Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of the levels of ncRNAs in different models of HD.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Transfection

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of protein and miRNA interactions of ncRNAs Meg3, Neat1, and Xist.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Expression of genes in HD associated with Huntington’s disease pathway (KEGG: 05016 and PANTHER: {"type":"entrez-protein","attrs":{"text":"P00029","term_id":"124076962","term_text":"P00029"}} P00029 ) and coded for protein interacting partners of NEAT1 or MEG3.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Expressing

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Summary of co-expressed genes of MEG3, NEAT1, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: MEG3, NEAT1 and XIST interacting protein enriched with HD pathway.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques:

Journal: RNA Biology

Article Title: Altered Levels of Long NcRNAs Meg3 and Neat1 in Cell And Animal Models Of Huntington’s Disease

doi: 10.1080/15476286.2018.1534524

Figure Lengend Snippet: Transcription factors that bind within 5 Kb upstream sequences of NEAT1, MEG3, and XIST.

Article Snippet: SiRNA mediated knockdown of Neat1 and Meg3 siRNAs against mouse NEAT1 (FlexiTube GeneSolution GS66961 for Neat1, Cat no: LPM15183A) and MEG3 (FlexiTube GeneSolution GS17263 for Meg3, Cat No: GS17263) were purchased from Qiagen and used, following the protocol described by the manufacturer. siRNAs were used at a final concentration of 40nM.

Techniques: Sequencing

Journal: Nature Communications

Article Title: Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1

doi: 10.1038/ncomms5589

Figure Lengend Snippet: ( a ) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min. Cell lysates were analysed using western blots with phospho-ERK1/2 (p-ERK1/2) or total ERK1/2. ( b ) Quantified time course in a showing ERK1/2 phosphorylation levels induced by 10 μM of each compound. ( c ) HEK293 cells expressing SEP-CB1Rs were exposed to 10μM CP55940, ORG27569, WIN 55212-2 and 2-AG for 5, 10 and 15 min with PTX pre-treatment, respectively. ( d ) Quantified time course in c showing ERK1/2 phosphorylation levels from cells pretreated with PTX. ( e , g , i , k ) HEK293 cells co-expressing SEP-CB1R and either control ( e ), β-arrestin-1 ( g ), β-arrestin-2 ( i ) siRNAs or β-arrestin-2 siRNA with PTX pretreatment ( k ) were exposed to 10 μM of CP55940, ORG27569, WIN 55212-2 and 2-AG, respectively, as indicated. ( f , h , j , l ). Graphs in f , h , j , l provide the quantified ERK1/2 phosphorylation levels induced by 10 μM of each compound as shown in e , g , i , k . Data represent the mean±s.e.m. of at least three independent experiments.

Article Snippet: Briefly, HEK293 cells that were 40–50% confluent in a six-well plate were transfected with the plasmid encoding rat CB1 and 2.6 mg of siRNA (Qiagen) sequences targeting β-arrestin 1, β-arrestin 2 or non-silencing RNA duplex for control.

Techniques: Expressing, Western Blot, Phospho-proteomics, Control

Journal: Nature Communications

Article Title: Ligand-specific endocytic dwell times control functional selectivity of the cannabinoid receptor 1

doi: 10.1038/ncomms5589

Figure Lengend Snippet: ( a ) HEK293 cells expressing SEP-CB1R were preincubated with 30μM dyngo-4a and imaged under TIRF before and after bath application of 10μM WIN55212-2. Representative kymograph shows prolonged SEP-CB1R dwell times in the presence of dyngo-4a. ( b ) HEK293 cells expressing SEP-CB1R were pre-incubated with 30μM dyngo-4a before exposure to agonists and compared with no dyngo-4a treatment. ( c ) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in b . Data are expressed as a percentage of the level of phosphorylation at 5 min for each compound without dyngo-4a pre-treatment. ( d ) HEK293 cells expressing SEP-CB1Rs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated with PTX pre-treatment. ( e ) HEK293 cells co-expressing SEP-CB1R and β-arrestin-1 siRNAs with and without dyngo-4a pretreatment were exposed to 10μM 2-AG as indicated. ( f ) Quantified time course in d , e showing ERK1/2 phosphorylation levels induced by 10μM 2-AG for 5, 15, 30, 45 and 60 min for cells either pre-treated with PTX or co-transfected with β-arrestin-1 siRNA. ( g ) HEK293 cells expressing SEP-CB1R were treated with PTX (or no PTX as a control), and then incubated with 30μM dyngo-4a before subsequent exposure to WIN 55,212-2. ( h ) Graph provides the quantified ERK1/2 phosphorylation induced by 10μM WIN 55,212-2 in the absence of PTX treatment shown in g . ( i ) HEK293 cells expressing SEP-CB1R with or without dominant-negative dynamin 2 K44A were exposed to agonists. ( j ) Graph provides the quantified ERK1/2 phosphorylation induced by 10 μM of each compound shown in i . All data are expressed as a percentage of the level of phosphorylation at 5 min for 2-AG without dyngo-4a pre-treatment and represent the mean±s.e.m. of at least three independent experiments.

Article Snippet: Briefly, HEK293 cells that were 40–50% confluent in a six-well plate were transfected with the plasmid encoding rat CB1 and 2.6 mg of siRNA (Qiagen) sequences targeting β-arrestin 1, β-arrestin 2 or non-silencing RNA duplex for control.

Techniques: Expressing, Incubation, Phospho-proteomics, Transfection, Control, Dominant Negative Mutation

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

doi: 10.1038/labinvest.2013.66

Figure Lengend Snippet: Expression of Toll-like receptor 4 (TLR4) in the lung tissue from rats at 18 h after liver ischemia/reperfusion (I/R) injury or sham operation. TLR4 mRNA ( a , real-time PCR) and protein ( b , western blot) expression levels in the lung tissue from rats at 18 h after liver I/R injury or sham operation. * P <0.05 compared with the control group; † P <0.05 compared with the non-targeting short hairpin RNA (shNT) group; # P <0.05 compared with the shNT+I/R group. Relative levels of TLR4 mRNA to the control group are normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and relative levels of TLR4 protein to the control group are normalized to β -actin. Data are expressed as means±s.d.. Blots shown here are from a representative experiment repeated three times with similar results ( n =6 per group). Control, control group; I/R, liver I/R injury group; shNT, negative control group; shNT+I/R, positive control group; shTLR4, TLR4 inhibition group; shTLR4+I/R, TLR4 inhibition group with liver I/R injury treatment.

Article Snippet: ShRNA targeting Rattus norvegicus TLR4 gene (shTLR4; GenBank accession no.: NM_019178 ) and non-targeting shRNA (shNT) sequences were designed, chemically synthesized, and inserted into the pGCSIL-GFP by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, shRNA, Negative Control, Positive Control, Inhibition

Journal: Laboratory Investigation; a Journal of Technical Methods and Pathology

Article Title: TLR4 as receptor for HMGB1-mediated acute lung injury after liver ischemia/reperfusion injury

doi: 10.1038/labinvest.2013.66

Figure Lengend Snippet: Acute lung injury and inflammation at 18 h after liver ischemia/reperfusion (I/R) injury or sham operation. ( a ) Morphological analysis of acute lung injury at 18 h after liver I/R injury ( × 100); ( b ) changes in lung histological scores; ( c ) P aO 2 /FiO 2 (P/F) ratios were obtained at baseline and 18 h after liver I/R injury; ( d ) interleukin (IL)-1 β levels in the lung tissue from rats at 18 h after liver I/R injury. * P <0.05, ** P <0.01 compared to the control group; # P <0.05 compared to non-targeting short hairpin RNA (shNT)+I/R group; § P <0.05 compared to baseline. Data are expressed as means±s.d. ( n =6 per group). Control, control group; I/R, liver I/R injury group; shNT, negative control group; shNT+I/R, positive control group; shTLR4, TLR4 inhibition group; shTLR4+I/R, TLR4 inhibition group with liver I/R injury treatment.

Article Snippet: ShRNA targeting Rattus norvegicus TLR4 gene (shTLR4; GenBank accession no.: NM_019178 ) and non-targeting shRNA (shNT) sequences were designed, chemically synthesized, and inserted into the pGCSIL-GFP by Shanghai Genechem Co. Ltd (Shanghai, China).

Techniques: shRNA, Negative Control, Positive Control, Inhibition

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: siRNA screen to identify Rab proteins affecting cell migration (A) U2OS cells transfected with control siRNA or pools of four different siRNAs against human Rabs were grown to confluency, scratch-wounded, and imaged every 3 h. The graph shows the relative wound density at 18 h after wounding represented as mean ± SEM of four independent experiments. The red solid line indicates the relative wound density (%) and the dashed red lines the boundaries of the SEM for cells transfected with control siRNA. (B) Representative images are shown for time 0 and 18 h after wounding for the two Rabs whose depletion had the strongest effect on wound closure. Scale bar: 200 μm. (C) U2OS cells transfected with control siRNA or a pool of four different siRNAs targeting Rab33b were imaged for 48 h. The rate of proliferation (measured as percentage of cell confluence) over time is shown as mean ± SEM of three independent experiments. (D) U2OS cells were transfected with control siRNA or each of the different individual four siRNAs present in the pool targeting Rab33b (Rab33b siRNA_1, siRNA_2, siRNA_3, or siRNA_4), grown to confluency, scratch-wounded, and imaged every 3 h. Representative images for time 0 and 24 h after wounding are shown. Scale bar: 200 μm. (E) Graph showing the relative wound density (%) over time for each sample in (d). The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, n.s. not significant, for t = 24h (two-tailed paired Student′s t-test). (F) Lysates from U2OS cells transfected with control siRNA, or with each of the different individual four siRNAs present in the pool targeting Rab33b were subjected to Western blot analysis with the indicated antibodies.

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

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

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b depletion promotes cell migration (A) U2OS cells transfected with either control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b or transiently transfected with GFP, or GFP-Rab33b T47N, were scratch-wounded and imaged every 3 h. Representative images for time 0 and 23 h after wounding are shown. Scale bar: 200 μm. (B) Graph showing relative wound density (%) for each sample in (a) over time. The graph represents the mean ± SEM of a minimum of three independent experiments. ∗p < 0.05, n.s., not significant, for t = 24h (two-tailed paired Student′s t-test). (C) Cell lysates from cells treated with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b were subjected to Western blot analysis with antibodies against Rab33b and tubulin (as loading control). (D) Representative track plots of the single-cell distances of migration for cells transfected with control siRNA, Rab33b siRNA_1, Rab33b siRNA_2, or treated with the same siRNAs against Rab33b and afterwards transfected with GFP-Rab33b. Individual tracks are shown so that each starts at the origin (distance 0). (E) Quantification of the mean ± SEM of the single cell speed from at least three independent experiments. n > 30 cells per condition and per experiment. ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figures S1 A–S1B

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

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

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: Rab33b is involved in the regulation of focal adhesion dynamics (A) U2OS cells silenced with control siRNA, Rab33b siRNA_1, or Rab33b siRNA_2, were fixed and stained with DAPI, rhodamine-conjugated phalloidin, and an antibody against vinculin. Scale bar: 5 μm. (B–C) Quantification of FA number per 100 μm 2 cell area (b) and size (c). The graphs represent the mean ± SEM for three independent experiments (n > 90 cells). ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (D) Control or Rab33b-depleted cells transfected with RFP-vinculin were imaged every 10 min for 40 min. Arrows show FA disassembly, and arrowheads show FA assembly. Scale bar: 10 μm. (E) Rainbow color representation of FA assembly and disassembly over time from cells shown in panel (d). Each time point is shown in a different color, as indicated in the bar. Insets show magnifications of the boxed areas. Scale bar: 10 μm. (F) Quantification of assembly and disassembly rates of FAs. The assembly and disassembly rate is shown as percentage of focal adhesion formation or disassembly per minute. The values represent the mean ± SEM from three independent experiments, in which 15 FAs were analyzed per cell (n > 5), per condition, and per experiment. ∗p < 0.05, n.s., not significant (two-tailed paired Student′s t-test). (G) Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and RFP-vinculin (red). Cells were imaged every 5 s with a Zeiss LSM880 confocal microscope. Magnifications of the boxed areas in the side panels show Rab33b-positive vesicles moving to focal adhesion sites. Scale bar: 5μm, inset: 1μm. See also Figure S2 and Video S1. Rab33b-positive vesicles are delivered to FAs, Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnification of the boxed area 1 in Figure 4g is shown and illustrates two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S2. Rab33b-positive vesicles are delivered to FAs,Live-cell imaging of U2OS cells co-transfected with GFP-Rab33b (green) and vinculin-RFP (red). Magnifications of the boxed area 2 in Figure 4g is shown and illustrate two examples of Rab33b-positive vesicles contacting focal adhesions. Cells were imaged every 5 s using a spinning disk confocal microscope. Related to Figure 4. , Video S3. Rab33b-positive vesicles are delivered to growing FAs during membrane protrusion,U2OS cells transiently transfected with GFP-Rab33b (green) and vinculin-RFP (red) were imaged every 30 s using a TIRF microscope with a penetration depth of 90 nm. The movie shows the recruitment of GFP-Rab33b-positive vesicles during membrane protrusion. Related to Figure 4. .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Staining, Two Tailed Test, Transfection, Live Cell Imaging, Microscopy

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet: VSV-G transport to the cell surface is inhibited by Rab33b depletion (A) U2OS cells silenced with control siRNA, siRNA against Rab33b, or silenced with Rab33b siRNA and transfected with RFP-Rab33b (red), were transfected with YFP-VSV-G (green) and incubated at 39°C for 16 h. Cells were then fixed either immediately (T0), 20 min (T20), or 90 min (T90) after a shift to 32°C. Scale bar: 10 μm. (B) Quantification of the VSV-G distribution 90 min after the shift to 32°C. 200 cells were analyzed from three independent experiments and the percentage of cells in which YFP-VSV-G was located at the Golgi, post-Golgi vesicles, and plasma membrane was determined. The graph shows the mean ± SEM ∗p < 0.05, ∗∗p < 0.01, n.s., not significant (two-tailed paired Student′s t-test). See also Figure S4 .

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transfection, Incubation, Two Tailed Test

Journal: iScience

Article Title: Rab33b-exocyst interaction mediates localized secretion for focal adhesion turnover and cell migration

doi: 10.1016/j.isci.2022.104250

Figure Lengend Snippet:

Article Snippet: Non-targeting control siRNA (sense sequence 5′-ACUUCGAGCGUGCAUGGCUTT-3′ and antisense 5′-AGCCAUGCACGCUCGAAGUTT-3′) was purchased from MWG-Biotech (Ebersberg, Germany).

Techniques: Transduction, Recombinant, Two Hybrid Screening, Sequencing, Plasmid Preparation, Software