 15-20 min until chromosome is aligned in control RNAi . Bars, 5 μm. Yellow arrow heads indicate the kinetochores unattached to microtubule in Ndc80 RNAi cells. The images from the movies were cropped and image intensity adjusted as required for better visualization of kinetochores and microtubules. (I) Selected frames from live imaging of parental (left panel) and Nuf2 KO (right panel) HeLa cells treated with Hoechst for 30 min prior to imaging to visualize the chromosomes. Images were captured every 2 min interval for around 30 min until all the chromosomes were aligned in parental cells. Bars, 5 μm.

Article Snippet: For RNA interference (RNAi) experiments, cells were transfected at 30-50% confluence using Dharmafect 2 (Dharmacon) according to the manufacturer’s instructions and analyzed 48-72 h after transfection.

Techniques: Immunofluorescence, Staining, Comparison, Control, Marker, Imaging, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

doi: 10.1101/2022.02.10.479964

Figure Lengend Snippet: (A) Cells were subjected to the indicated perturbations, treated with the indicated drugs/chemicals, and fixed according to the scheme. (B and C) Mitotic HeLa cells depleted of Ndc80 (B, panel) or knocked out of Nuf2 (C bottom panel) as compared to control RNAi (B, top panel) or parental control cells (B, bottom panel), and followed by the indicated drug treatments, were immunostained for α-tubulin (green), a kinetochore marker Zwint1 or CENPA (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. (D) Quantification of the frequency of mitotic cells with null kinetochores (see main text for more details) in samples from B and D. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ****P < 0.0001 (Student’s t test). (E) Immunofluorescence staining of mitotic RPE1 cells depleted of Nuf2 in comparison to control cells (top panel) and stained for α-tubulin (green), a kinetochore marker Zwint1 (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. (F) Quantification of the status of kMT attachments in cells from E. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ****P < 0.0001 (Student’s t test). (G) Selected frames from live imaging of HeLa cells stably expressing mCherry-Histone H2B to visualize the chromosomes in addition to GFP-α-tubulin and GFP-CENPA to visualize kMT attachment in control RNAi (top two panels) and Ndc80 RNAi cells (bottom two panels). Images were captured at every 1 min interval starting from the point of nuclear envelope breakdown for around 30 min until a proper mitotic spindle was formed during prometaphase in control RNAi cells. Bars, 5 μm. (H) Quantification of mitotic cells with null kinetochores from H. Error bars represent S.D. from three independent experiments. For each experiment, 50 mitotic cells were examined. (I) Immunofluorescence staining of MG132 + ZM treated mitotic HeLa cells depleted of endogenous Ndc80 and transfected with different Hec1-GFP constructs as indicated and stained for GFP (green), α-tubulin (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. (J) Quantification of mitotic cells with null kinetochores from A. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ****P < 0.0001 (Student’s t test).

Article Snippet: For RNA interference (RNAi) experiments, cells were transfected at 30-50% confluence using Dharmafect 2 (Dharmacon) according to the manufacturer’s instructions and analyzed 48-72 h after transfection.

Techniques: Control, Marker, Immunofluorescence, Staining, Comparison, Imaging, Stable Transfection, Expressing, Transfection, Construct

Journal: bioRxiv

Article Title: The Ndc80 complex is essential for the initial kinetochore-microtubule capture during early mitosis

doi: 10.1101/2022.02.10.479964

Figure Lengend Snippet: (A and B) Mitotic HeLa cells depleted of Ndc80 (A, bottom panel) or knocked out of Nuf2 (B, bottom panel) as compared to control RNAi (A, top panel) or parental control cells (B, bottom panel), and followed by the indicated drug treatments, were immunostained for α-tubulin (green), a kinetochore marker Zwint1 or CENPA (red) with the chromosomes counterstained using DAPI. Bars, 5 μm. (C) Quantification of the frequency of mitotic cells with null kinetochores (see main text for more details) in samples from A and B. Error bars represent S.D. from three independent experiments. For each experiment, 200 mitotic cells were examined. ****P < 0.0001 (Student’s t test). (D) Analyses of dynein localization to kinetochores in Ndc80-depleted cells with or without Aurora B inhibition. Prometaphase HeLa cells were either treated with Ndc80 siRNA alone (middle panel) or in combination with ZM (bottom panel) as compared to untreated controls (top panel), followed by fixation and immunostaining for the dynein intermediate chain (DIC, green), a kinetochore marker (CREST, red) with the chromosomes counterstained using DAPI. Bars, 5 μm. (E) Quantification of data in D. A total of 50 kinetochores were analyzed from at least 5 different prometaphase cells.

Article Snippet: For RNA interference (RNAi) experiments, cells were transfected at 30-50% confluence using Dharmafect 2 (Dharmacon) according to the manufacturer’s instructions and analyzed 48-72 h after transfection.

Techniques: Control, Marker, Inhibition, Immunostaining

Journal: The Journal of Biological Chemistry

Article Title: PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression

doi: 10.1074/jbc.RA120.013716

Figure Lengend Snippet: Akt3 depletion results in reduced mitochondrial function. A , co-transfection of a mitochondrial-directed GFP plus an RNAi directed against Akt3 ( Akt3i ) or scrambled control were assessed for mitochondrial fragmentation by immunofluorescence. 40× images are shown. Scale bar indicates 400 μ m . B , relative risk analysis fragmentation induced by Akt3 depletion using either RNAi or lentiviral transduction of an shRNA as compared with control. Inset shows an immunoblot of Akt3 knockdown under either RNAi or shRNA transduction. C , fluorescence microscopy images of ECs stained with MitoTracker Deep Red taken at equivalent exposure times. DAPI used as a nuclear stain. 40× images are shown. Scale bar indicates 400 μ m . D , quantification of background-corrected MitoTracker Deep Red staining, shown as -fold change relative to SCR control. E , quantitation of maximal FCCP-uncoupled respiration of HUVECs following transfection with RNAi directed against Akt3 ( Akt3i ) or scramble ( SCR ) control using an Xf96 extracellular flux analyzer. **, p < 0.05 relative to respective control. Error bars indicate S.E.

Article Snippet: RNAi was purchased from Dharmacon (Akt3) or Santa Cruz Biotechnology (Akt3 and PGC-1α).

Techniques: Cotransfection, Control, Immunofluorescence, Transduction, shRNA, Western Blot, Knockdown, Fluorescence, Microscopy, Staining, Quantitation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression

doi: 10.1074/jbc.RA120.013716

Figure Lengend Snippet: Sildenafil rescues mitochondrial function and angiogenesis inhibited by Akt3 depletion. A , quantitation of maximal FCCP-uncoupled respiration of ECs following transfection with RNAi directed against Akt3 ( Akt3i ) or scramble ( SCR ) plus or minus the addition of sildenafil (100 μ m ) for 48 h using an Xf96 extracellular flux analyzer. *, p < 0.02; **, p < 0.05 relative to respective control. B , real-time PCR analysis of the relative expression of HSP60 and HSP10 expression following transfection of SCR or Akt3 RNAi and sildenafil plus or minus Akt3i transfection. Expression is relative to S26 as an internal control. *, p < 0.05; **, p < 0.03. C , HUVEC transfected with either scrambled control ( SCR ) or RNAi directed against Akt3 ( Akt3i ) treated with or without sildenafil (100 μ m ) prior to plating on Matrigel. D , quantitation of branch points in the angiogenesis assays described in ( C ) p values < 0.5. All error bars in indicate S.E. 10× magnifications are shown. Scale bars indicate 100 μ m .

Article Snippet: RNAi was purchased from Dharmacon (Akt3) or Santa Cruz Biotechnology (Akt3 and PGC-1α).

Techniques: Quantitation Assay, Transfection, Control, Real-time Polymerase Chain Reaction, Expressing

Journal: The Journal of Biological Chemistry

Article Title: PDE5 inhibition rescues mitochondrial dysfunction and angiogenic responses induced by Akt3 inhibition by promotion of PRC expression

doi: 10.1074/jbc.RA120.013716

Figure Lengend Snippet: Sildenafil induces the expression of PRC expression independently of Akt3 depletion. A and B , real-time PCR of total RNA isolated from ECs treated with or without sildenafil (100 μ m ) using primers against PGC-1α ( A ) or PGC-1β ( B ). C , cells treated as in ( A ) with or without transfection using RNAi directed against Akt3 using primers against PRC in real-time PCR. D , ECs treated with paraquat (100 n m ) and total RNA used in real-time PCR with primers directed against PRC. All PCR is expressed as relative to S26 as an internal control. p < 0.05 for all real-time PCR. Error bars indicate S.E.

Article Snippet: RNAi was purchased from Dharmacon (Akt3) or Santa Cruz Biotechnology (Akt3 and PGC-1α).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Transfection, Control

Journal: Nature

Article Title: Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity

doi: 10.1038/s41586-020-03056-z

Figure Lengend Snippet: a , Confirmed hits from deconvolution siRNA screens grouped according to their requirement for SIN-induced autophagy, HSV-1ΔBBD-induced autophagy, or both. b , GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with indicated siRNAs (72 h) and mock-infected or infected with indicated virus for 4.5 h. MOI = 5 for HSV-1ΔBBD; 10 for SIN, Zika virus, WNV, CHIKV and IAV; and 20 for poliovirus and CVB3. Bars represent mean ± s.d. of three independent replicates (100-150 cells per sample). P -values, one-way ANOVA with Dunnetťs test for multiple comparisons. c , Survival of Snx5 +/+ and Snx5 −/− mice infected with SIN (strain dsTE12Q, 1,000 pfu i.c., 7-day-old mice), SIN.dnAtg5 (1,000 pfu i.c., 7-day-old mice), HSV-1ΔBBD (50,000 pfu i.c., 8 to 10-week-old mice), HSV-1ΔBBD-MR (50,000 pfu i.c., 8 to 10-week-old mice), WNV (1 pfu i.c., 5.5-day-old mice) or CHIKV (100,000 pfu s.c., 7-day-old mice). Results represent combined data for at least three independent experiments per virus; similar results obtained for each infection. P -values, log-rank test (two-sided).

Article Snippet: The high-content siRNA screens used a genome-wide human siRNA library (siGENOME of Dharmacon) containing 18,115 SMARTpools, in which each pool contains four siRNA oligonucleotides targeting an individual gene.

Techniques: Infection, Virus

Journal: Nature

Article Title: Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity

doi: 10.1038/s41586-020-03056-z

Figure Lengend Snippet: a , Flowchart summary of primary and deconvolution siRNA screens in HeLa/GFP-LC3 cells. Three filters were applied in the primary siRNA screens: (1) absence of cytotoxicity of siRNA pools in transfected cells; (2) lack of effect of siRNA pools on numbers of GFP-LC3 puncta in mock-infected cells (basal autophagy); and (3) significant reduction of numbers of GFP-LC3 puncta in cells infected with either SIN or HSV-1ΔBBD (virus-induced autophagy). In the deconvolution siRNA screens, positive hits were defined as those genes with two or more individual siRNA oligonucleotides from siRNA pools that reproduced the phenotype of the siRNA pools. b - d , Ranked distribution of median scores for each siRNA pool in primary siRNA screens. Dots shown in graphs denote median scores of GFP-LC3 puncta in mock-infected cells ( b ), SIN-infected cells ( c ) and HSV-1ΔBBD-infected cells ( d ). e , A representative receiver operating characteristic (ROC) curve of quality control plates for primary and deconvolution screens. The ROC mean ± s.d. of all the quality control plates was 0.97 ± 0.03, which indicates accuracy and specificity in the identification of cellular factors that regulate numbers of GFP-LC3 puncta. f , Scatter plot of median scores of 216 confirmed hits from the deconvolution siRNA screens (open circles) and 40 negative on-plate controls (open triangles). Scores are medians from triplicate assay plates of four individual siRNA oligonucleotides per gene. The 216 confirmed hits and 40 negative on-plate controls fall into two distinct clusters. g , Gene list from deconvolution screens for virus-induced autophagy during infection with Sindbis virus (S) and HSV-1ΔBBD (H). Numbers denote number of individual siRNA oligonucleotides that scored positive in each screen. Green, genes with 2 or more positive siRNA oligonucleotides (confirmed hits); magenta, genes with <2 positive siRNA oligonucleotides (non-confirmed hits). h , Enrichment analyses of gene sets including molecular function, biological process, cellular component and protein domain categories (terms) from DAVID Bioinformatics Resources. There were 174 terms (open triangles) that contain at least two confirmed hits and have hypergeometric test P -values less than 0.05; fourteen of these terms exhibited false discovery rate (FDR) adjusted P -values less than 0.05. The enrichment score was defined as −log 10 (hypergeometric test P -value). i , Fourteen highly enriched terms plotted as a network graph. Each node represents a gene set from a variety of categories (indicated in brackets) as follows: G, GOTERM; I, INTERPRO; P, PIR_SUPERFAMILY, U, UP_SEQ_FEATURE. The size of each node corresponds to the number of confirmed siRNA hits and the color intensity is scaled according to the enrichment score. The thickness and color intensity of lines connecting two nodes correspond to the extent of overlapping genes between two gene sets. See and to for further details.

Article Snippet: The high-content siRNA screens used a genome-wide human siRNA library (siGENOME of Dharmacon) containing 18,115 SMARTpools, in which each pool contains four siRNA oligonucleotides targeting an individual gene.

Techniques: Transfection, Infection, Virus, Control

Journal: Nature

Article Title: Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity

doi: 10.1038/s41586-020-03056-z

Figure Lengend Snippet: a , b , Representative fluorescent micrographs ( a ) and quantitation ( b ) of GFP-LC3 puncta in HeLa/GFP-LC3 cells that were either mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 50 and 25, respectively; 4.5 h) in the presence (pH 5.4) or absence (pH 7.4) of an acidic pulse (that induces viral entry at the plasma membrane) after viral attachment. Arrows in a denote representative GFP-LC3 puncta that would be scored as positive in b . Scale bars, 20 μm. Bars in b represent mean ± s.d. of triplicate samples (100-150 cells analyzed per sample). c , d , Viral entry efficiency in HeLa/GFP-LC3 cells treated similarly as in a and b . Bars in c and d represent mean ± s.d. of SIN minus-strand RNA levels ( c ) and HSV-1 immediate early gene ICP27 mRNA levels ( d ) of triplicate samples at indicated time points, respectively. e , Representative fluorescent micrographs of GFP-LC3 puncta in HeLa/GFP-LC3 cells that were treated with indicated siRNAs (72 h) and then either mock-infected or infected with indicated virus for 4.5 h (MOI = 10 for SIN, Zika virus, WNV, CHIKV and IAV; MOI = 5 for HSV-1ΔBBD; and MOI = 20 for poliovirus and CVB3). Scale bars, 20 μm. Arrows in e denote representative GFP-LC3 puncta that would be scored as positive in . f - i , Confirmation of gene knockdown in indicated siRNA-treated HeLa/GFP-LC3 cells (72 h) by western blot analyses of indicated proteins ( f - h ) or quantitative real-time PCR of SNX32 ( i ) for the experiment shown in . Bars in i represent mean ± s.d. of triplicate samples. j , Western blot detection of SNX5 and actin in wild-type HeLa/GFP-LC3 cells (WT), HeLa SNX5 KO /GFP-LC3 cells (KO), and two clones of reconstituted HeLa SNX5 KO /GFP-LC3/SNX5 cells used in the experiment shown in k . In b , one-way ANOVA with Dunnett’s test for multiple comparisons was used to compare means of SIN or HSV-1ΔBBD infection versus mock infection. In b - d , unpaired two-tailed t -tests were used to compare means of pH7.4 versus pH5.4 conditions. For e-h, j , similar results were observed in three independent experiments. In i , an unpaired two-tailed t -test was used to compare means of NC versus SNX32 knockdown. k , GFP-LC3 puncta in reconstituted HeLa SNX5 KO /GFP-LC3 cells mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 4.5 h). Bars represent mean ± s.d. of three independent replicates (100-150 cells per sample). P -values, one-way ANOVA with Dunnetťs test for multiple comparisons. For gel source data, see .

Article Snippet: The high-content siRNA screens used a genome-wide human siRNA library (siGENOME of Dharmacon) containing 18,115 SMARTpools, in which each pool contains four siRNA oligonucleotides targeting an individual gene.

Techniques: Quantitation Assay, Infection, Clinical Proteomics, Membrane, Virus, Knockdown, Western Blot, Real-time Polymerase Chain Reaction, Clone Assay, Two Tailed Test

Journal: Nature

Article Title: Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity

doi: 10.1038/s41586-020-03056-z

Figure Lengend Snippet: a - c , Representative fluorescent micrographs ( a ) and quantitation ( b , c ) of GFP-LC3 puncta (autophagosomes) in HeLa/GFP-LC3 cells treated with non-targeting control siRNA (NC) or ATG7 siRNA for 72 h and then mock-infected or infected with SIN or HSV-1ΔBBD (at a multiplicity of infection [MOI] of 10 and 5, respectively; 4.5 h) in the presence or absence of the lysosomal inhibitor bafilomycin A1 (Baf A1, 100 nM) for 1 h prior to fixation. Scale bars, 20 μm. Arrows denote representative autophagosomes that would be scored as positive in b and c . The box plot with whiskers from minimum to maximum in b represents 100-150 cells analyzed for one of triplicate samples per condition. Bars in c represent mean ± s.d. of triplicate samples (100-150 cells analyzed per sample). Similar results were observed in three independent experiments. Both analyses were performed for similar fluorescence microscopy experiments in , , and Extended Data Figs. 1, , , , , , and only bar graphs are shown. d , Western blot analyses of indicated proteins in HeLa/GFP-LC3 cells treated with NC or ATG7 siRNA for 72 h as in a - c . e - g , Representative western blot detection ( e ) and quantitation ( f , g ) of indicated proteins in lysates (prepared with Triton X-100 lysis buffer) of HeLa cells mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 7 h) with the presence or absence of Baf A1 (100 nM) for 2 h (from 5 hours post infection [hpi] to 7 hpi). Bars in f and g represent mean ± s.d. of LC3-II/LC3-I ratios ( f ) and p62/actin ratios ( g ) of three independent experiments, respectively. h , i , Representative western blot detection ( h ) and quantitation ( i ) of p62 and actin in lysates (prepared with SDS lysis buffer) of HeLa cells mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 7 h). Bars in i represent mean ± s.d. of p62/actin ratios of three independent experiments. j , Quantitation of GFP-LC3 puncta in HeLa/GFP-LC3 cells treated with NC or ATG13 siRNA for 72 h and then mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 4.5 h). k , Western blot analyses of indicated proteins in HeLa/GFP-LC3 cells treated with NC or ATG13 siRNA for 72 h as in j . l , Quantitation of GFP-LC3 puncta in HeLa/GFP-LC3 cells mock-infected or infected with SIN or HSV-1ΔBBD (MOI = 10 and 5, respectively; 4.5 h) and treated with PIK-III (5 μM), Baf A1 (100 nM) or DMSO control for 1 h (from 3.5 hpi to 4.5 hpi). Bars in j and l represent mean ± s.d. of triplicate samples (100-150 cells analyzed per sample). Similar results were observed in three independent experiments. Unpaired two-tailed t -tests were used to compare means of Baf A1- versus DMSO-treated cells, those of ATG13 siRNA- versus NC-treated cells, and those of PIK-III- versus DMSO-treated cells. One-way ANOVA with Dunnett’s test for multiple comparisons was used to compare means of SIN or HSV-1ΔBBD infection versus mock infection. For gel source data, see .

Article Snippet: The high-content siRNA screens used a genome-wide human siRNA library (siGENOME of Dharmacon) containing 18,115 SMARTpools, in which each pool contains four siRNA oligonucleotides targeting an individual gene.

Techniques: Quantitation Assay, Control, Infection, Fluorescence, Microscopy, Western Blot, Lysis, Two Tailed Test