Journal: Neurophotonics

Article Title: Inducing and monitoring photothrombotic stroke in anesthetic neuroprotection-free mice using functional photoacoustic microscopy

doi: 10.1117/1.NPh.13.2.025007

Figure Lengend Snippet: Experimental design and system setup. (a) Schematic design of the custom-built dual-wavelength OR-PAM system, illustrating optical and acoustic pathways. HWP, half-wave plate; PBS, polarizing beam splitter; CP, coupler; PM-SMF, polarization-maintaining single-mode fiber; BPF, band-pass filter; M, mirror; NDF, neutral density filter; DM, dichroic mirror; L, achromatic lens; OAC, optical/acoustic beam combiner; AL, acoustic lens; WT, water tank; UT, ultrasound transducer; AM, amplifier; DAQ, data acquisition unit; PC, personal computer; SP, scanning pathway. (b) Timeline of research including mouse grouping, skull-thinning, head-fixation training, PT induction, PAM imaging, behavioral tests, and pathological examination. (c) Schematic diagram of the head-restrained awake PAM setup. PA probe, photoacoustic probe. (d) Illustration of targeted PT induced in a single distal MCA branch using 532-nm laser illumination guided by PAM. Brain region abbreviations: M1/M2, primary/secondary motor cortex; S1/S2, primary/secondary somatosensory cortex; V1, visual cortex; AU, auditory cortex; RS, retrosplenial area; BC, barrel field; FL and HL, forelimb and hindlimb regions of the primary somatosensory area. The mouse brain atlas image is adapted from Ref. .

Article Snippet: The photoacoustic signals were amplified by 48 dB using a pair of amplifiers (ZFL-500LN, Mini-Circuits, Brooklyn, New York, United States) and digitized at 500 MHz via a data acquisition card (ATS9371, Alazar Technologies, Quebec, Canada).

Techniques: Imaging

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) Identification of stable IGS transcripts by Northern blot. The location of the oligonucleotide probe used for hybridization are indicated at the top of the image. B) Scheme for the positions of the IGS transcripts at the human rDNA gene (top) and the fragments identified by small RNA-seq in control cells (bottom). C) Inhibition of RNA pol I with 5 nM Act D for 2 hours or 0.1 μM CX-5461 for 24 hours, cDNA from total RNA was analysed by qRT-PCR and the signals were normalised to 18S rRNA. The ncRNA detected is marked under the bars. D) Inhibition of RNA pol II with 4 μM Act D for 2 hours or 5 μM α-amanitin for 24 hours, cDNA from total RNA was analysed by qRT-PCR and the signals were normalised to 18S rRNA. The detected ncRNA is marked under the bars. E) ChRIP analysis of the association of IGS19as, IGS32as and IGS38 to chromatin using antibodies against histone H3, the detected RNA is marked under the bars. Actin mRNA is used as a negative control. qRT-PCR data is shown as percentage enrichment over IgG. F) ChRIP analysis of the association of IGS32as and IGS38 to chromatin with histone modifications using antibodies against H3K9me2/3, H3K27me3, H3K27Ac, and UBF, as indicated. Actin mRNA is used as a negative control. qRT-PCR data is shown as percentage enrichment over IgG.= All experiments present means of at least 3 biological replicates. Error bars show the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, **p≤ 0.01,*** p≤ 0.001 and **** p≤ 0.0001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: Northern Blot, Hybridization, RNA Sequencing, Control, Inhibition, Quantitative RT-PCR, Negative Control, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) Analysis of the 47S rRNA level after knock down of IGS38 using LNA-DNA Gapmers at 10 and 30 nM. cDNA from total RNA was normalised to 18S rRNA and the RNA levels are expressed relative to levels in cells treated with control LNA-DNA. B) Analysis of the 47S rRNA level after knock down of IGS32as. cDNA from total RNA was normalised to 18S rRNA. RNA levels are expressed relative to levels in cells treated with control LNA-DNA. C) Overexpression of IGS38 with three different fragments: Fragment 1, 200 bp from location 38.759 bp to 38.957 bp; Fragment 2, 200 bp from location 38.958 kb to 39.156 kb; Fragment 3, 400 bp from 38.756 bp to 39.156 bp, analysed for 47S rRNA levels by qRT-PCR. Normalisation was done to 18S rRNA and the RNA levels are related to the level in cells transfected with control vector. D) ATAC-qPCR analysis to detect chromatin accessibility at the spacer promoter, enhancer region and the 47S rRNA promoter in IGS38 knock down cells, IGS32as knock down cells or control cells. Signal values were normalised to the 27 kb region and presented as knock downs related to control. E) ATAC-qPCR data zoomed in at the enhancer region and 47S rRNA promoter region in IGS38 knock down cells or control cells. F) RNA-FISH localisation of IGS38 RNA in the nucleolus. Probes against 47S rRNA were used as a positive control and a bacterial probe as a negative control. Fibrillarin was used as a nucleolar marker and the merge shows fibrillarin and the RNAs (IGS38 or 47S rRNA) together, DAPI marked the nucleus. The scale bar marks 10 μm. G) CHART analysis using specific probes against IGS38 and IGS32as ncRNA at the spacer promoter and the rRNA promoter. The actin promoter was used as a negative control region. Data is presented as fold enrichment after normalising to no oligo control. All experiments present means for at least 3 biological replicates. Error bars represent the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, **p≤ 0.01 and *** p≤ 0.001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: Knockdown, Control, Over Expression, Quantitative RT-PCR, Transfection, Plasmid Preparation, Positive Control, Negative Control, Marker, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) ChRIP analysis of IGS38 and IGS32as association with B-WICH bound chromatin using antibodies against WSTF and CSB. Actin was used as a negative control. qRT-PCR data is shown as percentage enrichment over IgG. B) RNA IP to analyse direct interactions between IGS38 and IGS32as with WSTF and CSB. qRT-PCR data is shown as percentage enrichment over IgG. C) ChIP-qPCR of WSTF occupancy at the spacer promoter and 47S rRNA promoter under IGS38 knock down cells, IGS32as knock down cells or control cells. Signals are calculated as percentage of input samples. D) qRT-PCR of WSTF and IGS38 levels in WSTF knock down cells related to control cells. The RNA is normalised to 18S rRNA. All experiments present means for at least 3 biological replicates. Error bars represent standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, **p≤ 0.01 and *** p≤ 0.001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: Negative Control, Quantitative RT-PCR, ChIP-qPCR, Knockdown, Control, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) ChIP-qPCR analysis of UBF occupancy at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. B) ChIP-qPCR analysis of histone H3 occupancy at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. C) ChIP-qPCR analysis of histone H2A.Z occupancy at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. D) ChIP-qPCR analysis of CTCF occupancy at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cellsE) ChIP-qPCR analysis of TTF1 occupancy at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. All signals from A) - E) are calculated as percentage of input samples. F) ChIP-qPCR analysis of the presence of the histone H3K27me3 at the spacer promoter and rRNA promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. The signal is relative to the histone H3 signal. All experiments present means for at least 3 biological replicates. Error bars represent the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, **p≤ 0.01 and *** p≤ 0.001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: ChIP-qPCR, Control, Knockdown, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) Graphical scheme of the locations of the primers used over the rDNA gene repeat: spacer promoter, rRNA promoter, ETS (1 kb), 4 kb (18S), 8 kb (28S), 13 kb (Transcription terminator site, TTS) and 27 kb (in the IGS). B) ChIP-qPCR analysis of RNA pol I occupancy at the spacer promoter and 47S rRNA gene promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. C) ChIP-qPCR analysis of RNA pol I occupancy in the gene body with primers indicated in (A) in control cells, IGS38 knock down cells and IGS32as knock down cells. D) ChIP-qPCR analysis of RRN3 occupancy at the spacer promoter and 47S rRNA gene promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. E) ChIP-qPCR analysis of TBP occupancy at the spacer promoter and 47S rRNA gene promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. F) ChIP-qPCR analysis of the SL1 component TAF1C occupancy at the spacer promoter and 47S rRNA gene promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. The signals in all panels are calculated as percentage of input samples. All experiments present means for at least 3 biological replicates. Error bars represent the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05 and **p≤ 0.01.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: ChIP-qPCR, Control, Knockdown, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) ChRIP of the association of IGS38 and IGS32as with TAF1C and RRN3 in chromatin. Actin was used as a negative control. RT-qPCR data is shown as percentage enrichment over IgG. B) RNA IP to analyse direct interactions between IGS38 and IGS32as with TAF1C, RRN3 and UBF. RT-qPCR data is shown as percentage enrichment over IgG. C) Model of the action of IGS38 and WSTF at the 47S rRNA gene promoter in growing cells (top) compared to IGS knock down cells (bottom). In growing cells IGS38 stabilises RNA Pol I in a transcription competent state, whereas in IGS38 knock down cells UBF levels are reduced, RRN3 is lost together with WSTF and concurrent the RNA pol I is accumulating at the promoter. Created by biorender ( https://biorender.com/shortURL ). D) Spacer RNA transcript expression at the spacer promoter (location 42235/-765), downstream of the TSS of the spacer-RNA (location 42370/-630), and at the T0 site (location 42807/-193), binding site of the pRNA. The qRT-PCR data was normalised to 18S rRNA and shown as fold change over Gapmer control. E) qRT-PCR of IGS38 KD and control cells to determine RNA levels of the of PAPAS expression at the position 42897/-103 at the 47S RNA promoter and 42951/-49 over the TSS of the 47S rRNA. The data was normalised to 18S rRNA and shown as fold change over Gapmer control. F) ChIP-qPCR analysis of CHD4 occupancy at the spacer promoter and 47S rRNA gene promoter in control cells, IGS38 knock down cells, and IGS32as knock down cells. The signals are calculated as percentage of input samples. All experiments present means for at least 3 biological replicates. Error bars represent the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, ** p≤ 0.01, *** p≤ 0.001 and **** p≤ 0.0001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: Negative Control, Quantitative RT-PCR, Knockdown, Expressing, Binding Assay, Control, ChIP-qPCR, Standard Deviation

Journal: bioRxiv

Article Title: IGS38, a lncRNA from the human rDNA intergenic spacer, regulates rRNA transcription by altering rDNA chromatin organisation and activating the transcription machinery

doi: 10.64898/2026.05.02.722362

Figure Lengend Snippet: A) qRT-PCR of the RNA levels of IGS19as, IGS32as, IGS38, PAPAS and 47S rRNA in cells exposed to hypotonic stressed and control cells. The data was normalised to 18S rRNA and shown as fold change over control. B) ChIP-qPCR analysis of WSTF, CHD4, Suv4-20h2 and Pol I occupancies at the 47S rRNA gene promoter in cells exposed to hypotonic and control cells. The signals are calculated as percentage of input samples. C) ChRIP of the association IGS38 and IGS32as with chromatin bound WSTF, CHD4 and Suv4-20h2 in cells exposed to hypotonic stress and control cells. RT-qPCR data is shown as percentage enrichment over IgG. D) ChRIP of the association of PAPAS with chromatin bound WSTF, CHD4 and Suv4-20h2 in cells exposed to hypotonic stress and control cells. The actin RNA was used as a control. RT-qPCR data is shown as percentage enrichment over IgG. E) Volcano plot for RNA sequencing of IGS38 knock down cells to control showing significant differentially expressed genes. F) Genes in the Gene Ontology term Interferon response genes, differentially expressed in IGS38 knock down cells compared to Gapmer control cells. G) Immunofluorescence in control and IGS38 knock down cells stained with J2-antibody against dsRNA (red), FITC-phallodin actin filaments in the cytoplasm (green), and DAPI staining the nucleus (blue). The size bars in the images represent 10 μm. H) The abundance of dsRNA by Dot Blot in Gapmer control cells, IGS38 knock down cells, and IGS32as knock down cells using the J2 antibody at 8 hours and 24 hours as indicated above the image in the left panel. Quantification of the dsRNA levels in the samples are shown at 8 hours and 24 hours are shown in the right panel. The median value in each box is marked. All experiments present means for at least 3 biological replicates. Error bars represent the standard deviation. P-values are calculated with unpaired student’s t-test: * p≤ 0.05, ** p≤ 0.01 and *** p≤ 0.001.

Article Snippet: Transcriptomic sequencing was performed on cell transfected with control scramble LNA-DNA Gapmers or cells transfected with IGS38 target LNA-DNA Gapmers after 24 hpt at a concentration of 30nM, using Eurofins Genomics service.

Techniques: Quantitative RT-PCR, Control, ChIP-qPCR, RNA Sequencing, Knockdown, Immunofluorescence, Staining, Dot Blot, Standard Deviation